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  • 1
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Adherence and invasion are thought to be key events in the pathogenesis of non-typeable Haemophilus influenzae (NTHi). The role of NTHi lipooligosaccharide (LOS) in adherence was examined using an LOS-coated polystyrene bead adherence assay. Beads coated with NTHi 2019 LOS adhered significantly more to 16HBE14 human bronchial epithelial cells than beads coated with truncated LOS isolated from an NTHi 2019 pgmB::ermr mutant (P = 0.037). Adherence was inhibited by preincubation of cell monolayers with NTHi 2019 LOS (P = 0.0009), but not by preincubation with NTHi 2019 pgmB::ermr LOS. Competitive inhibition studies with a panel of compounds containing structures found within NTHi LOS suggested that a phosphorylcholine (ChoP) moiety was involved in adherence. Further experiments revealed that mutations affecting the oligosaccharide region of LOS or the incorporation of ChoP therein caused significant decreases in the adherence to and invasion of bronchial cells by NTHi 2019 (P 〈 0.01). Analysis of infected monolayers by confocal microscopy showed that ChoP+ NTHi bacilli co-localized with the PAF receptor. Pretreatment of bronchial cells with a PAF receptor antagonist inhibited invasion by NTHi 2109 and two other NTHi strains expressing ChoP+ LOS glycoforms exhibiting high reactivity with an anti-ChoP antibody on colony immunoblots. These data suggest that a particular subset of ChoP+ LOS glycoforms could mediate NTHi invasion of bronchial cells by means of interaction with the PAF receptor.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 41 (2001), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Haemophilus influenzae incorporates choline obtained from environmental sources onto its lipopolysaccharide as phosphorylcholine (ChoP). The decoration of the bacterial surface with ChoP contributes to pathogenesis by allowing for mimicry of the host. As the main reservoir for choline in the host is phosphatidylcholine, we tested whether other choline-containing molecules associated with eukaryotic membranes could provide an alternative source of choline. H. influenzae was able to use glycerophosphorylcholine (GPC), an abundant degradation product of phospholipids, as efficiently as free choline. Utilization of GPC required glpQ, which expresses an enzyme with glycerophosphodiester phosphodiesterase activity. In the absence of free choline, this gene was required for adherent H. influenzae to obtain choline directly from epithelial cells in culture. GlpQ therefore allows choline to be transferred from the host to the bacterial cell surface.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The surface of Streptococcus pneumoniae is decorated with a family of choline-binding proteins (CBPs) that are non-covalently bound to the phosphorylcholine of the teichoic acid. Two examples (PspA, a protective antigen, and LytA, the major autolysin) have been well characterized. We identified additional CPBs and characterized a new CBP, CbpA, as an adhesin and a determinant of virulence. Using choline immobilized on a solid matrix, a mixture of proteins from a pspA-deficient strain of pneumococcus was eluted in a choline-dependent fashion. Antisera to these proteins passively protected mice challenged in the peritoneum with a lethal dose of pneumococci. The predominant component of this mixture, CbpA, is a 75-kDa surface-exposed protein that reacts with human convalescent antisera. The deduced sequence from the corresponding gene showed a chimeric architecture with a unique N-terminal region and a C-terminal domain consisting of 10 repeated choline-binding domains nearly identical to PspA. A cbpA-deficient mutant showed a 〉50% reduction in adherence to cytokine-activated human cells and failed to bind to immobilized sialic acid or lacto-N-neotetraose, known pneumococcal ligands on eukaryotic cells. Carriage of this mutant in an animal model of nasopharyngeal colonization was reduced 100-fold. There was no difference between the parent strain and this mutant in an intraperitoneal model of sepsis. These data for CbpA extend the important functions of the CBP family to bacterial adherence and identify a pneumococcal vaccine candidate.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Haemophilus influenzae undergoes spontaneous phase variation in colony morphology. Organisms from transparent colonies efficiently colonize the nasopharynx in an infant rat model of H. influenzae carriage, whereas organisms from more opaque colonies are deficient at colonization. A genetic approach relying on the transformability of H. influenzae was used to identify a locus contributing to opacity variation. By screening a library of chomosomal DNA from an opaque variant of strain Rd, it was possible to isolate a single clone capable of transforming a transparent Rd host to a more opaque phenotype. A region containing two genes, designated oapA and oapB, was identified. The deduced amino acid sequence of oapB has similarity to a consensus sequence for bacterial lipoproteins. Genetically defined mutations in oapA were transformed into the transparent Rd to confirm that this gene is required for expression of the transparent colony phenotype. Although oapA lacks a signal sequence, gene fusions to phoA show that OapA is secreted in H. influenzae and undergoes phase variation in expression. Mutagenesis of oapA in strain Rd, and type b strain Eagan, resulted in loss of the ability to colonize the nasopharynx of infant rats. The type b mutant, however, was as virulent as its parent strain when inoculated intraperitoneally. This suggests that the contribution of OapA to pathogenesis is limited to events associated with colonization of the mucosal surface.
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  • 5
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The lic1 locus of Haemophilus influenzae controls the incorporation of environmental choline into lipopolysaccharide (LPS) as phosphorylcholine (ChoP) as well as the phase variation of this structure. ChoP is the target of an acute phase reactant in serum, C-reactive protein (CRP), which mediates killing through the activation of complement when bound to the organism. Structural analysis of the oligosaccharide region of the H. influenzae LPS showed that ChoP is linked to different hexose residues on different chain extensions in strains Rd and Eagan. Differences in the molecular environment of ChoP affect the epitope defined by monoclonal antibody 12D9 and were associated with polymorphisms within LicD, a putative diphosphonucleoside choline transferase. Exchanging the licD genes between the two strains with ChoP on different chain extensions was sufficient to switch its position. Allelic variants with ChoP on a hexose on heptose III rather than heptose I were sensitive to CRP-mediated serum bactericidal activity regardless of the genetic background. Differences in CRP-mediated killing correlated with differences in the binding of CRP from human serum to whole bacteria. This suggests that, in addition to the mechanism involving phase variation, the structural rearrangements within the oligosaccharide contribute to evasion of innate and acquired immunity.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Oxford BSL : Blackwell Publishing Ltd
    Molecular microbiology 30 (1998), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Phase variation in colony morphology has been associated with the pathogenesis of infection caused by Haemophilus influenzae. This study shows that differences in colony opacity in non-typeable H. influenzae (NTHi) strain H233 involve phase changes in the lipopolysaccharide (LPS) and depend on the expression of lic1 and lic2, which contain translational switches based on intragenic tandem repeats of 5′-CAAT-3′. Genetic analysis showed that opaque organisms have an out-of-frame number of repeats in both lic1, required for the expression of phosphorylcholine (ChoP), and lic2, a putative galactosyl transferase that adds the terminal galactose on Galα1-4Gal. Defined variants in these loci were used to examine the contribution of individual LPS structures to resistance to serum bactericidal activity mediated by antibody and C-reactive protein (CRP). The addition of ChoP by lic1 was the only factor in serum killing involving CRP and complement. The terminal galactose moiety, in contrast, conferred resistance to killing by naturally acquired antibody and complement present in human serum. As Galα1-4Gal is also found on human glycolipids, it appears that decoration of the cell surface with this host-like antigen blocks antibody-mediated serum bactericidal activity. Genetic analysis of NTHi within the human respiratory tract demonstrated that Galα1-4Gal may not be expressed during carriage but may be advantageous for the organism in inflammatory states such as pneumonia.
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  • 7
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 16 (1995), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Streptococcus pneumoniae undergoes spontaneous phase variation in colony morphology. Differences in colony opacity have previously been shown to correlate with differences in the ability of organisms to colonize the mucosal surface of the nasopharynx in an animal model. The genetic basis of opacity variation was identified in transformation experiments. A DNA library, from a strain that varies at high frequency, was screened to identify a single clone capable of transforming a transparent recipient strain which varies at low frequency to an opaque phenotype. Analysis of this opacity locus revealed two genes, glpD and glpF, with similarity to genes required for glycerol metabolism in other bacteria. Following the pneumococcal glpF, repetitive intergenic elements, boxes A and C, were identified. These stem-loop-forming elements were not present in the same locus of the recipient strain. Although not required for phase variation in colony opacity, the box element was necessary for expression of phase variation at high frequency. Introduction of the box elements during transformation affected colony morphology, possibly by altering expression of a putative regulatory gene downstream from the box element. Mutagenesis within this region confirmed the contribution of the putative regulatory gene to the expression of colony opacity. Growth characteristics of strains generated in this study provide additional evidence for an association of differences in cell wall autolysis and variation in colony opacity.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Most clinical isolates of Streptococcus pneumoniae consist of heterogeneous populations of at least two colony phenotypes, opaque and transparent, selected for in the bloodstream and nasopharynx, respectively. Microarray analysis revealed 24 orfs that demonstrated differences in expression greater than twofold between variants of independent strains. Twenty-one of these showed increased expression in the transparent variants, including 11 predicted to be involved in sugar metabolism. A single genomic region contains seven of these loci including the gene that encodes the neuraminidase, NanA. In contrast to previous studies, there was no contribution of NanA to adherence of S. pneumoniae to epithelial cells or colonization in an animal model. However, we observed NanA-dependent desialylation of human airway components that bind to the organism and may mediate bacterial clearance. Targets of desialylation included human lactoferrin, secretory component, and IgA2 that were shown to be present on the surface of the pneumococcus in vivo during pneumococcal pneumonia. The efficiency of desialylation was increased in the transparent variants and enhanced for host proteins binding to the surface of S. pneumoniae. Because deglycosylation affects the function of many host proteins, NanA may contribute to a protease-independent mechanism to modify bound targets and facilitate enhanced survival of the bacterium.
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  • 9
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 50 (2003), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Haemophilus influenzae obtains choline from either its growth medium or host cell membrane lipids and expresses it on its lipopolysaccharide (LPS) in the form of phosphorylcholine (ChoP), which contributes to its pathogenesis by mimicry of host cell molecules. Two genes (licB and betT) revealed by whole genomic analysis as encoding potential choline transporters were tested for their role in LPS-ChoP synthesis. The betT gene in H. influenzae is similar to betT in Escherichia coli, which functions in choline transport for the generation of betaine in osmoprotection. The licB gene has homology to bacterial permeases including betT and is encoded in the lic1 locus, which is essential for the expression of LPS-ChoP. In the presence of high concentrations of choline, neither licB nor betT were necessary for expression of LPS-ChoP raising the possibility that other unidentified choline uptake mechanisms may exist in this species. However, under choline limiting conditions, including growth in human nasal airway surface fluid, the licB, but not betT, gene was required for choline transport and synthesis of LPS-ChoP suggesting that LicB functions as a high affinity choline permease. The betT, but not licB, gene was shown to function in osmoprotection in H. influenzae, similar to the role of betT in E. coli. Further analysis demonstrated growth condition dependent differences in the regulation of transcription of the licB and betT genes. We conclude that H. influenzae may have multiple mechanisms for choline uptake and distinct pathways for choline utilization in LPS-ChoP biosynthesis and osmoregulation.
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  • 10
    Publication Date: 2019-01-11
    Description: The past two decades have witnessed an alarming expansion of staphylococcal disease caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The factors underlying the epidemic expansion of CA-MRSA lineages such as USA300, the predominant CA-MRSA clone in the United States, are largely unknown. Previously described virulence and antimicrobial resistance genes that promote the dissemination of CA-MRSA are carried by mobile genetic elements, including phages and plasmids. Here, we used high-resolution genomics and experimental infections to characterize the evolution of a USA300 variant plaguing a patient population at increased risk of infection to understand the mechanisms underlying the emergence of genetic elements that facilitate clonal spread of the pathogen. Genetic analyses provided conclusive evidence that fitness (manifest as emergence of a dominant clone) changed coincidently with the stepwise emergence of (i) a unique prophage and mutation of the regulator of the pyrimidine nucleotide biosynthetic operon that promoted abscess formation and colonization, respectively, thereby priming the clone for success; and (ii) a unique plasmid that conferred resistance to two topical microbiocides, mupirocin and chlorhexidine, frequently used for decolonization and infection prevention. The resistance plasmid evolved through successive incorporation of DNA elements from non-S. aureus spp. into an indigenous cryptic plasmid, suggesting a mechanism for interspecies genetic exchange that promotes antimicrobial resistance. Collectively, the data suggest that clonal spread in a vulnerable population resulted from extensive clinical intervention and intense selection pressure toward a pathogen lifestyle that involved the evolution of consequential mutations and mobile genetic elements.
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
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