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  • 1
    Electronic Resource
    Electronic Resource
    IN VIVO GENETIC ANALYSIS OF BACTERIAL VIRULENCE (1999)
    Palo Alto, Calif. : Annual Reviews
    Annual Review of Microbiology 53 (1999), S. 129-154 
    Details
    ISSN: 0066-4227
    Source: Annual Reviews Electronic Back Volume Collection 1932-2001ff
    Topics: Biology
    Notes: Abstract In vitro assays contribute greatly to our understanding of bacterial pathogenesis, but they frequently cannot replicate the complex environment encountered by pathogens during infection. The information gained from such studies is therefore limited. In vivo models, on the other hand, can be difficult to use, and this has to some extent diminished the incentive to perform studies in living animals. However, several recently developed techniques permit in vivo examination of many genes simultaneously. Most of these methods fall into two broad classes: in vivo expression technology and signature-tagged mutagenesis. In vivo expression technology is a promoter-trap strategy designed to identify genes whose expression is induced in a specific environment, typically that encountered in a host. Signature-tagged mutagenesis uses comparative hybridization to isolate mutants unable to survive specified environmental conditions and has been used to identify genes critical for survival in the host. Both approaches have so far been used exclusively for investigating pathogen-host interactions, but they should be easily adaptable to the study of other processes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1146/annurev.micro.53.1.129
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  • 2
    Electronic Resource
    Electronic Resource
    Complementary activities of SseJ and SifA regulate dynamics of the Salmonella typhimurium vacuolar membrane (2002)
    Oxford, UK : Blackwell Science Ltd.
    Molecular microbiology 44 (2002), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Salmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS) of Salmonella typhimurium is required for bacterial replication within host cells. It acts by translocating effector proteins across the membrane of the Salmonella-containing vacuole (SCV). The SifA effector is required to maintain the integrity of the SCV membrane, and for the formation in epithelial cells of Salmonella-induced filaments (Sifs), which are tubular extensions of SCVs. We have investigated the role in S. typhimurium virulence of the putative SPI-2 effector genes sifB, srfJ, sseJ and sseI. An S. typhimurium strain carrying a mutation in sseJ was mildly attenuated for systemic virulence in mice, but strains carrying mutations in either srfJ, sseI or sifB had very little or no detectable virulence defect after intraperitoneal inoculation. Expression of SseJ in HeLa cells resulted in the formation of globular membranous compartments (GMCs), the composition of which appears to be similar to that of SCV membranes and Sifs. The formation of GMCs was dependent on the serine residue of the predicted acyltransferase/lipase active site of SseJ. Transiently expressed SseJ also inhibited Sif formation by wild-type bacteria, and was found to associate with Sifs, SCV membranes and simultaneously expressed SifA. Intracellular vacuoles containing sseJ mutant bacteria appeared normal but, in contrast to a sifA mutant, a sifA sseJ double mutant strain did not lose its vacuolar membrane, indicating that loss of vacuolar membrane around sifA mutant bacteria requires the action of SseJ. Collectively, these results suggest that the combined action of SseJ and SifA regulate dynamics of the SCV membrane in infected cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2002.02912.x
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  • 3
    Electronic Resource
    Electronic Resource
    A functional genomic analysis of type 3 Streptococcus pneumoniae virulence (2001)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 40 (2001), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Streptococcus pneumoniae remains a serious cause of morbidity and mortality in humans, but relatively little is known about the molecular basis of its pathogenesis. We used signature-tagged mutagenesis together with an analysis of S. pneumoniae genome sequence to identify and characterize genes required for pathogenesis. A library of signature-tagged mutants was created by insertion–duplication mutagenesis, and 1786 strains were analysed for their inability to survive and replicate in murine models of pneumonia and bacteraemia. One hundred and eighty-six mutant strains were identified as attenuated, and 56 were selected for further genetic characterization based on their ability to excise the integrated plasmid spontaneously. The genomic DNA inserts of the plasmids were cloned in Escherichia coli and sequenced. These sequences were subjected to database searches, including the S. pneumoniae genome sequence, which allowed us to examine the chromosomal regions flanking these genes. Most of the insertions were in probable operons, but no pathogenicity islands were found. Forty-two novel virulence loci were identified. Five strains mutated in genes involved in gene regulation, cation transport or stress tolerance were shown to be highly attenuated when tested individually in a murine respiratory tract infection model. Additional experiments also suggest that induction of competence for genetic transformation has a role in virulence.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2001.02335.x
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  • 4
    Electronic Resource
    Electronic Resource
    A Streptococcus pneumoniae pathogenicity island encoding an ABC transporter involved in iron uptake and virulence (2001)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 40 (2001), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Restricted iron availability is a major obstacle to growth and survival of pathogenic bacteria during infection. In contrast to Gram-negative pathogens, little is known about how Gram-positive pathogens obtain this essential metal. We have identified two Streptococcus pneumoniae genetic loci, pit1 and pit2, encoding homologues of ABC iron transporters that are required for iron uptake by this organism. S. pneumoniae strains containing disrupted copies of either pit1 or pit2 had decreased sensitivity to the iron-dependent antibiotic streptonigrin, and a strain containing disrupted copies of both pit1 and pit2 was unable to use haemoglobin as an iron source and had a reduced rate of iron uptake. The pit2− strain was moderately and the pit1−/pit2− strain strongly attenuated in virulence in mouse models of pulmonary and systemic infection, showing that the pit loci play a critical role during in vivo growth of S. pneumoniae. The pit2 locus is contained within a 27 kb region of chromosomal DNA that has several features of Gram-negative bacterial pathogenicity islands. This probable pathogenicity island (PPI-1) is the first to be described for S. pneumoniae, and its acquisition is likely to have played a significant role in the evolution of this important human pathogen.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2001.02414.x
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  • 5
    Electronic Resource
    Electronic Resource
    Signature-tagged and directed mutagenesis identify PABA synthetase as essential for Aspergillus fumigatus pathogenicity (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Signature-tagged mutagenesis (STM) is a method that has been used to screen for genes required for in vivo survival of pathogenic bacteria, but has not been used to investigate a eukaryotic pathogen in an animal model of disease. We have adapted STM to identify genes required for in vivo growth of the opportunistic fungal pathogen Aspergillus fumigatus. Using a mouse model of invasive pulmonary aspergillosis, we have isolated several mutant strains with defects in their ability to replicate in vivo. One strain unable to cause lethal infection was further characterized and found to have an insertion into the promoter of a gene (pabaA) encoding para-aminobenzoic acid synthetase, an enzyme catalyzing a late step in the biosynthesis of folate. The complete inability of this strain, and other pabaA− strains constructed in this study by targeted gene deletion, to cause lethal infection in mice confirms the importance of the folate synthesis pathway for in vivo survival of this pathogen. The successful application of STM to A. fumigatus demonstrates that in vivo genetic analysis of eukaryotic pathogens is feasible and could result in the identification of potential targets, such as para-aminobenzoic acid synthetase, for novel antifungal therapies.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2000.01953.x
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  • 6
    Electronic Resource
    Electronic Resource
    Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages (1998)
    Oxford BSL : Blackwell Science Ltd, UK
    Molecular microbiology 30 (1998), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The type III secretion system of Salmonella pathogenicity island 2 (SPI-2) is required for systemic infection of this pathogen in mice. Cloning and sequencing of a central region of SPI-2 revealed the presence of genes encoding putative chaperones and effector proteins of the secretion system. The predicted products of the sseB, sseC and sseD genes display weak but significant similarity to amino acid sequences of EspA, EspD and EspB, which are secreted by the type III secretion system encoded by the locus of enterocyte effacement of enteropathogenic Escherichia coli. The transcriptional activity of an sseA::luc fusion gene was shown to be dependent on ssrA, which is required for the expression of genes encoding components of the secretion system apparatus. Strains carrying non-polar mutations in sseA, sseB or sseC were severely attenuated in virulence, strains carrying mutations in sseF or sseG were weakly attenuated, and a strain with a mutation in sseE had no detectable virulence defect. These phenotypes were reflected in the ability of mutant strains to grow within a variety of macrophage cell types: strains carrying mutations in sseA, sseB or sseC failed to accumulate, whereas the growth rates of strains carrying mutations in sseE, sseF or sseG were only modestly reduced. These data suggest that, in vivo, one of the functions of the SPI-2 secretion system is to enable intracellular bacterial proliferation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1998.01047.x
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  • 7
    Electronic Resource
    Electronic Resource
    The Aspergillus fumigatus chsC and chsG genes encode Class III chitin synthases with different functions (1996)
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 20 (1996), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Two genes, designated chsC and chsG were isolated from DNA libraries of the opportunistic fungal pathogen, Aspergillus fumigatus. The genes were characterized with respect to their nucleotide sequences and mutant phenotypes. The complete deduced amino acid sequences of chsC and chsG show that the products of both genes are Class III zymogen-type enzymes. A mutant strain constructed by disruption of chsC is phenotypically indistinguishable from the wild-type strain, but chsG− and chsC− chsG− strains have reduced colony radial growth rate and chitin synthase activity, conidiate poorly and produce highly branched hyphae. Despite these defects, the double-mutant strain retained the ability to cause pulmonary disease in neutropenic mice. However, in comparison to the wild-type strain, there was a decrease in mortality and delay in the onset of illness in mice inoculated with the double-mutant strain, which was associated with smaller and more highly branched fungal colonies in lung tissue.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1996.5571084.x
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  • 8
    Electronic Resource
    Electronic Resource
    pH-dependent secretion of SseB, a product of the SPI-2 type III secretion system of Salmonella typhimurium (1999)
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 33 (1999), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The type III secretion system of Salmonella pathogenicity island 2 (SPI-2) is required for bacterial replication inside macrophages. SseB has been considered a putative target of the secretion system on the basis of its similarity with EspA, a protein secreted by the type III secretion system of enteropathogenic Escherichia coli (EPEC). EspA forms a filamentous structure on the bacterial cell surface and is involved in translocation of proteins into the eukaryotic cytosol. In this paper, we show that SseB is a secreted protein that associates with the surface of the bacterial cell and might, therefore, also be required for delivery of SPI-2 effector proteins to the eukaryotic cell cytosol. SseB begins to accumulate inside the bacterial cell when the culture enters early stationary phase. However, SseB is only secreted if the bacteria are grown at low pH or if the pH is shifted after growth from 7.0 to below pH 5.0. The secretion occurs within minutes of acidification and is totally dependent on a functional SPI-2 type III secretion system. As the pH of the Salmonella-containing vacuole inside host cells has been shown to acidify to between pH 4.0 and 5.0, and as SPI-2 gene expression occurs inside host cells, low pH might be a physiological stimulus for SPI-2-mediated secretion in vivo.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1999.01527.x
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  • 9
    Electronic Resource
    Electronic Resource
    Functional analysis of ssaJ and the ssaK/U operon, 13 genes encoding components of the type III secretion apparatus of Salmonella Pathogenicity Island 2 (1997)
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 24 (1997), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We have investigated the structure and transcriptional organization of 13 genes of Salmonella Pathogenicity Island 2 (SPI2) that encode components of the second type III secretion apparatus of Salmonella typhimurium. ssaK, L, M, V, N, O, P, Q, R, S, T, U constitute one operon of 10 kb. ssaJ lies upstream of ssaK and is the terminal gene of another operon. The deduced products of ssaJ, ssaK, ssaV, ssaN, ssaO, ssaQ, ssaR, ssaS, ssaT, and ssaU show greatest similarity to the Yersinia spp. genes yscJ, yscL, lcrD, yscN, yscO, yscQ, yscR, yscS, yscT, and yscU, respectively. The products of the ssaL, ssaM and ssaP genes do not have significant similarity to products of other type III secretion systems, and might be important for the specific function of the SPI2 type III secretion system. Bacterial strains carrying different ssa mutations display minor alterations in terms of serum sensitivity when compared with the wild-type strain, but none are defective in replication within macrophage-like RAW 264.7 cells. However, some of the ssa mutant strains invade HEp2 cells less efficiently and are less cytotoxic to RAW 264.7 macrophages than the wild-type strain. We show that the invasion defect is correlated with a lack of SipC in culture supernatants of these mutant strains. SipC is a product of the SPI1 type III secretion system of S. typhimurium, and is important for epithelial cell invasion. Therefore, mutations in SPI2 can affect the SPI1 secretion system, which raises the possibility of an interaction between the two type III secretion systems.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1997.3271699.x
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  • 10
    Electronic Resource
    Electronic Resource
    Identification of Staphylococcus aureus virulence genes in a murine model of bacteraemia using signature-tagged mutagenesis (1997)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 26 (1997), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Signature-tagged mutagenesis with transposon Tn917 was used to identify genes of Staphylococcus aureus required for virulence in a murine model of bacteraemia. Screening 1248 mutant strains in pools of 96 resulted in the provisional identification of 50 mutants attenuated in virulence. Subsequent individual analysis of many of these mutants confirmed that they are attenuated in virulence. DNA sequence analysis of regions flanking their transposon insertion points revealed that approximately half of them represent genes with unknown function, while most of the remainder are involved in nutrient biosynthesis and cell surface metabolism. Three mutants were found with transposon insertions in different positions in femA, and one mutant had an insertion in femB. Both femA and femB are involved in the formation of cell wall peptidoglycan pentaglycine cross-bridges. A further mutation occurred in a previously unknown gene that shares significant similarity to femB. Mutations were also obtained in recA and lsp (encoding the S. aureus prolipoprotein signal peptidase). On the basis of sequence similarities to proteins of known function, the products of other genes are probably involved in the synthesis of diaminopimelic acid (a component of peptidoglycan), maintenance of surface adhesins and cell surface membrane transport, showing that many components of the S. aureus cell surface are critical for the survival and replication of this pathogen in blood.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1997.5911966.x
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