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  • Articles  (7,862)
  • Blackwell Publishing Ltd
  • American Institute of Physics (AIP)
  • Medicine  (7,862)
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  • 1
    Electronic Resource
    Electronic Resource
    Blue light negatively regulates the sexual filamentation via the Cwc1 and Cwc2 proteins in Cryptococcus neoformans (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Cryptococcus neoformans is a heterothallic basidiomycetous yeast that primarily infects immunocompromised individuals. Dikaryotic hyphae resulting from the fusion of the MATa and MATα mating type strains represent the filamentous stage in the sexual life cycle of C. neoformans. In this study we demonstrate that the production of dikaryotic filaments is inhibited by blue light. To study blue light photoresponse in C. neoformans, we have identified and characterized two genes, CWC1 and CWC2, which are homologous to Neurospora crassa wc-1 and wc-2 genes. Conserved domain analyses indicate that the functions of Cwc1 and Cwc2 proteins may be evolutionally conserved. To dissect their roles in the light response, the CWC1 gene deletion mutants are created in both mating type strains. Mating filamentation in the bilateral cross of cwc1 MATa and MATα strains is not sensitive to light. The results indicate that Cwc1 may be an essential regulator of light responses in C. neoformans. Furthermore, overexpression of the CWC1 or CWC2 gene requires light activation to inhibit sexual filamentation, suggesting both genes may function together in the early step of blue light signalling. Taken together, our findings illustrate blue light negatively regulates the sexual filamentation via the Cwc1 and Cwc2 proteins in C. neoformans.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2005.04549.x
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  • 2
    Electronic Resource
    Electronic Resource
    Retrotransposition strategies of the Lactococcus lactis Ll.LtrB group II intron are dictated by host identity and cellular environment (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Group II introns are mobile retroelements that invade their cognate intron-minus gene in a process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. Previous studies of the Lactococcus lactis intron Ll.LtrB indicated that in its native host, as in Escherichia coli, retrohoming occurs by the intron RNA reverse splicing into double-stranded DNA (dsDNA) through an endonuclease-dependent pathway. However, in retrotransposition in L. lactis, the intron inserts predominantly into single-stranded DNA (ssDNA), in an endonuclease-independent manner. This work describes the retrotransposition of the Ll.LtrB intron in E. coli, using a retrotransposition indicator gene previously employed in our L. lactis studies. Unlike in L. lactis, in E. coli, Ll.LtrB retrotransposed frequently into dsDNA, and the process was dependent on the endonuclease activity of the intron-encoded protein. Further, the endonuclease-dependent insertions preferentially occurred around the origin and terminus of chromosomal DNA replication. Insertions in E. coli can also occur through an endonuclease-independent pathway, and, as in L. lactis, such events have a more random integration pattern. Together these findings show that Ll.LtrB can retrotranspose through at least two distinct mechanisms and that the host environment influences the choice of integration pathway. Additionally, growth conditions affect the insertion pattern. We propose a model in which DNA replication, compactness of the nucleoid and chromosomal localization influence target site preference.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2005.04554.x
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  • 3
    Electronic Resource
    Electronic Resource
    Nck-independent actin assembly is mediated by two phosphorylated tyrosines within enteropathogenic Escherichia coli Tir (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Enteropathogenic Escherichia coli (EPEC) stimulates tyrosine-kinase signalling cascades to trigger localized actin assembly within mammalian cells. During actin ‘pedestal’ formation, the EPEC effector protein Tir is translocated into the plasma membrane, becomes phosphorylated on tyrosine-474 (Y474) and promotes recruitment of the mammalian adaptor protein Nck to efficiently activate N-WASP-Arp2/3-mediated actin polymerization. Tir also triggers localized actin assembly in the absence of Nck, but the Tir sequences involved in this signalling cascade have not been defined. To identify and characterize the phosphotyrosines that contribute to Nck-independent pedestal formation, we investigated the regulation of Tir tyrosine phosphorylation and found that phosphorylation is stimulated by Tir clustering. In addition to Y474, residue Y454 is also phosphorylated, although at lower efficiency. These tyrosines differentially contribute to actin polymerization in a fashion reminiscent of actin ‘tail’ formation mediated by the vaccinia virus envelope protein A36R, which utilizes two similarly spaced phosphotyrosines to recruit the adaptors Nck and Grb2, respectively, in order to stimulate N-WASP. Neither phosphorylated Y454 nor Y474 directly bind Grb2, but Tir derivatives harbouring these residues ultimately recruit N-WASP and Arp2/3 independently of Nck, suggesting that EPEC exploits additional phosphotyrosine-binding adaptors capable of initiating actin assembly.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2005.04558.x
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  • 4
    Electronic Resource
    Electronic Resource
    The TyrR regulon (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 55 (2005), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The TyrR protein of Escherichia coli can act both as a repressor and as an activator of transcription. It can interact with each of the three aromatic amino acids, with ATP and, under certain circumstances, with the C-terminal region of the α-subunit of RNA polymerase. TyrR protein is a dimer in solution but in the presence of tyrosine and ATP it self-associates to form a hexamer. Whereas TyrR dimers can, in the absence of any aromatic amino acids, bind to certain recognition sequences referred to as ‘strong TyrR boxes’, hexamers can bind to extended sequences including lower-affinity sites called ‘weak TyrR boxes’, some of which overlap the promoter. There is no single mechanism for repression, which in some cases involves exclusion of RNA polymerase from the promoter and in others, interference with the ability of bound RNA polymerase to form open complexes or to exit the promoter. When bound to a site upstream of certain promoters, TyrR protein in the presence of phenylalanine, tyrosine or tryptophan can interact with the α-subunit of RNA polymerase to activate transcription. In one unusual case, activation of a non-productive promoter is used to repress transcription from a promoter on the opposite strand. Regulation of individual transcription units within the regulon reflects their physiological function and is determined by the position and nature of the recognition sites (TyrR boxes) associated with each of the promoters. The intracellular levels of the various forms of the TyrR protein are also postulated to be of critical importance in determining regulatory outcomes. TyrR protein remains a paradigm for a regulator that is able to interact with multiple cofactors and exert a range of regulatory effects by forming different oligomers on DNA and making contact with other proteins. A recent analysis identifying putative TyrR boxes in the E. coli genome raises the possibility that the TyrR regulon may extend beyond the well-characterized transcription units described in this review.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2004.04385.x
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  • 5
    Electronic Resource
    Electronic Resource
    Sequential binding of SeqA protein to nascent DNA segments at replication forks in synchronized cultures of Escherichia coli (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 55 (2005), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: To demonstrate that sequestration A (SeqA) protein binds preferentially to hemimethylated GATC sequences at replication forks and forms clusters in Escherichia coli growing cells, we analysed, by the chromatin immunoprecipitation (ChIP) assay using anti-SeqA antibody, a synchronized culture of a temperature-sensitive dnaC mutant strain in which only one round of chromosomal DNA replication was synchronously initiated. After synchronized initiation of chromosome replication, the replication origin oriC was first detected by the ChIP assay, and other six chromosomal regions having multiple GATC sequences were sequentially detected according to bidirectional replication of the chromosome. In contrast, DNA regions lacking the GATC sequence were not detected by the ChIP assay. These results indicate that SeqA binds hemimethylated nascent DNA segments according to the proceeding of replication forks in the chromosome, and SeqA releases from the DNA segments when fully methylated. Immunofluorescence microscopy reveals that a single SeqA focus containing paired replication apparatuses appears at the middle of the cell immediately after initiation of chromosome replication and the focus is subsequently separated into two foci that migrate to 1/4 and 3/4 cellular positions, when replication forks proceed bidirectionally an approximately one-fourth distance from the replication origin towards the terminus. This supports the translocating replication apparatuses model.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2004.04389.x
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  • 6
    Electronic Resource
    Electronic Resource
    The human malaria parasite Plasmodium falciparum possesses two distinct dihydrolipoamide dehydrogenases (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 55 (2005), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Plasmodium falciparum genome contains genes encoding three α-ketoacid dehydrogenase multienzyme complexes (KADHs) that have central metabolic functions. The parasites possess two distinct genes encoding dihydrolipoamide dehydrogenases (LipDH), which are indispensable subunits of KADHs. This situation is reminiscent of that in plants, where two distinct LipDHs are found in mitochondria and chloroplasts, respectively, that are part of the organelle-specific KADHs. In this study, we show by reverse transcription polymerase chain reaction (RT-PCR) that the genes encoding subunits of all three KADHs, including both LipDHs, are transcribed during the erythrocytic development of P. falciparum. Protein expression of mitochondrial LipDH and mitochondrial branched chain α-ketoacid dihydrolipoamide transacylase in these parasite stages was confirmed by Western blotting. The localization of the two LipDHs to the parasite's apicoplast and mitochondrion, respectively, was shown by expressing the LipDH N-terminal presequences fused to green fluorescent protein in erythrocytic stages of P. falciparum and by immunofluorescent colocalization with organelle-specific markers. Biochemical characterization of recombinantly expressed mitochondrial LipDH revealed that the protein has kinetic and physicochemical characteristics typical of these flavo disulphide oxidoreductases. We propose that the mitochondrial LipDH is part of the mitochondrial α-ketoglutarate dehydrogenase and branched chain α-ketoacid dehydrogenase complexes and that the apicoplast LipDH is an integral part of the pyruvate dehydrogenase complex which occurs only in the apicoplast in P. falciparum.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2004.04398.x
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  • 7
    Electronic Resource
    Electronic Resource
    Regulation of Sin recombinase by accessory proteins (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Sin recombinase from Staphylococcus aureus acts selectively on directly repeated resH sites, assembling an intertwined synapse in which exactly three supercoils are trapped between the points of strand exchange. Resolution requires the two Sin binding sites in resH (site I, where strand exchange occurs, and site II) and a non-specific DNA-bending protein (e.g. Hbsu). We show that a single amino acid substitution in Sin (I100T) is sufficient to relax the normal requirements for site II and Hbsu. Using this hyperactive protein, and the variant recombination site resHAT, we investigate the roles of site II and Hbsu in synapsis and strand exchange. We conclude that Sin bound at site II, and Hbsu, act together to control site I alignment and the topology of the synapse, and to stimulate strand exchange.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2004.04550.x
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  • 8
    Electronic Resource
    Electronic Resource
    Protein kinase C modulates light responses in Neurospora by regulating the blue light photoreceptor WC-1 (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Neurospora protein kinase C (NPKC) is a regulator of light responsive genes. We have studied the function of NPKC in light response by investigating its biochemical and functional interaction with the blue light photoreceptor white-collar 1 (WC-1), showing that activation of NPKC leads to a significant decrease in WC-1 protein levels. Furthermore, we show that WC-1 and NPKC interact in a light-regulated manner in vivo, and that protein kinase C (PKC) phosphorylates WC-1 in vitro. We designed dominant negative and constitutively active forms of PKC which are able to induce either a large increase of WC-1 protein level or a strong reduction respectively. Moreover, these changes in PKC activity result in an altered light response. As WC-1 is a key component of Neurospora circadian clock and regulates the clock oscillator component FRQ we investigated the effect of NPKC-mutated forms on FRQ levels. We show that changes in PKC activity affect FRQ levels and the robustness of the circadian clock. Together these data identify NPKC as a novel component of the Neurospora light signal transduction pathway that modulates the circadian clock.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2005.04545.x
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  • 9
    Electronic Resource
    Electronic Resource
    Co-regulation of Salmonella enterica genes required for virulence and resistance to antimicrobial peptides by SlyA and PhoP/PhoQ (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Analysis of the transcriptome of slyA mutant Salmonella enterica serovar Typhimurium revealed that many SlyA-dependent genes, including pagC, pagD, ugtL, mig-14, virK, phoN, pgtE, pipB2, sopD2, pagJ and pagK, are also controlled by the PhoP/PhoQ regulatory system. Many SlyA- and PhoP/PhoQ-co-regulated genes have functions associated with the bacterial envelope, and some have been directly implicated in virulence and resistance to antimicrobial peptides. Purified His-tagged SlyA binds to the pagC and mig-14 promoters in regions homologous to a previously proposed ‘SlyA-box’. The pagC promoter lacks a consensus PhoP binding site and does not bind PhoP in vitro, suggesting that the effect of PhoP on pagC transcription is indirect. Stimulation of pagC expression by PhoP requires SlyA. Levels of SlyA protein and mRNA are not significantly changed under low-magnesium PhoP-inducing conditions in which pagC expression is profoundly elevated, however, indicating that the PhoP/PhoQ system does not activate pagC expression by altering SlyA protein concentration. Models are proposed in which PhoP may control SlyA activity via a soluble ligand or SlyA may function as an anti-repressor to allow PhoP activation. The absence of almost all SlyA-activated genes from the Escherichia coli K12 genome suggests that the functional linkage between the SlyA and PhoP/PhoQ regulatory systems arose as Salmonella evolved its distinctive pathogenic lifestyle.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2005.04553.x
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  • 10
    Electronic Resource
    Electronic Resource
    Granulocytes govern the transcriptional response, morphology and proliferation of Candida albicans in human blood (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Survival in blood and escape from blood vessels into tissues are essential steps for the yeast Candida albicans to cause systemic infections. To elucidate the influence of blood components on fungal growth, morphology and transcript profile during bloodstream infections, we exposed C. albicans to blood, blood fractions enriched in erythrocytes, polymorphonuclear or mononuclear leukocytes, blood depleted of neutrophils and plasma. C. albicans cells exposed to erythrocytes, mononuclear cells, plasma or blood lacking neutrophils were physiologically active and rapidly switched to filamentous growth. In contrast, the presence of neutrophils arrested C. albicans growth, enhanced the fungal response to overcome nitrogen and carbohydrate starvation, and induced the expression of a large number of genes involved in the oxidative stress response. In particular, SOD5, encoding a glycosylphosphatidylinositol (GPI)-anchored superoxide dismutase localized on the cell surface of C. albicans, was strongly expressed in yeast cells that were associated with neutrophils. Mutants lacking key genes involved in oxidative stress, morphology or virulence had significantly reduced survival rates in blood and the neutrophil fraction, but remained viable for at least 1 h of incubation when exposed to erythrocytes, mononuclear cells, plasma or blood lacking neutrophils. These data suggest that C. albicans genes expressed in blood were predominantly induced in response to neutrophils, and that neutrophils play a key role during C. albicans bloodstream infections. However, C. albicans is equipped with several genes and transcriptional programmes, which may help the fungus to counteract the attack of neutrophils, to escape from the bloodstream and to cause systemic infections.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2005.04557.x
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  • 11
    Electronic Resource
    Electronic Resource
    The mutation G145S in PrfA, a key virulence regulator of Listeria monocytogenes, increases DNA-binding affinity by stabilizing the HTH motif (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Listeria monocytogenes, a Gram-positive, facultative intracellular human pathogen, causes systemic infections with high mortality rate. The majority of the known pathogenicity factors of L. monocytogenes is regulated by a single transcription factor, PrfA. Hyperhaemolytic laboratory strains of L. monocytogenes express the constitutively active mutant PrfAG145S inducing virulence gene overexpression independent of environmental conditions. PrfA belongs to the Crp/Fnr family of transcription factors generally activated by a small effector, such as cAMP or O2. We present the crystal structures of wild-type PrfA, the first Gram-positive member of the Crp/Fnr family, and of the constitutively active mutant PrfAG145S. Cap (Crp) has previously been described exclusively in the cAMP-induced (DNA-free and -bound) conformation. By contrast, the PrfA structures present views both of the non-induced state and of the mutationally activated form. The low DNA-binding affinity of wild-type PrfA is supported both structurally (partly disordered helix–turn–helix motif, overall geometry of the HTH α-helices deviates from Cap) and by surface plasmon resonance analyses (KD = 0.9 µM). In PrfAG145S the HTH motifs dramatically rearrange to adopt a conformation comparable to cAMP-induced Cap and hence favourable for DNA binding, supported by a DNA-binding affinity of 50 nM. Finally, the hypothesis that wild-type PrfA, like other Crp/Fnr family members, may require an as yet unidentified cofactor for activation is supported by the presence of a distinct tunnel in PrfA, located at the interface of the β-barrel and the DNA-binding domain.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2005.04561.x
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  • 12
    Electronic Resource
    Electronic Resource
    Enteropathogenic Escherichia coli EspG disrupts microtubules and in conjunction with Orf3 enhances perturbation of the tight junction barrier (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: EspG, a secreted effector of enteropathogenic Escherichia coli (EPEC), as well as its homologue Orf3, has been shown to disrupt microtubules (MTs) in fibroblasts and non-polarized epithelial cells. The roles of MTs and the effects of MT disruption in these cell types differ significantly. The aim of this study was to investigate the effects of EspG on polarized, host target intestinal epithelial cells. Immunofluorescent labelling of tubulin showed that EPEC caused progressive fragmentation and loss of the MT network in cells harbouring attached organisms. Immunoblots of proteins extracted from EPEC-infected cells showed a corresponding loss of α-tubulin. Type III secretion system (TTSS)-deficient strains had no effect on MT suggesting TTSS dependence. Mutation of espG, but not espF or map, ablated EPEC's effects on MTs for up to 6 h. Ectopic expression of EspG in HeLa cells caused MT disruption. While deletion of espG alone had no effect on the EPEC-induced decrease in transepithelial electrical resistance (TER), mutation of both espG and orf3 significantly delayed the kinetics of this response. Complementation of the double mutant with espG alone restored the kinetics of TER drop to that of wild type. Herein, we describe a previously unrecognized phenotype for the EPEC effectors EspG and Orf3.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2005.04571.x
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  • 13
    Electronic Resource
    Electronic Resource
    Erwinia chrysanthemi requires a second iron transport route dependent of the siderophore achromobactin for extracellular growth and plant infection (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 55 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Full virulence of the pectinolytic enterobacterium Erwinia chrysanthemi strain 3937 depends on the production in planta of the catechol-type siderophore chrysobactin. Under iron-limited conditions, E. chrysanthemi synthesizes a second siderophore called achromobactin belonging to the hydroxy/carboxylate class of siderophore. In this study, we cloned and functionally characterized a 13 kb long operon comprising seven genes required for the biosynthesis (acs) and extracellular release (yhcA) of achromobactin, as well as the gene encoding the specific outer membrane receptor for its ferric complex (acr). The promoter of this operon was negatively regulated by iron. In a fur null mutant, transcriptional fusions to the acsD and acsA genes were constitutively expressed. Band shift assays showed that the purified E. chrysanthemi Fur repressor protein specifically binds in vitro to the promoter region of the acsF gene confirming that the metalloregulation of the achromobactin operon is achieved directly by Fur. The temporal production of achromobactin in iron-depleted bacterial cultures was determined: achromobactin is produced before chrysobactin and its production decreases as that of chrysobactin increases. Pathogenicity tests performed on African violets showed that achromobactin production contributes to the virulence of E. chrysanthemi. Thus, during infection, synthesis of these two different siderophores allows E. chrysanthemi cells to cope with the fluctuations of iron availability encountered within plant tissues. Interestingly, iron transport mediated by achromobactin or a closely related siderophore probably exists in other phytopathogenic bacterial species such as Pseudomonas syringae.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2004.04383.x
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  • 14
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    Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 55 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The minimization of a genome is necessary to identify experimentally the minimal gene set that contains only those genes that are essential and sufficient to sustain a functioning cell. Recent developments in genetic techniques have made it possible to generate bacteria with a markedly reduced genome. We developed a simple system for formation of markerless chromosomal deletions, and constructed and characterized a series of large-scale chromosomal deletion mutants of Escherichia coli that lack between 2.4 and 29.7% of the parental chromosome. Combining deletion mutations changes cell length and width, and the mutant cells with larger deletions were even longer and wider than the parental cells. The nucleoid organization of the mutants is also changed: the nucleoids occur as multiple small nucleoids and are localized peripherally near the envelope. Inhibition of translation causes them to condense into one or two packed nucleoids, suggesting that the coupling of transcription and translation of membrane proteins peripherally localizes chromosomes. Because these phenotypes are similar to those of spherical cells, those may be a consequence of the morphological change. Based on the nucleoid localization observed with these mutants, we discuss the cellular nucleoid dynamics.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2004.04386.x
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  • 15
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    New insertion sequences of Sulfolobus: functional properties and implications for genome evolution in hyperthermophilic archaea (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 55 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Analyses of complete genomes indicate that insertion sequences (ISs) are abundant and widespread in hyperthermophilic archaea, but few experimental studies have measured their activities in these hosts. As a way to investigate the impact of ISs on Sulfolobus genomes, we identified seven transpositionally active ISs in a widely distributed Sulfolobus species, and measured their functional properties. Six of the seven were found to be distinct from previously described ISs of Sulfolobus, and one of the six could not be assigned to any known IS family. A type II ‘Miniature Inverted-repeat Transposable Element’ (MITE) related to one of the ISs was also recovered. Rates of transposition of the different ISs into the pyrEF region of their host strains varied over a 250-fold range. The Sulfolobus ISs also differed with respect to target-site selectivity, although several shared an apparent preference for the pyrEF promoter region. Despite the number of distinct ISs assayed and their molecular diversity, only one demonstrated precise excision from the chromosomal target region. The fact that this IS is the only one lacking inverted repeats and target-site duplication suggests that the observed precise excision may be promoted by the IS itself. Sequence searches revealed previously unidentified partial copies of the newly identified ISs in the Sulfolobus tokodaii and Sulfolobus solfataricus genomes. The structures of these fragmentary copies suggest several distinct molecular mechanisms which, in the absence of precise excision, inactivate ISs and gradually eliminate the defective copies from Sulfolobus genomes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2004.04391.x
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  • 16
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    The malaria parasite Plasmodium falciparum has only one pyruvate dehydrogenase complex, which is located in the apicoplast (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 55 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The relict plastid (apicoplast) of apicomplexan parasites synthesizes fatty acids and is a promising drug target. In plant plastids, a pyruvate dehydrogenase complex (PDH) converts pyruvate into acetyl-CoA, the major fatty acid precursor, whereas a second, distinct PDH fuels the tricarboxylic acid cycle in the mitochondria. In contrast, the presence of genes encoding PDH and related enzyme complexes in the genomes of five Plasmodium species and of Toxoplasma gondii indicate that these parasites contain only one single PDH. PDH complexes are comprised of four subunits (E1α, E1β, E2, E3), and we confirmed four genes encoding a complete PDH in Plasmodium falciparum through sequencing of cDNA clones. In apicomplexan parasites, many nuclear-encoded proteins are targeted to the apicoplast courtesy of two-part N-terminal leader sequences, and the presence of such N-terminal sequences on all four PDH subunits as well as phylogenetic analyses strongly suggest that the P. falciparum PDH is located in the apicoplast. Fusion of the two-part leader sequences from the E1α and E2 genes to green fluorescent protein experimentally confirmed apicoplast targeting. Western blot analysis provided evidence for the expression of the E1α and E1β PDH subunits in blood-stage malaria parasites. The recombinantly expressed catalytic domain of the PDH subunit E2 showed high enzymatic activity in vitro indicating that pyruvate is converted to acetyl-CoA in the apicoplast, possibly for use in fatty acid biosynthesis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2004.04407.x
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  • 17
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    Identification of the infectious source of an unusual outbreak of histoplasmosis, in a hotel in Acapulco, state of Guerrero, Mexico (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Three isolates of Histoplasma capsulatum were identified from mice lung, liver, and spleen inoculated with soil samples of the X hotel's ornamental potted plants that had been fertilized with organic material known as compost. The presence of H. capsulatum in the original compost was detected using the dot-enzyme-linked immunosorbent assay. Nested-PCR, using a specific protein Hcp100 coding gene sequence, confirmed the fungal identification associated with an unusual histoplasmosis outbreak in Acapulco. Although, diversity between the H. capsulatum isolate from the hotel and some clinical isolates from Guerrero (positive controls) was observed using random amplification of polymorphic DNA based-PCR, sequence analyses of H-anti and ole fragment genes revealed a high homology (92–99%) between them.
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    Analysis and expression of STE13ca gene encoding a putative X-prolyl dipeptidyl aminopeptidase from Candida albicans (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Candida albicans STE13ca gene was identified by its homology to the Saccharomyces cerevisiae STE13 gene that encodes for the dipeptidyl aminopeptidase A (DAP A) involved in the maturation of α-factor mating pheromone. Our study revealed that C. albicans ATCC 10231 depicts dipeptidyl aminopeptidase activity. We also analyzed the expression of the STE13ca gene homologue from this pathogenic yeast. This gene of 2793 pb is homozygotic and encodes for a predicted protein of 930 amino acids with a molecular weight of 107,035 Da. The predicted protein displays significant sequence similarity to S. cerevisiae Ste13p. This C. albicans gene is located in chromosome R. STE13ca gene increases its levels of expression in conditions of nutritional stress (proline as nitrogen source) and during formation of the germinal tube, suggesting a basic biological function for the STE13ca in this yeast.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1016/j.femsim.2005.05.020
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    Cryptococcus neoformans: A sugar-coated killer with designer genes (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Cryptococcus neoformans has become a common central nervous system pathogen as the immunocompromised populations enlarge world-wide. This encapsulated yeast has significant advantages for the study of fungal pathogenesis and these include: (1) a clinically important human pathogen; (2) a tractable genetic system; (3) advanced molecular biology foundation; (4) understanding of several virulence phenotypes; (5) well-studied pathophysiology; and (6) robust animal models. With the use of a sequenced genome and site-directed mutagenesis to produce specific null mutants, the virulence composite of C. neoformans has begun to be identified one gene at a time. Studies into capsule production, melanin synthesis, high temperature growth, metabolic pathways and a variety of signaling pathways have led to understandings of what makes this yeast a pathogen at the molecular level. Multiple principles of molecular pathogenesis have been demonstrated in virulence studies with C. neoformans. These include evolutionary differences between the varieties of C. neoformans in their genes for virulence, quantitative impact of genes on the virulence composite, species and site-specific importance of a virulence gene, gene expression correlation with its functional importance or phenotype and the impact of a pathogenesis gene on the host immune response. C. neoformans has now become a primary model to study molecular fungal pathogenesis with the goal of identifying drug targets or vaccine strategies.
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    Brucella melitensis infection associated with Guillain–Barré syndrome through molecular mimicry of host structures (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Brucella melitensis is a facultative intracellular bacterium that can survive inside macrophages and the causative agent of brucellosis. In the present study, we found that a lipooligosaccharide of B. melitensis has a GM1 ganglioside-like structure and shows a strong antibody response in mice. The cholera toxin B subunit, which binds to GM1 ganglioside specifically, reacted with the surface of B. melitensis. Immunization with B. melitensis induced the production of anti-GM1 ganglioside antibodies in mice and serum from immunized mice showed a cross-reaction with Guillain–Barré syndrome (GBS)-associated Campylobacter jejuni, but not non-GBS-associated C. jejuni. When B. melitensis was treated with a neuraminidase, antibody responses disappeared. B. melitensis immunization induced the production of anti-GM1 ganglioside antibodies in BALB/c mice but not in C57BL/6 and ddY mice, and for BALB/c mice, immunization with B. melitensis induced much greater production of anti-GM1 ganglioside than GBS-associated C. jejuni. Flaccid limb weakness was observed in B. melitensis immunized mice. These results suggest that B. melitensis is a new etiological agent for GBS and that immunological responses between it and GBS-associated C. jejuni in the mouse model may be different.
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    Host defense effector molecules in mucosal secretions (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Mucosal secretions contain a range of defense effector molecules including antimicrobial peptides and proteinase inhibitors. These molecules play a central role in host defense against infection, and in a variety of immune and inflammatory reactions. The aim of this study was to analyze the levels of neutrophil defensins, the cathelicidin hCAP-18/LL-37, and the proteinase inhibitors secretory leukocyte proteinase inhibitor, SKALP/elafin and cystatin M/E in various mucosal secretions and urine. We show here that especially seminal plasma is characterized by high concentrations of hCAP-18/LL-37, SLPI, SKALP/elafin and cystatin M/E. The results of this study demonstrate that each mucosal secretion is characterized by a unique profile of effector molecules, which may supply individual mucosal secretions with specific properties related to the control of local infection and inflammation.
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    Determination of the cytotoxic effect of Clostridium histolyticum culture supernatant on HeLa cells in the presence of protease inhibitors (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Clostridium histolyticum culture supernatant contains numerous enzymes, which exert a cytotoxic effect on host cells. This includes lethal toxin, clostripain and high-potassium-sensitive toxin. Since the number of C. histolyticum infections increased during the last several years, it seems worthwhile to evaluate whether protease inhibitors, used for the treatment of many diseases, could influence toxicity, and thus, pathogenicity of C. histolyticum. In this study we evaluated in vitro the influence of four common protease inhibitors: aprotinin, phenylmethylsulphonyl fluoride (PMSF), l-1-chloro-3-[4-tosylamido]-7-amino-2-heptanone-HCl (TLCK) and chymostatin on the toxicity of C. histolyticum supernatant towards human epithelial HeLa cells. We show that aprotinin has no effect, while PMSF, TLCK and chymostatin potentiate the cytotoxic activity of C. histolyticum, probably by hindering natural defence mechanisms of cells. In addition, PMSF and TLCK block clostripain enzymatic activity, while chymostatin leaves it intact. Elevated cytotoxicity of the supernatant is not related to the quantity of high-potassium-sensitive toxin, as was reported previously, since desalted supernatant still exerted its strong toxic effect. Our results show that addition of protease inhibitors for treating diseases complicated by concurrent C. histolyticum infection must require special attention.
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    Secretion of matrix metalloproteinase-9 by macrophages, in vitro, in response to Helicobacter pylori (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We have previously shown that matrix metalloproteinase-9 (MMP-9) activity is greatly enhanced within the active chronic inflammation of Helicobacter pylori infected individuals, of which a major fraction derives from macrophages in the tissue. Here, we have investigated the ability of macrophages to secrete MMPs in response to H. pylori. Human macrophages secrete MMP-9 in response to live and inactivated H. pylori, as well as to specific bacterial products. Protein kinase C, phosphatiolylinositol 3-kinase and calcium uptake channels all play a role in MMP-9 secretion, whereas neither tumour necrosis factor alpha, interleukin-8, nor interleukin-1β autocrine stimulation appear to contribute. We conclude that human macrophages have the ability to react directly against several H. pylori derived factors, utilising several signalling pathways.
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    URL: http://dx.doi.org/10.1016/j.femsim.2005.03.008
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    In vitro and in silico analysis of signal peptides from the human blood fluke, Schistosoma mansoni (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Proteins secreted by and anchored on the surfaces of parasites are in intimate contact with host tissues. The transcriptome of infective cercariae of the blood fluke, Schistosoma mansoni, was screened using signal sequence trap to isolate cDNAs encoding predicted proteins with an N-terminal signal peptide. Twenty cDNA fragments were identified, most of which contained predicted signal peptides or transmembrane regions, including a novel putative seven-transmembrane receptor and a membrane-associated mitogen-activated protein kinase. The developmental expression pattern within different life-cycle stages ranged from ubiquitous to a transcript that was highly upregulated in the cercaria. A bioinformatics-based comparison of 100 signal peptides from each of schistosomes, humans, a parasitic nematode and Escherichia coli showed that differences in the sequence composition of signal peptides, notably the residues flanking the predicted cleavage site, might account for the negative bias exhibited in the processing of schistosome signal peptides in mammalian cells.
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    Expanding the bactericidal action of the food color additive phloxine B to gram-negative bacteria (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Phloxine B (D&C red no. 28) is a color additive for food, drugs, and cosmetics. It has been previously shown to have anti-Staphylococcus aureus activities. In this work, the effect of Phloxine B on various gram-negative bacteria and other gram-positive bacteria including Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus subtilis, Bacillus aureus, Salmonella, Escherichia coli and Shigella was studied, along with the mechanism of anti-microbial activity. In the presence of fluorescent light, the viable count for gram-positive bacteria, (Bacillus spp. and S. aureus) decreased in a dose and time dependent manner when incubated with Phloxine B. The viability of gram-positive bacteria was reduced by 99.99% in 40 min, while there was no effect on gram-negative bacteria (Salmonella choleraesuis, E. coli and Shigella flexneri). However, the use of ethylenediaminetetraacetic acid (EDTA) expands the spectrum of activity for Phloxine B to include gram-negative bacteria. EDTA increased membrane-permeability by releasing lipopolysaccharide. Overall, in an Agar diffusion test the light-dependent bactericidal activity of 1 μg of Phloxine B had a potency of 0.64 units of chloramphenicol and 0.5 units of tetracycline when tested on B. cereus, and had a potency of 0.7 units of chloramphenicol and 0.2 units of tetracycline when tested on S. aureus. The data suggest that the dye may have some potential anti-microbial applications.
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    Distinct bacterial dissemination and disease outcome in mice subcutaneously infected with Borrelia burgdorferi in the midline of the back and the footpad (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Subcutaneous inoculation of mice with Borrelia burgdorferi, the causative agent of Lyme disease, results in established infection and the development of acute arthritis and carditis, hallmarks of human disease. Because conflicting results may originate from the site of subcutaneous inoculation, we addressed the dissemination capacity of spirochetes injected in the shoulder region versus the footpad. Spirochetes inoculated in the footpad disseminated to a lesser extent to distant organs, such as the ear and the heart. This resulted in distinct degrees of joint and cardiac inflammation at the peak of the disease. The differences eventually leveled out. These results suggest that caution must be exercised in the interpretation of results obtained with routes of inoculation that do not closely represent the natural site of infection.
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    Expression and secretion of the protective antigen of Bacillus anthracis in Bacillus brevis (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 μg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 °C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.
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    Book Review (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1016/j.femsim.2005.06.002
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    Identification of glyceraldehyde-3-phosphate dehydrogenase of epithelial cells as a second molecule that binds to Porphyromonas gingivalis fimbriae (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Binding of Porphyromonas gingivalis to the host cells is an essential step in the pathogenesis of periodontal disease. P. gingivalis binds to and invades epithelial cells, and fimbriae are thought to be involved in this process. In our earlier studies, two major epithelial cell components of 40 and 50 kDa were identified as potential fimbrial receptors. Sequencing of a cyanogen bromide digestion fragment of the 50-kDa component resulted in an internal sequence identical to keratin I molecules, and hence this cytokeratin represents one of the epithelial cell receptors for P. gingivalis fimbriae. In this study, the 40-kDa component of KB cells was isolated and its amino-terminal sequence determined. The N-terminal amino sequence was found to be GKVKVGVNGF and showed perfect homology with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, purified P. gingivalis fimbriae were found to bind to rabbit muscle GAPDH. Antibodies directed against internal peptide 49–68 and 69–90 of fimbrillin were shown to inhibit the binding of P. gingivalis and of fimbriae to epithelial cells. Antibodies against these peptides also inhibited the binding of fimbriae to GAPDH. Our results confirmed that the amino-terminal domain corresponding to amino residues 49–68 of the fimbrillin protein is the major GAPDH binding domain. These studies point to GAPDH as a major receptor for P. gingivalis major fimbriae and, as such, GAPDH likely plays a role in P. gingivalis adherence and colonization of the oral cavity, as well as triggering host cell processes involved in the pathogenesis of P. gingivalis infections.
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    Identification and characterization of the TonB region and its role in transferrin-mediated iron acquisition in Haemophilus parasuis (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Haemophilus parasuis is the causative agent of Glässer's disease, which is responsible for considerable economic losses in the pig-rearing industry. The aim of the study reported here was the identification, sequencing and molecular characterization of the TonB region that includes tonB, exbBD, and tbpBA genes in H. parasuis. In addition, two fusion proteins were generated. One of them (pGEX-6P-1-GST-TbpB) contained the first 501 amino acids of H. parasuis TbpB protein, while the second (pBAD-Thio-TbpB-V5-His) included the first 102 amino acids of H. parasuis TbpB N-terminus domain. A panel of 14 hybridomas secreting monoclonal antibodies was raised against the two recombinant TbpB fusion proteins. Furthermore, to assess whether the expression of the H. parasuis ExbB, TbpB, and TbpA proteins was upregulated under conditions of restricted availability of iron, a rabbit polyclonal antibody against H. parasuis TbpB-His fusion protein was produced. A rabbit polyclonal antibody against serotype 7 of Actinobacillus pleuropneumoniae ExbB and TbpA proteins was also used for the detection of the homologous proteins in H. parasuis. Overall, the data indicate that H. parasuis, like other members of the Pasteurellaceae family, possesses the genetic elements of the TonB region for iron acquisition and the transferrin-binding proteins encoded under this region are upregulated under restricted iron availability.
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    Comparative evaluation of intranasal and subcutaneous route of immunization for development of mucosal vaccine against experimental tuberculosis (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Activation of mucosal immunity in the respiratory tract is crucial for protection against respiratory infections. Whether the intranasal route of vaccination imparts better protection against pulmonary tuberculosis than that of subcutaneous vaccination remains a debatable issue. In this study, we have investigated the effect of the routes of immunization on the induction of immunoprotection against experimental tuberculosis employing mycobacterial culture filtrate proteins complexed with dimethyldioctadecylammonium bromide. Vaccination via intranasal and subcutaneous routes triggered immune activation in the spleen and cervical lymph node, while the former route of vaccination lead to higher antigen-specific lymphocyte proliferation, interferon-γ, interleukin-12 and interleukin-4 responses in cervical lymph node and induction of antigen-specific IgA responses at mucosal level of the respiratory tract. Mice vaccinated via the intranasal route were found to be better protected against experimental tuberculosis particularly in lung compared to subcutaneous-immunized mice. These results emphasize the importance of the intranasal route vaccination in tuberculosis.
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    Identification of Helicobacter pylori and the cagA genotype in gastric biopsies using highly sensitive real-time PCR as a new diagnostic tool (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The CagA protein is one of the virulence factors of Helicobacter pylori, and two major subtypes of CagA have been observed, the Western and East Asian type. CagA is injected from the bacteria into gastric epithelial cells, undergoes tyrosine phosphorylation, and binds to Src homology 2 domain-containing protein-tyrosine phosphatase SHP-2. The East Asian type CagA binds to SHP-2 more strongly than the Western type CagA. Here, we tried to distinguish the CagA type by highly sensitive real-time PCR with the objective of establishing a system to detect H. pylori and CagA subtypes from gastric biopsies. We designed primers and probe sets for Western or East Asian-cagA at Western-specific or East Asian-specific sequence regions, respectively, and H. pylori 16S rRNA. We could detect the H. pylori 16S rRNA gene, Western and East Asian-cagA gene from DNA of gastric biopsies. The sensitivity and specificity for H. pylori infection was 100% in this system. In Thai patients, 87.8% (36/41) were cagA-positive; 26.8% (11/41) were Western-cagA positive and 53.7% (22/41) were East Asian-cagA positive, while 7.3% (3/41) reacted with both types of cagA. These results suggest that this real-time PCR system provides a highly sensitive assessment of CagA type as a new diagnostic tool for the pathogenicity of H. pylori infection.
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    Detection of Mycobacterium leprae in nasal mucosa biopsies by the polymerase chain reaction (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Several discoveries about leprosy indicate that Mycobacterium leprae transmission mainly occurs by inhalation, and the nose is major port of entry and exit. The present study evaluated the clinical application of PCR for detection of M. leprae DNA in nasal mucosa biopsies in untreated leprosy patients (52) and their contacts (99) from the State Reference Center in Sanitary Dermatology and Leprosy, Uberlândia, MG, Brazil. PCR detection of a 372-base pair DNA fragment from M. leprae was accomplished in 36 (69.2%) patients, from which 34 (91.9%) of them were multibacillaries. Furthermore, PCR was positive in 3 (16.7%) of 18 slit-skin smear negative, 4 (25.0%) of 16 skin lesion BI negative, 8 (33.3%) of 24 nasal mucosa BI negative patients, and 10 of 99 contacts (10.1%). The presence of bacilli in 10.1% of the contacts may potentially reflect an occult leprosy, and these patients must be accompanied, followed by a chemoprophylaxy treatment. Considering all PCR results against clinical and BI classification of patients and controls, we have found a sensitivity of 69.2%, a specificity of 89.9%, and an accuracy of 82.8%. It has been demonstrated here through PCR of nasal biopsies that the bacillus invades the mucosa, passing through the nasal inferior turbinate to reach peripheral blood. Therefore, the molecular investigation of invasive nasal biopsies by PCR tests has proven to be useful in defining patients of higher risk of transmission and risk-group contacts, which is an important step to reach the World Health Organization objective towards the elimination of leprosy as a public health problem.
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    Identification of a new sialic acid-binding protein in Helicobacter pylori (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: A novel sialic acid-specific lectin has been isolated from Helicobacter pylori lysate using fetuin–agarose affinity chromatography followed by cleavage of the α(2,3) and α(2,6) linkages of sialic acids using neuraminidase. The protein had a molecular weight of 17.5 kDa on sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and was identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to be protein of unknown function with gene number HP0721. Recombinant HP0721 was shown to bind to fetuin–agarose and sialic acid-containing glycosphingolipids on thin-layer plates suggesting this protein may represent another sialic acid-specific adhesin of H. pylori. A H. pylori mutant defective for HP0721 was generated and its ability to bind to human AGS cells assayed.
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    Helicobacter pylori antibodies and gastric cancer: a gender-related difference (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Helicobacter pylori has been proposed as a causative agent of gastric cancer. The aim of this study was to define serum antibodies response against different H. pylori antigens in patients with gastric cancer. Serum samples were collected from 115 Lithuanian patients with non-cardia gastric cancer and 110 age- and sex-matched controls without cancer. Heat-stable, low-molecular-mass, and outer membrane proteins were used as antigens to analyze serum IgG antibody response against H. pylori by enzyme-linked immunosorbent assay. Seroprevalence of H. pylori using low-molecular-mass antigen was significantly higher in gastric cancer patients, compared to controls (77% versus 57%, p 〈 0.05). Significant differences in the prevalence of H. pylori infection between gastric cancer patients and controls were found in females using all three studied antigens: heat-stable (98% versus 84%, p 〈 0.05), low-molecular-mass (88% versus 48%, p 〈 0.05) and outer membrane proteins (78% versus 57%, p 〈 0.05). In males, no significant differences were revealed between gastric cancer patients and controls. There may be other cofactors in addition to H. pylori that are important for the development of gastric cancer. H. pylori seems, however, to be a more important for development of gastric cancer in females than in males or males may have more confounding risk factors for gastric cancer than females.
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    Characterization of the PCR inhibitory effect of bile to optimize real-time PCR detection of Helicobacter species (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The inhibitory effect of human and porcine bile samples to detect Helicobacter DNA was studied by adding different concentrations of bile samples to PCR mixtures of six thermostable DNA polymerases containing cagA specific primers and Helicobacter pylori DNA. PCR products were amplified by using the Rotorgene system and SYBR Green I. Among the six DNA polymerases tested, rTth had the lowest sensitivity to bile inhibitors, whereas Taq and Tfl had the highest sensitivity. Bile proteins did not inhibit AmpliTaq DNA polymerase, whereas the fraction containing mainly bile acids and their salts inhibited the amplification capacity of AmpliTaq. Heating human bile at 98 °C and adding casein and formamide to the reaction mixture reduced the PCR inhibitory effect of bile. Therefore, a pre-PCR treatment based on dilution and heating of bile, adding casein and formamide to the reaction mixture of rTth DNA polymerase was found efficient to amplify DNA directly in bile.
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    A genetic locus of Helicobacter pylori inversely associated with gastric intestinal metaplasia (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The genomic contents of Helicobacter pylori strain C1 from a patient with gastric cancer and strain 98587 from a patient with duodenal ulcer disease were compared using a rapid subtractive hybridisation approach. A total of 11 tester-specific sequences representing gene specificity, DNA rearrangement and sequence variation were identified. This included two novel sequences, clone P32 and clone F5, which have no significant homologue in the database. H. pylori strains positive for clone P32 were less prevalent in patients with gastric intestinal metaplasia (12.5%) than in duodenal ulcer (39.1%) (p= 0.036), or chronic gastritis (38.1%) (p= 0.036). The results suggest that H. pylori clone P32 is potentially a useful marker for distinguishing intestinal metaplasia associated strains from others.
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    High relative content of lysophospholipids of Helicobacter pylori mediates increased risk for ulcer disease (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Helicobacter pylori phospholipase A (OMPLA) degrades bacterial membrane phospholipids to lysophospholipids. High levels of lysophospholipids are associated with higher hemolytic activity, increased release of urease and vacA and better adherence to epithelial cells in vitro. The phospholipase A gene (pldA) displays phase variation due to a slippage in a homopolymeric tract. The aim of this study was to determine if the relative amount of lysophospholipids in the cell wall is associated with ulcer disease, and to further investigate the significance of pldA phase variation. H. pylori isolates of 40 patients were examined. The relative lysophospholipid content of each isolate was determined and the pldA gene was sequenced. The study indicated that H. pylori can regulate its OMPLA activity by phase variation in the pldA gene or by protein level regulation among phase variants in the pldA‘ON’ status. We found a significant difference between the relative amount of lysophospholipids of the ulcer group and the non-ulcer group (p= 0.022). When the lysophospholipid/phospholipid ratios were compared with outcome, the OR for ulcer disease was 9.0 (95% CI 1.6–49.4; p= 0.014). Isolates with a high OMPLA activity are significantly associated with patients with ulcer disease.
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    Ribotyping of Burkholderia mallei isolates (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: In this study, the subspecies differentiation of 25 isolates of Burkholderia mallei was attempted based on their ribotype polymorphisms. The isolates were from human and equine infections that occurred at various times around the world. DNA samples from each isolate were digested separately with PstI and EcoRI enzymes and probed with an Escherichia coli-derived 18-mer rDNA sequence to identify diagnostic fragments. Seventeen distinct ribotypes were identified from the combined data obtained with the two restriction enzymes. The results demonstrate the general utility of ribotyping for the subspecies identification of B. mallei isolates.
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    Expression of a peroxiredoxin–glutaredoxin by Haemophilus influenzae in biofilms and during human respiratory tract infection (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Evidence is mounting that nontypeable Haemophilus influenzae grows as a biofilm in the middle ear of children with otitis media and the airways of adults with chronic obstructive pulmonary disease. To begin to assess antigens expressed by H. influenzae in biofilms, cell envelopes of bacteria grown as a biofilm were compared to those grown planktonically. A ∼30 kDa peroxiredoxin–glutaredoxin was present in greater abundance during growth in biofilms. Mutants deficient in expression of peroxiredoxin–glutaredoxin were constructed by homologous recombination in four clinical isolates. The mutants showed a 25–50% reduction in biofilm formation compared to the corresponding parent strains. To study in vivo expression of peroxiredoxin–glutaredoxin during human respiratory tract infection, paired pre- and post-exacerbation serum from adults with chronic obstructive pulmonary disease and H. influenzae in sputum were assayed using an enzyme-linked immunosorbent assay and purified recombinant peroxiredoxin–glutaredoxin. Eight from 18 (44.4%) paired serum samples showed a significant increase in antibody to peroxiredoxin–glutaredoxin from pre- to post-infection. These results indicate that (1) peroxiredoxin–glutaredoxin is present in greater abundance in H. influenzae biofilms compared to planktonically grown bacteria; (2) peroxiredoxin–glutaredoxin is involved in biofilm formation by H. influenzae and the degree of involvement varies among strains; and (3) peroxiredoxin–glutaredoxin is expressed by H. influenzae during infection of the human respiratory tract and is recognized by the human immune system.
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    cagA gene variants in Malaysian Helicobacter pylori strains isolated from patients of different ethnic groups (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Helicobacter pylori infection of a distinct subtype of cagA may lead to different pathological manifestation. The aim of this study is to determine the presence of cagA gene and its variants in H. pylori infection among different ethnic groups and its effect on gastroduodenal diseases. Overall detection of cagA among the 205 clinical isolates of H. pylori was 94%. Variations in size of the 3′ region of cagA gene were examined among 192 Malaysian H. pylori cagA-positive strains. Results showed that three cagA variants differing in fragment length of PCR products were detected and designated as type A (621–651 bp), type B (732–735 bp) and type C (525 bp). Although there was no association between any of the cagA subtypes with peptic ulcer disease (p 〉 0.05), an association between cagA subtypes with a specific ethnic group was observed. Specific-cagA subtype A strains were predominantly isolated from Chinese compared to Malays and Indians (p 〈 0.0005), and cagA subtype B strains were predominantly isolated from Malays and Indians compared to Chinese (p 〈 0.05). The cagA type A strains of H. pylori is commonly found in the Chinese patients who have a higher risk of peptic ulcer disease, thus indicating that it could be used as an important clinical biomarker for a more severe infection.
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    In vivo expression of the 25-kDa laminin-binding protein of Helicobacter pylori (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The gastroduodenal pathogen Helicobacter pylori has been shown to inhibit the interaction between the extracellular matrix protein laminin and its receptor on gastric epithelial cells, potentially contributing to a loss of mucosal integrity. As a 25-kDa outer membrane protein of H. pylori in association with the bacterial lipopolysaccharides (LPS) mediates attachment to laminin, the aim of this study was to determine whether the 25-kDa protein is produced by H. pylori in infected hosts. We examined the immune response to the 25-kDa laminin binding protein in 12 paediatric patients; samples from a H. pylori-negative healthy adult were used as controls. In immunoblotting, antibodies to a 25-kDa protein were found in the serum and saliva of H. pylori-positive individuals only, and using the positive sera and saliva, laminin binding to the 25-kDa protein was inhibited. Thus, the 25-kDa laminin-binding protein is produced by H. pylori in infected hosts.
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    Detection and differentiation of Burkholderia pseudomallei, Burkholderia mallei and Burkholderia thailandensis by multiplex PCR (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Burkholderia pseudomallei, a Gram-negative bacterium that causes melioidosis may be differentiated from closely related species of Burkholderia mallei that causes glanders and non-pathogenic species of Burkholderia thailandensis by multiplex PCR. The multiplex PCR consists of primers that flank a 10-bp repetitive element in B. pseudomallei and B. mallei amplifying PCR fragment of varying sizes between 400–700 bp, a unique sequence in B. thailandensis amplifying a PCR fragment of 308 bp and the metalloprotease gene amplifying a PCR fragment of 245 bp in B. pseudomallei and B. thailandensis. The multiplex PCR not only can differentiate the three Burkholderia species but can also be used for epidemiological typing of B. pseudomallei and B. mallei strains.
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    Protective efficacy of a recombinant plague vaccine when co-administered with another sub-unit or live attenuated vaccine (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Vaccines against bioterrorism agents offer the prospect of providing high levels of protection against airborne pathogens. However, the diversity of the bioterrorism threat means that it may be necessary to use several vaccines simultaneously. In this study we have investigated whether there are changes to the protective immune response to a recombinant sub-unit plague vaccine when it is co-administered with other sub-unit or live attenuated vaccines. Our results indicate that the co-administration of these vaccines did not influence the protection afforded by the plague vaccine. However, the co-administration of the plague sub-unit vaccine with a live vaccine resulted in markedly increased levels of IgG2a subclass antibodies, and markedly reduced levels of IgG1 subclass antibodies, to the plague sub-unit vaccine. This finding might have implications when considering the co-administration of other vaccine combinations.
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    Anti-Helicobacter pylori activity of Plumbago zeylanica L. (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: It has been shown that the presence of infection by Helicobacter pylori is strongly associated with gastric cancer and peptic ulceration. In western medicine, a 3-fold therapeutic regimen, emphasizing the use of antibiotics, is typically used to suppress H. pylori activity. However, antibiotic drug resistance frequently develops as a consequence of such treatment. In our previous study, 50 Taiwanese folk medicinal plants were screened for their anti-H. pylori activities. The results revealed that Plumbago zeylanica L. had the highest inhibitory effects against H. pylori. In this study, therefore, we have focused on establishing the anti-H. pylori activities of P. zeylanica L. Water and the organic solvents ethanol, ethyl acetate and acetone were used for P. zeylanica L. extraction, obtaining yields of 1.66–6.84% (w/w). Excluding the water extract, higher anti-H. pylori activity was demonstrated for all the extracts, both using the agar diffusion and dilution methods. The ethyl acetate extract exhibited the lowest minimum inhibitory concentrations against five H. pylori strains, of which ranged from 0.32 to 1.28 mg ml−1, followed, in ascending order, by the acetone, ethanol and water analogs. Bactericidal activity was determined for P. zeylanica L. extracts, with the lowest minimum bactericidal concentrations (5.12–20.48 mg ml−1) demonstrated for the ethyl acetate, followed, in ascending order, by the acetone and ethanol analogs. Bactericidal activity appeared to be in a dose-dependent manner. Through a broad pH range (2–7), bactericidal activity was not affected when extract concentrations were greater than or equal to the minimum bactericidal concentration. High stability was demonstrated for the ethyl acetate P. zeylanica L. extract within pH range of 1–7, exhibiting all pH treatments bactericidal activity.
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    A study on the bacterial photo-toxicity of phenothiazinium based photosensitisers (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: “Comet assay” showed light activated (3.15 J cm−2 over 30 min) phenothiazinium based photosensitisers (PhBPs) to induce photo-damage of Staphylococcus aureus DNA, as indicated by DNA “tails” between 80 and 120 μm. In general, PhBPs exhibited significant singlet oxygen yields (ΦΔPhBP 〉 0.7), suggesting the use of type II mechanisms of photo-oxidation. However, the photodynamic action of PhBPs on DNA showed generally insignificant production of 7,8-dihydro-8-oxo-2′-deoxyguanosine, normally a major product of type II DNA photo-oxidation. These combined results show DNA to be a major site of action of PhBPs and suggest that this action may involve type II attack on a nucleoside(s) other than guanosine.
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    Structure of the O-polysaccharide and serological studies of the lipopolysaccharide of Proteus penneri 60 classified into a new Proteus serogroup O70 (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: An alkali-treated lipopolysaccharide of Proteus penneri strain 60 was studied by chemical analyses and 1H, 13C and 31P NMR spectroscopy, and the following structure of the linear pentasaccharide–phosphate repeating unit of the O-polysaccharide was established:〈displayedItem type="mathematics" xml:id="mu1" numbered="no"〉〈mediaResource alt="image" href="urn:x-wiley:09288244:FIM351:FIM_351_mu1"/〉 Rabbit polyclonal O-antiserum against P. penneri 60 reacted with both core and O-polysaccharide moieties of the homologous LPS. Based on the unique O-polysaccharide structure and serological data, we propose to classify P. penneri 60 into a new, separate Proteus serogroup O70. A weak cross-reactivity of P. penneri 60 O-antiserum with the lipopolysaccharide of Proteus vulgaris O8, O15 and O19 was observed and discussed in view of the chemical structures of the O-polysaccharides.
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    Mucosal immunisation with novel Streptococcus pneumoniae protein antigens enhances bacterial clearance in an acute mouse lung infection model (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Streptococcus pneumoniae contains many proteins that have not been evaluated as potential protective vaccine antigens. In this study we isolated proteins from a serotype 3 strain of S. pneumoniae for use in mouse immunisation studies. Separation of the protein mix was achieved by SDS–PAGE electrophoresis followed by electro-elution to isolate individual proteins. This procedure successfully separated 21 fractions from which six proteins were selected based on purity and quantity and were initially denoted by their molecular masses: 14-, 34-, 38-, 48-, 57- and 75-kDa. The immunogenicity of these proteins was investigated in a mucosal immunisation model in mice involving a primary inoculation to the intestinal Peyer's patches followed by an intra-tracheal boost two weeks later. The immune response was assessed by enhancement of pulmonary clearance of infection, recruitment of phagocytes to the lungs and induction of an antibody response. Two of the proteins, the 14-kDa identified as a L7/L12 ribosomal protein, and the 34-kDa identified as glyceraldehyde-3-phosphate dehydrogenase resulted in up to 99% and 94%, respectively, enhanced clearance of infection within 5 h following pulmonary challenge with S. pneumoniae. This study has shown that novel pneumococcal proteins have the potential to be vaccine candidates to enhance clearance of an acute mucosal S. pneumoniae infection.
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    Mechanisms of Porphyromonas gingivalis-induced monocyte chemoattractant protein-1 expression in endothelial cells (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Monocyte chemoattractant protein-1 (MCP-1) is expressed in vascular endothelial cells of inflamed gingival tissues and plays an important role in periodontal pathogenesis. Endothelial cells produce high levels of MCP-1 in response to Porphyromonas gingivalis, an important periodontal pathogen. The present study investigated the mechanisms involved in MCP-1 production by human umbilical vein endothelial cells (HUVEC) following infection with P. gingivalis. In contrast to P. gingivalis, Bacteroides forsythus only weakly stimulated MCP-1 production while Treponema denticola could not induce MCP-1 in HUVEC. The MCP-1 production was independent of endogenous interleukin (IL)-1α as IL-1 receptor antagonist treatment did not reduce MCP-1 production by P. gingivalis. Meanwhile, antioxidant treatment and inhibition of NAD(P)H oxidase significantly reduced MCP-1 production. Pharmacological inhibition of p38 mitogen-associated protein (MAP) kinase, c-Jun N-terminal kinase (JNK), nuclear factor-κB (NF-κB) or activator protein-1 (AP-1) also substantially attenuated P. gingivalis-induced MCP-1 expression by HUVEC. Indeed, activation of NF-κB and AP-1 was observed in P. gingivalis-infected HUVEC. These results suggest that MCP-1 expression is upregulated in P. gingivalis-infected endothelial cells via reactive oxygen species, p38 MAP kinase, JNK, NF-κB, and AP-1.
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    Role of anti-β-glucan antibody in host defense against fungi (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We have recently detected an anti-β-glucan antibody in normal human and normal mouse sera. The anti-β-glucan antibody showed reactivity to pathogenic fungal Aspergillus and Candida cell wall glucan. Anti-β-glucan antibody could bind whole Candida cells. It also enhanced the candidacidal activity of macrophages in vitro. The anti-β-glucan antibody titer of DBA/2 mice intravenously administered either Candida or Aspergillus solubilized cell wall β-glucan decreased remarkably dependent on dose. Moreover, in deep mycosis patients, the anti-β-glucan antibody titer decreased, and this change correlated with clinical symptoms and other parameters such as C-reactive protein. It was suggested that the anti-β-glucan antibody formed an antigen–antibody complex and participated in the immune response as a molecule recognizing pathogenic fungi.
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    Evaluation of SIV-lipopeptide immunizations administered by the intradermal route in their ability to induce antigen specific T-cell responses in rhesus macaques (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Numerous clinical and experimental observations have shown that cellular immunity, in particular CD8+ T-lymphocytes, plays an important role in the control of HIV infection. We have focused on a lipopeptide vaccination strategy that has been shown to induce polyepitopic T-cell responses in both animals and humans, in order to deliver simian immunodeficiency virus (SIV) antigens to rhesus macaques. Given the relevance of antigen administration route in the development of an effective cellular immune response, this study was designed to assess SIV lipopeptide immunizations administered either by the intradermal (ID) or the intramuscular (IM) routes in their ability to elicit GAG and NEF multispecific T-lymphocytes in the rhesus macaque. Antigen specific T-cell responses were observed between 7 and 11 weeks following vaccination in both groups. Macaques immunized by the IM route yielded antigen-specific IFN-γ secreting lymphocytes in response to no more than two pools of peptides derived from SIV-NEF. In contrast, among the four ID-immunized macaques, two presented multi-specific T-cell responses to as many as four pools of SIV-NEF and/or GAG peptides. Responses persisted 16 weeks following the vaccination protocol in one of the ID-vaccinated macaques. The induction of such responses is of great clinical relevance in the development of an effective HIV vaccine. Given the crucial role of CD8+ T-lymphocytes in HIV/SIV containment, vaccination through the intradermal route should merit high consideration in the development of an AIDS vaccine.
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    Th1-type immune response to a Coccidioides immitis antigen delivered by an attenuated strain of the non-invasive enteropathogen Vibrio cholerae (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The antigen-2 or proline rich antigen (Ag2/PRA) from Coccidioides immitis, known to protect mice against experimental Coccidioidomycosis, was expressed in the genetically attenuated cholera vaccine candidate Vibrio cholerae 638 and its thymine auxotrophic derivative 638T. Intranasal immunization of mice with strains producing Ag2/PRA induced serum vibriocidal antibody and Ag2/PRA-specific total IgG responses in outbred Swiss Webster and inbred BALB/c mice. Analysis of IgG subclasses showed a predominance of IgG2a subclass antibodies. Lymphocytes from immunized mice stimulated with pure Ag2/PRA showed a significant proliferative response with production of interferon-γ. Positive selection for plasmid maintenance in vivo did not enhance immune response to Ag2/PRA. These results demonstrate that genetically attenuated strains of the non-invasive pathogen V. cholerae can be used to express and deliver foreign antigens to stimulate a Th1 type of immune response.
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    Antibody response to GroEL varies in patients with acute Mycoplasma pneumoniae infection (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Although heat-shock proteins represent major antigens in a wide spectrum of bacterial infections, their immunogenicity is not known for Mycoplasma pneumoniae. M. pneumoniae is a major human respiratory pathogen and it has been suggested that its groEL gene might be dispensable in vitro. Using the specific monoclonal antibody 2C2/C3 we found an abundant synthesis of about 58 kDa GroEL in M. pneumoniae reference strains and in 15 clinical isolates examined at low and higher passages. In patients with acute respiratory disease caused by M. pneumoniae immunoblot analyses showed relatively low prevalence of systemic antibodies against its GroEL protein. Whereas all patients had strong antibody response to the P1 adhesin, only 5 of 29 patients (17.2%) had antibodies to GroEL. Among them, patient RI raised an early and very strong antibody response to GroEL. During the convalescent phase, levels of his serum IgG (mainly IgG2) to GroEL increased and were higher than levels of IgG to P1.
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    Induction of CD69 expression and Th1 cytokines release from human peripheral blood lymphocytes after in vitro stimulation with Alloiococcus otitidis and three middle ear pathogens (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Alloiococcus otitidis is a recently discovered pathogen of otitis media. However, only a limited number of studies are available about the pathogenic and immunological role of A. otitidis. The aim of this study was to investigate the activation and the cytokine production of human peripheral blood lymphocytes at the early immune response after stimulation with A. otitidis. After stimulation of whole human peripheral blood lymphocytes for 18 h with whole killed A. otitidis or the three major middle ear pathogens (Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis), the expression of CD69 and the production of cytokines were analyzed. The expression of CD69 on T cells and B cells was dose-dependently enhanced after stimulation with A. otitidis. The release of interleukin (IL)-12 was induced after stimulation with A. otitidis, whereas the release of IL-4 was not induced after stimulation with A. otitidis. In addition, the release of interferon (IFN)-γ was induced after stimulation with A. otitidis. Although the release of IFN-γ started within 18 h after stimulation with A. otitidis, intracellular production of IFN-γ was not observed in either CD4+ T cells or CD8+ T cells within 18 h upon stimulation. The patterns of CD69 expression and T helper-type 1 (Th1)-promoting cytokines production were similarly shown when human peripheral blood lymphocytes were stimulated with the other three major pathogens. Our results suggest that A. otitidis has sufficient immunogenic potential to modulate a host immune response, like the other three major middle ear pathogens, and also suggest that the immunogenicity of A. otitidis is very similar, at the early immune response, to that of the three major middle ear pathogens.
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    URL: http://dx.doi.org/10.1016/j.femsim.2004.10.014
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    Population genetic analysis of Pseudomonas aeruginosa using multilocus sequence typing (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: To study the population genetic structure of Pseudomonas aeruginosa, we developed a multilocus sequence typing scheme. The sequences of internal fragments of seven housekeeping genes were obtained for 34 P. aeruginosa isolates from patients hospitalized in five different European cities. Twenty-six different allelic profiles were identified. The mean allelic diversity was 0.854 (range: 0.606–0.978), which was about six times greater than the results obtained with the multilocus enzyme electrophoresis method. Linkage disequilibrium was measured with the index of association. An index of 1.95 ± 0.24 was calculated when all the strains were considered. This index was 1.76 ± 0.27 when only one strain per sequence type was considered. Both results were different from 0, indicating linkage among loci, which means that the population structure of our set of P. aeruginosa isolates is clonal. The clonal structure of the population was also suggested by the congruence of the topology of the different trees obtained from the seven housekeeping genes. These results are in contrast to previous studies, finding a non clonal population structure. Since a small number of isolates was analyzed in this study, there might be a bias of selection which includes the possibility that they belong to widely disseminated epidemic clones. Another possibility is that recombination did not occurred homogeneously throughout the genome of P. aeruginosa, so that part of it has a clonal structure, while the remaining part of the genome is more frequently subject to recombination.
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    URL: http://dx.doi.org/10.1016/j.femsim.2004.06.024
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    Similar bifidogenic effects of prebiotic-supplemented partially hydrolyzed infant formula and breastfeeding on infant gut microbiota (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The aim of the study was to assess the quantitative and qualitative differences of the gut microbiota in infants. We evaluated gut microbiota at the age of 6 months in 32 infants who were either exclusively breast-fed, formula-fed, nursed by a formula supplemented with prebiotics (a mixture of fructo- and galacto-oligosaccharides) or breast-fed by mothers who had been given probiotics. The Bifidobacterium, Bacteroides, Clostridium and Lactobacillus/Enterococcus microbiota were assessed by the fluorescence in situ hybridization, and Bifidobacterium species were further characterized by PCR. Total number of bifidobacteria was lower among the formula-fed group than in other groups (P= 0.044). Total amounts of the other bacteria were comparable between the groups. The specific Bifidobacterium microbiota composition of the breast-fed infants was achieved in infants receiving prebiotic supplemented formula. This would suggest that early gut Bifidobacterium microbiota can be modified by special diets up to the age of 6 months.
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    Elevated levels of plasma VEGF in patients with dengue hemorrhagic fever (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Vascular endothelial growth factor (VEGF) can be produced by monocytes and endothelial cells. It plays important role in angiogenesis and vascular permeability. The phenomenon of extensive plasma leakage into various serous cavities of the body is a cardinal symptom of dengue hemorrhagic fever (DHF). This study was performed to investigate the role of VEGF in patients with DHF. Plasma samples collected from the 53 dengue fever (DF) patients (including 14 patients with DHF), and 5 additional subjects with non-dengue febrile illness as controls were tested for VEGF levels using commercial enzyme-linked immunosorbent assay (ELISA) kits. The results showed that median plasma levels of VEGF in the patients with DHF (54.6 pg ml−1) were significantly higher than those of DF (14.6 pg ml−1) and control group (27.1 pg ml−1) (P 〈 0.05). In addition, VEGF levels in DF patients were not significantly different from those of control patients with non-dengue febrile illness (P= 0.17). Multiple regression analysis was used to analyze the clinical variables independently associated with VEGF levels. The data showed that D-dimer levels were significantly associated with VEGF levels. In this study, plasma VEGF levels in patients with DHF were significantly higher than values from DF patients. The association between increased plasma VEGF levels and increased plasma D-dimer levels in the patients with dengue illness suggests that activation of the fibrinolytic system may play a role in VEGF production in the patients with DF.
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    Towards more virulent and antibiotic-resistant Salmonella? (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Salmonella are well-known pathogens. Virulence determinants can be present on the chromosome, usually encoded on pathogenicity islands, or on plasmids and bacteriophages. Antibiotic resistance determinants usually are encoded on plasmids, but can also be present on the multidrug resistance region of Salmonella Genomic Island 1 (SGI1). Virulence plasmids show a remarkable diversity in the combination of virulence factors they encode, which appears to adapt them to specific hosts and the ability to cause gastroenteritidis or systemic disease. The appearance of plasmids with two replicons may help to extend the host range of these plasmids and thereby increase the virulence of previously non- or low pathogenic serovars. Antibiotic resistance among Salmonella is also increasing. This increase is not only in the percentage isolates resistant to a particular antibiotic, but also the development of resistance against newer antibiotics. The increased occurrence of integrons is particularly worrying. Integrons can harbour a varying set of antibiotic resistance encoding gene cassettes. Gene cassettes can be exchanged between integrons. Although the gene cassettes currently present in Salmonella integrons encode for older antibiotics (however, some still frequently used) gene cassettes encoding resistance against the newest antibiotics has been documented in Enterobacteriaceae. Furthermore, β-lactamases with activity against broad-spectrum cephalosporins, which are often used in empiric therapy, have been found associated with integrons. So, empiric treatment of Salmonella infections becomes increasingly more difficult. The most worrisome finding is that virulence and resistance plasmids form cointegrates. These newly formed plasmids can be selected by antibiotic pressure and thereby for virulence factors. Taken together these trends may lead to more virulent and antibiotic-resistant Salmonella.
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    Enhanced resistance to Gram-positive bacterium and increased susceptibility to bacterial endotoxin in mice sensitized with Propionibacterium acnes: involvement of Toll-like receptor (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Mice sensitized with Propionibacterium acnes showed an enhanced resistance against infection with Listeria monocytogenes in contrast to the increased susceptibility to LPS-induced endotoxin shock. The enhanced protection to L. monocytogenes was mediated by activated innate immunity but not by generation of Listeria-specific acquired immunity. After infection with L. monocytogenes, the elimination of bacteria was observed earlier in accordance with a higher level of endogenous cytokine production in P. acnes-sensitized mice than in control mice. Peritoneal cells from P. acnes-sensitized mice produced a larger amount of IL-12p70 and nitric oxide after stimulation with heat-killed L. monocytogenes or peptideglycan purified from Staphylococcus aureus. RT-PCR analysis showed that the expression of TLR2 but not TLR1, TLR4 nor TLR6 was induced by injection of P. acnes in peritoneal cells. These results indicated that P. acnes-sensitization could induce the activation of innate immunity against L. monocytogenes through increased recognition of bacterial components by TLR2.
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    Interaction of viral proteins with metal ions: role in maintaining the structure and functions of viruses (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Metal ions are integral part of some viral proteins and play an important role in their survival and pathogenesis. Zinc, magnesium and copper are the commonest metal ion that binds with viral proteins. Metal ions participate in maturation of genomic RNA, activation and catalytic mechanisms, reverse transcription, initial integration process and protection of newly synthesized DNA, inhibition of proton translocation (M2 protein), minus- and plus-strand transfer, enhance nucleic acid annealing, activation of transcription, integration of viral DNA into specific sites and act as a chaperone of nucleic acid. Metal ions are also required for nucleocapsid protein-transactivation response (TAR)–RNA interactions. In certain situations more than one metal ion is required e.g. RNA cleavage by RNase H. This review underscores the importance of metal ions in the survival and pathogenesis of a large group of viruses and studies on structural basis for metal binding should prove useful in the early design and development of viral inhibitors.
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    Epitope mapping of Burkholderia pseudomallei serine metalloprotease: identification of serine protease epitope mimics (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Filamentous phage random peptide libraries were used to identify the epitopes of Burkholderia pseudomallei protease by panning against IgG polyclonal sera that exhibited protease neutralizing properties. The isolated fusion peptides presented a consensus peptide sequence, T KSMA LSG, which closely resembles part of the active site sequence, 435GTSMAT PHVAG 445, of B. pseudomallei serine metalloprotease. By comparing the consensus sequence, TKSMALSG, with the predicted three-dimensional molecular model of B. pseudomallei serine metalloprotease, it appears that the potential antibody binding epitope was buried within the molecule. This active site was conformational whereby one continuous sub-region (SMA) was located between two discontinuous sub-regions, supplied by the flanking residues in the same polypeptide. All phages selected from the biopanning with IgG polyclonal sera showed good binding towards the polyclonal antibodies when compared to the negative control. In addition, these peptide-bearing phages showed competitive inhibition of B. pseudomallei serine metalloprotease binding to the polyclonal IgG.
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    Nitric oxide production and inducible nitric oxide synthase expression induced by Prevotella nigrescens lipopolysaccharide (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We have examined the effects of lipopolysaccharide (LPS) from Prevotella nigrescens, one of the causative agents of inflammatory periodontal disease and endodontic infections, on the production of nitric oxide (NO) and expression of inducible nitric oxide synthase (iNOS) in the murine macrophage cell line RAW264.7. We also attempted to throw light on the signaling mechanisms involved in P. nigrescens LPS-induced NO production. We found that P. nigrescens LPS can induce iNOS expression and stimulate the release of NO without additional stimuli and demonstrated an important role of the transcription factor NF-κB and microtubule polymerization in NO production. The production of NO required l-arginine and protein tyrosine kinase but not activation of protein kinase C. The ability of P. nigrescens LPS to promote the production of NO may be important in the pathogenesis of inflammatory periodontal disease and endodontic infections.
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    Pathogenic enteric Escherichia coli in children with and without diarrhea in Maputo, Mozambique (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: A study was conducted on the circulation of potentially diarrheagenic Escherichia coli in two groups of children, both under the age of seven. The first group (548 children) suffered from mild diarrhea and attended the Xipamanine Health Center of Maputo, in Mozambique. The second group (380 children) included randomly chosen, asymptomatic, children from the same population. A total of 503 E. coli strains were isolated from the two groups of children (n= 375 and 128, respectively). All E. coli strains were genotypically and phenotypically screened. The presence of virulence-associated genes was assessed by a set of multiplex PCR specific for st and lt genes of enterotoxic Escherichia coli (ETEC), eae and bfpA genes of enteropathogenic E. coli (EPEC), stx1 and stx2 of enterohemorrhagic E. coli (EHEC), ial of enteroinvasive E. coli (EIEC) and the species-specific gene uidA. Adhesion and citotoxicity of isolated E. coli were evaluated in vitro on different cell cultures. A total of 37 isolates harbored virulence-associated genes: 18 were classified as ETEC, (15 from symptomatic, and three from asymptomatic children), 16 as EPEC (respectively, 13 and 3) and three EIEC in the symptomatic group. No stx1 or stx2 genes, associated with enterohemorrhagic E. coli were found. On the basis of the adhesion pattern on HeLa cells, 167 E. coli were classified as diffusely adhering, (125 in patients and 42 in controls) and 67 as enteroaggregative, (50 and 17, respectively). To the best of our knowledge, this is the first report in the literature on the circulation of potentially diarrheagenic E. coli in Mozambique.
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    Candida guilliermondii isolated from HIV-infected human secretes a 50 kDa serine proteinase that cleaves a broad spectrum of proteinaceous substrates (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Non-albicans Candida species cause 35–65% of all candidemias in the general population, especially in immunosuppressed individuals. Here, we describe a case of a 19-year-old HIV-infected man with pneumonia due to a yeast-like organism. This clinical yeast isolate was identified as Candida guilliermondii through mycological tests. C. guilliermondii was cultivated in brain heart infusion medium for 48 h at 37 °C. After sequential centrifugation and concentration steps, the free-cell culture supernatant was obtained and extracellular proteolytic activity was assayed firstly using gelatin-SDS–PAGE. A 50 kDa proteolytic enzyme was detected with activity at physiological pH. This activity was completely blocked by 10 mM phenylmethylsulphonyl fluoride (PMSF), a serine proteinase inhibitor, suggesting that this extracellular proteinase belongs to the serine proteinase class. E-64, a strong cysteine proteinase inhibitor, and pepstatin A, a specific aspartic proteolytic inhibitor, did not interfere with the 50 kDa proteinase. Conversely, a zinc-metalloproteinase inhibitor (1,10-phenanthroline) restrained the proteinase activity released by C. guilliermondii by approximately 50%. Proteinases are a well-known class of enzymes that participate in a vast context of yeast–host interactions. In an effort to establish a functional implication for this extracellular serine-type enzyme, we investigated its capacity to hydrolyze some serum proteins and extracellular matrix components. We demonstrated that the 50 kDa exocellular serine proteinase cleaved human serum albumin, non-immune human immunoglobulin G, human fibronectin and human placental laminin, generating low molecular mass polypeptides. Collectively, these results showed for the first time the ability of an extracellular proteolytic enzyme other than aspartic-type proteinases in destroying a broad spectrum of relevant host proteins by a clinical species of non-albicans Candida.
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    Characterization and immunogenicity of Kingella kingae outer-membrane proteins (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 43 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: In recent years, Kingella kingae has emerged as an important pediatric pathogen but the antigenicity of the organism and the host immune response have not been studied. Outer membrane proteins (OMPs) of 57 K. kingae isolates were characterized and the immune response of 19 children with invasive infections was studied by immunoblotting. Kingella kingae OMPs were remarkably similar disregarding place and time of isolation and associated clinical condition (asymptomatic carriage, bacteremia, endocarditis, septic arthritis or osteomyelitis). Most OMPs were immunogenic but the specific bands that reacted in each strain and the intensity of the reactions varied substantially. When convalescent sera were reacted with heterologous strains, bands that either were not recognized by the homologous serum or were not present in the homologous strain were visualized. These results demonstrate that OMPs of K. kingae are highly conserved but suggest that some epitopes are polymorphic, resulting in a variable pattern of immune response.
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    Respiration metabolism of Group B Streptococcus is activated by environmental haem and quinone and contributes to virulence (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Group B Streptococcus (GBS) is a common constituent of the vaginal microflora, but its transmission to newborns can cause life-threatening sepsis, pneumonia and meningitis. Energy metabolism of this opportunist pathogen has been deduced to be strictly fermentative. We discovered that GBS undergoes respiration metabolism if its environment supplies two essential respiratory components: quinone and haem. Respiration metabolism led to significant changes in growth characteristics, including a doubling of biomass and an altered metabolite profile under the tested conditions. The GBS respiratory chain is inactivated by: (i) withdrawing haem and/or quinone, (ii) treating cultures with a respiration inhibitor or (iii) inactivating the cydA gene product, a subunit of cytochrome bd quinol oxidase, in all cases resulting in exclusively fermentative growth. cydA inactivation reduced GBS growth in human blood and strongly attenuated virulence in a neonatal rat sepsis model, suggesting that the animal host may supply the components that activate GBS respiration. These results suggest a role of respiration metabolism in GBS dissemination. Our findings show that environmental factors can increase the flexibility of GBS metabolism by activating a newly identified respiration chain. The need for two environmental factors may explain why GBS respiration metabolism was not found in previous studies.
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    Patterns of nucleosomal organization in the alc regulon of Aspergillus nidulans: roles of the AlcR transcriptional activator and the CreA global repressor (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We have studied the chromatin organization of three promoters of the alc regulon of Aspergillus nidulans. No positioned nucleosomes are seen in the aldA (aldehyde dehydrogenase) promoter under any physiological condition tested by us. In the alcA (alcohol dehydrogenase I) and alcR (coding for the pathway-specific transcription factor) promoters, a pattern of positioned nucleosomes is seen under non-induced and non-induced repressed conditions. While each of these promoters shows a specific pattern of chromatin restructuring, in both cases induction results in loss of nucleosome positioning. Glucose repression in the presence of inducer results in a specific pattern of partial positioning in the alcA and alcR promoters. Loss of nucleosome positioning depends absolutely on the AlcR protein and it is very unlikely to be a passive result of the induction of transcription. In an alcR loss-of-function background and in strains carrying mutations of the respective AlcR binding sites of the alcA and alcR promoters, nucleosomes are fully positioned under all growth conditions. Analysis of mutant AlcR proteins establishes that all domains needed for transcriptional activation and chromatin restructuring are included within the first 241 residues. The results suggest a two-step process, one step resulting in chromatin restructuring, a second one in transcriptional activation. Partial positioning upon glucose repression shows a specific pattern that depends on the CreA global repressor. An alcR loss-of-function mutation is epistatic to a creA loss-of-function mutation, showing that AlcR does not act by negating a nucleosome positioning activity of CreA.
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    Measure for measure in the control of type III secretion hook and needle length (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Bacterial flagella and injectisomes are supramolecular structures that are responsible for motility and for delivering toxic proteins into the cytosol of eukaryotic cells, respectively. They look very similar to each other. Both systems are called type III secretion pathways, and their components share substantial sequence similarities. One remarkable feature of the type III systems is that the length of their substructure is fairly well controlled by a secretion switching machinery, which consists of at least two proteins, a length control protein and an integral membrane secretion component. Here, we review how and why the length of these structures must be accurately controlled.
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    The unique structure of archaeal ‘hami’, highly complex cell appendages with nano-grappling hooks (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Proteinaceous, hair-like appendages known as fimbriae or pili commonly extend from the surface of prokaryotic cells and serve important functions such as cell adhesion, biofilm formation, motility and DNA transfer. Here we show that a novel group of archaea from cold, sulphidic springs has developed cell surface appendages of an unexpectedly high complexity with a well-defined base-to-top organization. It represents a new class of filamentous cell appendages, for which the term ‘hamus’ is proposed. Each archaeal cell is surrounded by a halo of about 100 hami, which mediate strong adhesion of the cells to surfaces of different chemical composition. The hami are mainly composed of 120 kDa subunits and remained stable in a broad temperature and pH range (0–70°C; 0.5–11.5). Electron microscopy and cryo-electron tomography revealed that the hamus filament possesses a helical basic structure. At periodic distances, three prickles emanate from the filament, giving it the character of industrially produced barbwire. At its distal end the hami carry a tripartite, barbed grappling hook (60 nm in diameter). The architecture of this molecular hook is reminiscent of man-made fishhooks, grapples and anchors. It appears that nature has developed a perfect mechanical nano-tool in the course of biological evolution, which also might prove useful in the field of nanobiotechnology.
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    A truncated H-NS-like protein from enteropathogenic Escherichia coli acts as an H-NS antagonist (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
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    N-terminal signal region of FliK is dispensable for length control of the flagellar hook (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The length of the flagellar hook is regulated; it is 55 ± 6 nm long in Salmonella. Five genes involved in hook-length regulation are fliK, flhB, fliG, fliM and fliN. The last four genes encode structural components of the protein export apparatus in the flagellar base, whereas FliK is soluble and secreted during flagellar assembly. The role of FliK, however, remains ambiguous. We constructed two kinds of FliK variants: N-terminally truncated FliK protein and FliK N-terminally fused with cyan fluorescent protein (CFP-FliK). Both N-terminally truncated FliK missing the first 99 amino acids (aa) and CFP-FliK fusion variants partially complemented a fliK null (polyhook) mutant to produce cells with filaments, allowing cells to swim; the hooks, however, were not normal but were polyhooks. When the N-terminally defective FliK variants were expressed at high levels, the average polyhook length was shortened coming close to the length of the wild-type hook, independently of the sizes of the FliK variants. These FliK variants were not secreted. CFP-FliK fusion proteins were observed to homogeneously distribute in the cytoplasm. We conclude that FliK does not need to be exported to control hook length and is unlikely to be a ruler; instead, we conclude that FliK controls hook length by the timely switching of secretion modes of the flagellar type III secretion system  by  the  FliK  C-terminal  domain,  and  that  the  N-terminal region is dispensable for hook length control.
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    Strange organelles –Plasmodium mitochondria lack a pyruvate dehydrogenase complex (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 55 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Our understanding of the Plasmodium mitochondrion and apicoplast has been greatly assisted by the genome sequence project. Sequence data have seeded recent research showing that the apicoplast is  the  site  of  several  anabolic  pathways  including fatty acid synthesis. The discovery of an active apicoplast pyruvate dehydrogenase complex implies this enzyme generates the acetyl-CoA needed for fatty acid synthesis. However, the absence of a corresponding mitochondrial complex suggests that energy generation in Plasmodium is considerably different from pathways described in other eukaryotes.
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    A bacterial hormone (the SCB1) directly controls the expression of a pathway-specific regulatory gene in the cryptic type I polyketide biosynthetic gene cluster of Streptomyces coelicolor (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Gamma-butyrolactone signalling molecules are produced by many Streptomyces species, and several have been shown to regulate antibiotic production. In Streptomyces coelicolor A3(2) at least one γ-butyrolactone (SCB1) has been shown to stimulate antibiotic production, and genes encoding proteins that are involved in its synthesis (scbA) and binding (scbR) have been characterized. Expression of these genes is autoregulated by a complex mechanism involving the γ-butyrolactone. In this study, additional genes influenced by ScbR were identified by DNA microarray analysis, and included a cryptic cluster of genes for a hypothetical type I polyketide. Further analysis of this gene cluster revealed that the pathway-specific regulatory gene, kasO, is a direct target for regulation by ScbR. Gel retardation and DNase I footprinting analyses identified two potential binding sites for ScbR, one at −3 to −35 nt and the other at −222 to −244 nt upstream of the kasO transcriptional start site. Addition of SCB1 eliminated the DNA binding activity of ScbR at both sites. The expression of kasO was growth phase regulated in the parent (maximal during transition phase), undetectable in a scbA null mutant, and constitutively expressed in a scbR null mutant. Addition of SCB1 to the scbA mutant restored the expression of kasO, indicating that ScbR represses kasO until transition phase, when presumably SCB1 accumulates in sufficient quantity to relieve kasO repression. Expression of the cryptic antibiotic gene cluster was undetectable in a kasO deletion mutant. This is the first report with comprehensive in vivo and in vitro data to show that a γ-butyrolactone-binding protein directly regulates a secondary metabolite pathway-specific regulatory gene in Streptomyces.
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    Translation elongation factor EF-Tu is a target for Stp, a serine-threonine phosphatase involved in virulence of Listeria monocytogenes (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Listeria monocytogenes is a pathogen that causes listeriosis, a severe food-borne infection. This bacterium, in order to survive and grow in the multiple conditions encountered in the host and the environment, has evolved a large number of regulatory elements, in particular many signal transduction systems based on reversible phosphorylation. The genome sequence has revealed genes for 16 putative two-component systems, four putative tyrosine phosphatases, three putative serine-threonine kinases and two putative serine-threonine phosphatases. We found that one of the latter genes, stp, encodes a functional Mn2+-dependent serine-threonine phosphatase similar to PPM eukaryotic phosphatases (Mg2+-or Mn2+-dependent protein phosphatase) and is required for growth of L. monocytogenes in a murine model of infection. We identified as the first target for Stp, the elongation factor EF-Tu. Post-translational phosphorylation of EF-Tu had been shown to prevent its binding to amino-acylated transfer RNA as well as to kirromycin, an antibiotic known to inhibit EF-Tu function. Accordingly, an stp deletion mutant is less sensitive to kirromycin. These results suggest an important role for Stp in regulating EF-Tu and controlling bacterial survival in the infected host.
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    Defining the role of PfCRT in Plasmodium falciparum chloroquine resistance (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Recent studies have highlighted the importance of a parasite protein referred to as the chloroquine resistance transporter (PfCRT) in the molecular basis of Plasmodium falciparum resistance to the quinoline antimalarials. PfCRT, an integral membrane protein with 10 predicted transmembrane domains, is a member of the drug/metabolite transporter superfamily and is located on the membrane of the intra-erythrocytic parasite's digestive vacuole. Specific polymorphisms in PfCRT are tightly correlated with chloroquine resistance. Transfection studies have now proven that pfcrt mutations confer verapamil-reversible chloroquine resistance in vitro and reveal their important role in resistance to quinine. Available evidence is consistent with the view that PfCRT functions as a transporter directly mediating the efflux of chloroquine from the digestive vacuole.
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    Reticulocyte-binding protein homologue 1 is required for sialic acid-dependent invasion into human erythrocytes by Plasmodium falciparum (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 55 (2005), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Apicomplexan parasite responsible for the most virulent form of malaria, Plasmodium falciparum, invades human erythrocytes through mutiple ligand–receptor interactions. Some strains of P. falciparum are sensitive to neuraminidase treatment of the host erythrocyte and these parasites have been termed sialic acid-dependent as they utilize receptors containing sialic acid. In contrast, other strains can efficiently invade neuraminidase-treated erythrocytes and hence are sialic acid-independent. The molecular interactions that allow P. falciparum to differentially utilize receptors for merozoite invasion are not understood. The P. falciparum reticulocyte-binding protein homologue (PfRh or PfRBL) family have been implicated in the invasion process but their exact role is unknown. PfRh1, a member of this protein family, appears to be expressed in all parasite lines analysed but there are marked differences in the level of expression between different strains. We have used targeted gene disruption of the PfRh1 gene in P. falciparum to show that the encoded protein is required for sialic acid-dependent invasion of human erythrocytes. The ΔPfRh1 parasites are able to invade normally; however, they utilize a pattern of ligand–receptor interactions that are more neuraminidase-resistant. Current data suggest a strategy based on the differential function of specific PfRh proteins has evolved to allow P. falciparum parasites to utilize alternative receptors on the erythrocyte surface for evasion of receptor polymorphisms and the host immune system.
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    Differential ability of σs and σ70 of Escherichia coli to utilize promoters containing half or full UP-element sites (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 55 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The σs subunit of RNA polymerase (RNAP) is the master regulator of the general stress response in Escherichia coli. Nevertheless, the selectivity of promoter recognition by the housekeeping σ70-containing and σ5-containing RNAP holoenzymes (Eσ70 and Eσs respectively) is not yet fully clarified, as they both recognize nearly identical −35 and −10 promoter consensus sequences. In this study, we show that in a subset of promoters, Eσs favours the presence of a distal UP-element half-site, and at the same time is unable to take advantage of a proximal half-site or a full UP-element. This is reflected by the frequent occurrence of distal UP-element half-sites in natural σs-dependent promoters and the absence of proximal half-sites. Eσ70, however, exhibits the opposite preference. The presence of the −35 element is a prerequisite for this differential behaviour. In the absence of the −35 element, half or full UP-element sites play no role in sigma selectivity, but the distal subsite leads to an equivalent, if not greater, transcriptional stimulation than the proximal one for both sigma factors. Finally, experiments using single amino acid substitutions of σs indicate that the foundation for this preference lies in an inability of σs to interact with the a subunit C-terminal domain.
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    Heterologous expression of Aquifex aeolicus ribosome recycling factor in Escherichia coli is dominant lethal by forming a complex that lacks functional co-ordination for ribosome disassembly (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 55 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Recycling the post-termination ribosomal complex requires the co-ordinated effort of the ribosome, ribosome  recycling  factor  (RRF)  and  elongation  factor EF-G. Although Aquifex aeolicus RRF (aaRRF) binds Escherichia coli ribosomes as efficiently as E. coli RRF, the resulting complex is non-functional and dominant lethal in E. coli, even in the presence of homologous A. aeolicus EF-G. These findings suggest that the E. coli post-termination ribosomal complex with aaRRF lacks functional co-ordination with EF-G required for ribosome recycling. A chimeric EF-G (E. coli domains I–III, A. aeolicus domains IV–V) or an A. aeolicus EF-G with distinct mutations in the domain I–II interface could activate aaRRF. Furthermore, novel mutations that localize to one surface of the  L-shape  structure  of  aaRRF  restored  activity  in E. coli. These aaRRF mutations are spatially distinct from mutations previously described and suggest a novel active centre for coupling EF-G's G domain motor action to ribosome disassembly.
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    A kinase for light and time (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The eukaryotic filamentous fungus Neurospora crassa has proven to be a dependable model system for the analysis of light-regulated gene expression and circadian rhythmicity. The molecular basis of the core circadian clock is a transcription/translation-based feedback loop in which a heterodimer of the white-collar 1 (WC-1) and white-collar 2 (WC-2) proteins act as a transcription factor to promote expression of the negative elements in the clock, frq mRNA and FRQ protein. Additionally, WC-1 is a flavoprotein that acts as the primary responder to environmental light and, in conjunction with WC-2, is the major photoreceptor for the clock as well as other light-regulated processes. Protein kinase C acts as a light-dependent regulator of the WC-1 protein.
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    Understanding the control of Pseudomonas aeruginosa alginate synthesis and the prospects for management of chronic infections in cystic fibrosis (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Decades of research have been dedicated to the study of the opportunistic pathogen Pseudomonas aeruginosa, a Gram-negative, environmental bacterium that secretes the exopolysaccharide alginate during chronic lung infection of cystic fibrosis (CF) patients. Although P. aeruginosa utilizes a variety of factors to establish a successful infection in the lungs of CF patients, alginate has stood out as one of the best-studied prognostic indicators of chronic lung infection. While the genetics, biosynthesis and regulation of alginate are well understood, questions still remain concerning its role in biofilm development and its potential as a therapeutic target. The purpose of this review is to provide a brief summary of alginate biosynthesis and regulation, and to highlight recent discoveries in the areas of alginate production, biofilm formation and vaccine design. This information is placed in context with a proposed P. aeruginosa infectious pathway, highlighting avenues for the use of existing therapies as well as the potential for novel agents to reduce or eliminate chronic infections in CF patients.
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    The gene NCE103 (YNL036w) from Saccharomyces cerevisiae encodes a functional carbonic anhydrase and its transcription is regulated by the concentration of inorganic carbon in the medium (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Carbonic anhydrase (CA) catalyses the rapid interconversion between CO2 and HCO3–. Despite its wide distribution among living organisms, the presence of CA in fungi has been controversially discussed. Using mass spectrometric analysis of 18O exchange from doubly labelled CO2, we were able to measure CA activity in intact cells of Saccharomyces cerevisiae. Intracellular CA activity was lacking in the Δnce103 mutant, indicating that NCE103 encodes a functional CA. This was proven by overexpressing and purification of the NCE103 gene product showing a specific activity of around 6900 units per mg protein. Interestingly, the in vivo CA activity was 10–20 times higher in cells grown on low inorganic carbon (Ci; air containing 0.035% CO2) than in high-Ci cells (grown on 5% CO2). The enhanced CA activity of low-Ci cells was inducible after transferring high-Ci cells to air. Northern blot analysis revealed that that expression of NCE103 is transcriptionally regulated by low Ci which was also demonstrated by fusing the NCE103 promoter to β-galactosidase as a reporter gene. Inactivation of NCE103 results in a high CO2 requiring mutant indicating that a functional CA is an important prerequisite for S. cerevisiae to grow under low-Ci conditions
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    Regulation of azole drug susceptibility by Candida albicans protein kinase CK2 (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 56 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Fluconazole resistance of the fungal pathogen Candida albicans can arise through several mechanisms, but the responsible genes and pathways are poorly understood. We report here that mutations in CKA2, identified through an insertional mutagenesis screen, confer fluconazole resistance. CKA2 and its homologue CKA1 specify catalytic subunits of protein kinase CK2. Although cka1 mutations have little effect on fluconazole resistance, CKA1 overexpression suppresses the fluconazole resistance of a cka2 mutant. This observation, along with synthetic cka1–cka2 interactions, argues that Cka1p and Cka2p carry out similar functions. cka2 mutants overexpress CDR1 and CDR2, two fluconazole efflux transporter genes, and a cdr1 mutation decreases resistance of a cka2 mutant, as expected if CDR1 and CDR2 overexpression is responsible for fluconazole resistance of the cka2 mutant. The protein phosphatase calcineurin is required for azole tolerance, and we find that the calcineurin inhibitor cyclosporin reverses fluconazole resistance of cka2 mutants. In addition, a mutation in CRZ1, which specifies a homologue of the Saccharomyces cerevisiae transcription factor that is a major target of calcineurin, suppresses fluconazole resistance  of  cka2 mutants.  Expression  analysis  of Cka2p-responsive genes argues that Cka2p and Crz1p act through distinct mechanisms. Several clinical fluconazole-resistant isolates overexpress some Cka2p-responsive genes. We suggest that a Cka2p-dependent regulatory pathway is altered by clinically derived azole resistance mutations.
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    Disulphide trapping of an in vivo energy-dependent conformation of Escherichia coli TonB protein (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 55 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: In Escherichia coli, the TonB system transduces the protonmotive force (pmf) of the cytoplasmic membrane to support a variety of transport events across the outer membrane. Cytoplasmic membrane proteins ExbB and ExbD appear to harvest pmf and transduce it to TonB. Experimental evidence suggests that TonB shuttles to the outer membrane, apparently to deliver conformationally stored potential energy to outer membrane transporters. In the most recent model, discharged TonB is then recycled to the cytoplasmic membrane to be re-energized by the energy coupling proteins, ExbB/D. It has been suggested that the carboxy-terminal 75 amino acids of active TonB could be represented by the rigid, strand-exchanged, dimeric crystal structure of the corresponding fragment. In contrast, recent genetic studies of alanine substitutions have suggested instead that in vivo the carboxy-terminus of intact TonB is dynamic and flexible. The biochemical studies presented here confirm and extend those results by demonstrating that individual cys substitution at aromatic residues in one monomeric subunit can form spontaneous dimers in vivo with the identical residue in the other monomeric subunit. Two energized TonBs appear to form a single cluster of 8–10 aromatic amino acids, including those found at opposite ends of the crystal structure. The aromatic cluster requires both the amino-terminal energy coupling domain of TonB, and ExbB/D (and cross-talk analogues TolQ/R) for in vivo formation. The large aromatic cluster is detected in cytoplasmic membrane-, but not outer membrane-associated TonB. Consistent with those observations, the aromatic cluster can form in the first half of the energy transduction cycle, before release of conformationally stored potential energy to ligand-loaded outer membrane transporters. The model that emerges is one in which, after input of pmf mediated through ExbB/D and the TonB transmembrane domain, the TonB carboxy-terminus can form a meta-stable high-energy conformation that is not represented by the crystal structure of the carboxy-terminus.
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    cAMP blocks MAPK activation and sclerotial development via Rap-1 in a PKA-independent manner in Sclerotinia sclerotiorum (2005)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 55 (2005), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Sclerotinia sclerotiorum is a filamentous ascomycete phytopathogen able to infect an extremely wide range of cultivated plants. Our previous studies have shown that increases in cAMP levels result in the impairment of the development of the sclerotium, a highly differentiated structure important in the disease cycle of this fungus. cAMP also inhibits the activation of a S. sclerotiorum mitogen-activated protein kinase (MAPK), which we have previously shown to be required for sclerotial maturation; thus cAMP-mediated sclerotial inhibition is modulated through MAPK. However, the mechanism(s) by which cAMP inhibits MAPK remains unclear. Here we demonstrate that a protein kinase A (PKA)-independent signalling pathway probably mediates MAPK inhibition by cAMP. Expression of a dominant negative form of Ras, an upstream activator of the MAPK pathway, also inhibited sclerotial development and MAPK activation, suggesting that a conserved Ras/MAPK pathway is required for sclerotial development. Evidence from bacterial toxins that specifically inhibit the activity of small GTPases, suggested that Rap-1 or Ras is involved in cAMP action. The Rap-1 inhibitor, GGTI-298, restored MAPK activation in the presence of cAMP, further suggesting that Rap-1 is responsible for cAMP-dependent MAPK inhibition. Importantly, inhibition of Rap-1 is able to restore sclerotial development blocked by cAMP. Our results suggest a novel mechanism involving the requirement of Ras/MAPK pathway for sclerotial development that is negatively regulated by a PKA-independent cAMP signalling pathway. Cross-talk between these two pathways is mediated by Rap-1.
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    Coccidioidomycosis and blastomycosis: Advances in molecular diagnosis (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Clinical isolates of Coccidioides spp. and Blastomyces dermatitidis can be identified by chemiluminescent DNA probes and PCR assays targeting multicopy genes. In fixed tissue samples, cells of the two fungi are specified by in situ hybridization and PCR assays targeting 18S rDNA but sequencing of the products is mandatory. Nested PCR assays targeting genes encoding species- or genus-specific proteins like proline rich antigen of Coccidioides spp. and B. dermatitidis adhesin facilitate amplification of specific DNA from fixed tissue samples. The value of DNA amplification from native specimens of suspected cases of coccidioidomycosis or blastomycosis still needs to be determined.
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    Electrophoresis karyotype and chromosome-length polymorphism of Histoplasma capsulatum clinical isolates from Latin America (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Intact chromosomes of 19 clinical isolates of Histoplasma capsulatum recently obtained in Argentina, Mexico and Guatemala and the laboratory reference strain G186B from Panama were analyzed using pulsed-field gel electrophoresis. Chromosomal banding patterns of the human isolates revealed 5–7 bands, ranging from 1.3 to 10 Mbp in size. Strain G186B showed five bands of approximately 1.1, 2.8, 3.3, 5.4 and 9.7 Mbp. Thirteen different electrokaryotypes were identified, indicating that the genome of H. capsulatum varies widely in nature, as observed previously in laboratory strains. No definite association was found between electrokaryotype and geographical or clinical source.
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    Towards a molecular diagnosis of invasive aspergillosis and disseminated candidosis (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: A lot of in-house polymerase chain reaction assays have been reported for diagnosis of invasive aspergillosis and disseminated candidosis. Encouraging results have been published to anticipate the diagnosis over the conventional microbiological methods. However, the absence of standardized methods has led to diverging results. As a consequence, these tests are not recognized as consensual diagnostic criteria, in contrast with some antigenemia detection kits. The major breakthrough for improving the results of these methods is the emergence of real-time technologies. This markedly improves the reliability of the PCR results by dramatically decreasing the risk of false positive results due to PCR products carryover. Moreover, using the quantitative results provided by this technique, this allows to rapidly compare the efficiency of primers, probes, and DNA extraction methods. Therefore, the hope is to identify the more specific and sensitive parameters to implement comparative studies. Automated DNA extraction should also be useful to achieve this goal.Whatever sophisticated technology is used, we still have to define the meaning of detecting nucleic acids in a given clinical sample. This seems simple in normally sterile anatomical sites but less obvious for example in respiratory specimens for invasive aspergillosis or in blood for candidosis in heavily colonized patients. Additional studies of the kinetics of fungal DNA are needed. The development of real-time technology should improve our knowledge in order to give the clinicians informative clues for making a decision.
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    Biosynthesis of glycoproteins in the pathogenic fungus Candida albicans: Activation of dolichol phosphate mannose synthase by cAMP-mediated protein phosphorylation (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Following incubation with ATP and a cAMP-dependent protein kinase under optimal conditions of lipid acceptor, phospholipid and metal ion requirements, the transfer activity of partially purified dolichol phosphate mannose synthase (DPMS) increased about 60% and this activation correlated with a 50% increase in Vmax with no alteration in the apparent Km for GDP-Manose. Phosphorylation with [γ-32P]ATP resulted in the labeling of several polypeptides, one of which exhibited the molecular weight of the enzyme (30 kDa) and was also recognized using a specific anti-DPMS monoclonal antibody. This and the fact that the phosphate label could be removed by an alkaline phosphatase indicate that Candida DPMS may be regulated by phosphorylation–dephosphorylation, a mechanism that has been proposed for the enzyme in other organisms.
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    Interaction of pathogenic fungi with host cells: Molecular and cellular approaches (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: This review provides an overview of several molecular and cellular approaches that are likely to supply insights into the host–fungus interaction. Fungi present intra- and/or extracellular host–parasite interfaces, the parasitism phenomenon being dependent on complementary surface molecules. The entry of the pathogen into the host cell is initiated by the fungus adhering to the cell surface, which generates an uptake signal that may induce its cytoplasmatic internalization. Furthermore, microbial pathogens use a variety of their surface molecules to bind to host extracellular matrix (ECM) components to establish an effective infection. On the other hand, integrins mediate the tight adhesion of cells to the ECM at sites referred to as focal adhesions and also play a role in cell signaling. The phosphorylation process is an important mechanism of cell signaling and regulation; it has been implicated recently in defense strategies against a variety of pathogens that alter host-signaling pathways in order to facilitate their invasion and survival within host cells. The study of signal transduction pathways in virulent fungi is especially important in view of their putative role in the regulation of pathogenicity. This review discusses fungal adherence, changes in cytoskeletal organization and signal transduction in relation to host–fungus interaction.
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    Editorial: Molecular genetic approaches to the study of human pathogenic fungi (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1016/j.femsim.2005.06.004
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    Homology, disruption and phenotypic analysis of CaGS Candida albicans gene induced during macrophage infection (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 45 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: During macrophage infection Candida albicans expresses differentially several genes whose functions are associated with its survival strategy. Among others, we have isolated CaGS gene, which is homologous to SNF3, a glucose sensor of Saccharomyces cerevisiae. To elucidate its potential role during infection, CaGS has been disrupted and the resulting phenotype analyzed on different solid media. The null mutant lost the ability to form hyphae on a medium with low glucose concentration and serum. Furthermore, this mutant does not disrupt macrophage in in vitro infections. We believe that this putative glucose sensor is involved in hyphal development during macrophage infection.
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    A swine toll-like receptor 2-expressing transfectant as a potential primary screening system for immunobiotic microorganisms (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
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    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Toll-like receptor 2 (TLR2) has been shown to mediate cell signaling in response to microbial cell wall components, such as peptidoglycan, lipoteichoic acid, microbial lipoprotein, and zymosan. In this study, we cloned the swine TLR2 and used it to transfect Chinese hamster ovary K-1 cells. We demonstrated that the swine TLR2-expressing transfectant can bind not only zymosan from yeast cell wall components but also intact lactic acid bacteria, resulting in the activation of nuclear factor-κB. These findings suggest that the swine TLR2-expressing transfectant can be very useful for the primary screening of immunobiotic microorganisms.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1016/j.femsim.2004.12.009
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    Distribution of “classic” virulence factors among Salmonella spp. (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Whether an infection with Salmonella spp. leads to a disease largely depends on the virulence of the strain and the constitution of the host. The virulence of the strain is determined by so-called virulence factors. Whereas a number of virulence factors of Salmonella have been identified only recently, others have been studied for decades. These latter virulence factors i.e., virulence-plasmids, toxins, fimbriae and flagella are therefore referred to as “classic” virulence factors. Here we present an overview on the distribution of (genes coding for) these virulence factors among Salmonella spp. The pathogenicity islands of Salmonella are also reviewed, all be it briefly, since they contain a major part of the virulence genes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1016/j.femsim.2005.02.002
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  • 94
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    Correlation of the Helicobacter pylori adherence factor BabA with duodenal ulcer disease in four European countries (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Helicobacter pylori strains harboring the vacAs1, cagA and babA2 have been associated with ulcer disease (UD). We compared the prevalence of these different genotypes and adhesive properties in H. pylori infected patients with UD in four European countries. Genomic DNA was isolated from 314 H. pylori strains: Germany (GER; n= 92), Sweden (SWE, n= 74), Portugal (POR, n= 91) and Finland (FIN, n= 57). The frequencies of babA2 genotype varied from 35% to 60%. Triple-positive strains (vacAs1+, cagA+ and babA2+) were significantly associated with UD in GER and POR and were closely correlated with UD in FIN, but not in SWE. Classification as triple-positive strains had a higher specificity for detection of UD in GER, POR and FIN than type1 or cagA+ strains. In vitro adhesion assays revealed that Swedish strains showed high adhesion properties and were thus correlated with the diagnosis of UD, although PCR detected the babA2 gene at lower frequencies and failed to show a correlation with UD. This finding appears to reflect allelic variations of the babA2 gene in SWE, although adhesive properties of the strains are retained.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1016/j.femsim.2004.10.010
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  • 95
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    Novel plasmids for gene expression analysis and for genetic manipulation in the gastric pathogen Helicobacter pylori (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: To facilitate gene expression analysis in the human gastric pathogen Helicobacter pylori, we constructed the plasmids pHPLAC-KAN and pHPLAC-CAT containing a promoterless Escherichia coli lacZ gene located upstream from the antibiotic resistance genes aphA-3 or cat, respectively. The suitability of the plasmids for H. pylori mutagenesis and gene expression analysis was evaluated by plasmid integration into the genome of H. pylori strain 1061 by single homologous recombination, using the rpl9 gene encoding ribosomal protein L9 as target. By monitoring β-galactosidase production from the resulting rpl9::lacZ fusion, it was demonstrated that H. pylori rpl9 displays the classical growth phase-dependent regulation of components of the protein synthesis machinery, as β-galactosidase production dropped fivefold in the stationary growth phase. The plasmids described in this study extend our methodological repertoire for genetic modification and molecular analysis of H. pylori, and may also be of use for other bacteria, as the resistance cassettes and the lacZ gene are active in the related Campylobacter species.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1016/j.femsim.2004.10.016
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  • 96
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    Restriction of DNA encoding selectable markers decreases the transformation efficiency of Helicobacter pylori (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Helicobacter pylori populations recovered from the human stomach display extensive recombination and quasispecies development, and this suggests frequent exchange of DNA between different strains in vivo. In vitro, however, most H. pylori strains display restriction to the uptake of non-self DNA, as measured using selectable markers, regardless of their competency for transformation with self DNA. We have examined the effect of different selectable markers on double-crossover recombination efficiencies in three reference strains (1061, 26695 & SS1) and one clinical isolate (CHP1) of H. pylori. All strains were efficiently transformable to kanamycin or chloramphenicol resistance by using self-genomic DNA from isogenic mutants bearing the aphA3 or cat cassettes, respectively. However, strains 26695 and CHP1 showed a 3–5-log reduction in transformation efficiency by non-self recombinant DNA containing aphA3, when compared to cat. Strain 1061 readily accepted either cassette, and strain SS1 was poorly tolerant of any non-self DNA. Genome-wide random mutagenesis of these strains was only achievable with a selectable marker that allowed high transformation efficiency. Digestion of 32P-labelled cassettes by H. pylori lysates mirrored the transformation results and indicated that in some strains these cassettes are the targets of enzymatic restriction.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1016/j.femsim.2004.10.019
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  • 97
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    CD4+CD25+ suppressor T cells regulate pathogen induced inflammation and disease (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: A key suppressor role has recently been ascribed to the natural CD4+CD25+ regulatory T cells (Treg), the removal of which leads to the development of autoimmune disease and aggravated pathogen-induced inflammation in otherwise normal hosts. The repertoire of antigen specificities of Treg is as broad as that of naïve T cells, recognizing both self and non-self antigens, enabling Treg to control a broad range of immune responses. Although widely acknowledged to play a role in the maintenance of self-tolerance, recent studies indicate that Treg can be activated and expanded against bacterial, viral and parasite antigens in vivo. Such pathogen-specific Treg can prevent infection-induced immunopathology but may also increase the load of infection and prolong pathogen persistence by suppressing protective immune responses. This review discusses the role of Treg in the prevention of exaggerated inflammation favoring chronicity in bacterial or fungal infections and latency in viral infections. Special attention is given to the role of Treg in the modulation of gastric inflammation induced by Helicobacter pylori infection. Findings in both experimentally infected mice and humans with natural infection indicate that Treg are important in protecting the H. pylori-infected host against excessive gastric inflammation and disease symptoms but on the negative side promote bacterial colonization at the gastric and duodenal mucosa which may increase the risk in H. pylori-infected individuals to develop duodenal ulcers.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1016/j.femsim.2004.10.017
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  • 98
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    The presence of the cag pathogenicity island is associated with increased superoxide anion radical scavenging activity by Helicobacter pylori (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Reactive oxygen species (ROS) generated by Helicobacter pylori infection have been suggested to be important factors in induction of gastric malignancies. Utilizing electron spin resonance spectrometry, H. pylori-dependent radical formation and hydroxyl- and superoxide-anion radical scavenging activity was investigated. In contrast to previous reports, we found that H. pylori does not produce ROS, but displays superoxide scavenging activity. This scavenging activity was increased in cag-positive H. pylori strains when compared to strains lacking an intact cag pathogenicity island, and was dependent on enzyme activity. We hypothesize that the increased scavenging activity of cag-positive H. pylori strains is an adaptation to the increased inflammatory response associated with the cag-positive genotype of H. pylori.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1016/j.femsim.2004.10.018
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  • 99
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    Helicobacter pylori VacA cytotoxin interacts with fibronectin and alters HeLa cell adhesion and cytoskeletal organization in vitro (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Helicobacter pylori vacuolating cytotoxin VacA causes multiple effects on epithelial cell function and morphology, but the effects of VacA on signal transduction pathways and the cytoskeleton have not been investigated in detail. In this study, we analyzed the effects of native VacA on HeLa and AGS cell adhesion to fibronectin and laminin under serum-free conditions. Confocal microscopic examination revealed increased number of cells with rounded morphology and inhibition of actin fiber formation, in the presence of VacA. VacA binds to fibronectin in vitro in a dose-dependent manner. This interaction was partly inhibited by a peptide containing an arginine-glycine-aspartic acid motif. The adhesion of HeLa cells to fibronectin, but not to laminin, was decreased in the presence of VacA. Thus, VacA may interact with fibronectin and influence integrin receptor-induced cell signaling and cytoskeleton-dependent cell functions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1016/j.femsim.2004.10.020
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  • 100
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    Mutational analysis of the Helicobacter pylori carbonic anhydrases (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 44 (2005), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: In the gastric microenvironment, Helicobacter pylori is exposed to bicarbonate, urea and acid. Here it is demonstrated that both H. pylori carbonic anhydrases (CAs) are required for maintaining urease activity and therefore influence H. pylori urea resistance at neutral pH. Furthermore, the β-CA is required for acid resistance as indicated by a growth defect of the corresponding mutant at low pH. The α- and β-CA mutants as well as the double mutant were more resistant to bicarbonate, indicating that both enzymes are involved in bicarbonate metabolism. These phenotypes support important CA-functions in H. pylori urea and bicarbonate metabolism and acid resistance. Thus, both CA enzymes might be required for survival in the gastric niche.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1016/j.femsim.2004.10.021
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