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  • American Society for Microbiology  (15)
  • 1
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    Online Resource
    Characterization of Haemophilus ducreyi cdtA, cdtB , and cdtC Mutants in In Vitro and In Vivo Systems
    American Society for Microbiology ; 2001
    In:  Infection and Immunity Vol. 69, No. 9 ( 2001-09), p. 5626-5634
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 69, No. 9 ( 2001-09), p. 5626-5634
    Abstract: Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that is encoded by the cdtABC gene cluster and can be detected in culture supernatant fluid by its ability to kill HeLa cells. The cdtA, cdtB , and cdtC genes of H. ducreyi were cloned independently into plasmid vectors, and their encoded proteins expressed singly or in various combinations in an Escherichia coli background. All three gene products had to be expressed in order for E. coli -derived culture supernatant fluids to demonstrate cytotoxicity for HeLa cells. Isogenic H. ducreyi cdtA and cdtB mutants were constructed and used in combination with the wild-type parent strain and a previously described H. ducreyi cdtC mutant (M. K. Stevens, J. L. Latimer, S. R. Lumbley, C. K. Ward, L. D. Cope, T. Lagergard, and E. J. Hansen, Infect. Immun. 67:3900–3908, 1999) to determine the relative contributions of the CdtA, CdtB, and CdtC proteins to CDT activity. Expression of CdtA, CdtB, and CdtC appeared necessary for H. ducreyi -derived culture supernatant fluid to exhibit cytotoxicity for HeLa cells. Whole-cell sonicates and periplasmic extracts from the cdtB and cdtC mutants had no effect on HeLa cells, whereas these same fractions from a cdtA mutant had a very modest cytotoxic effect on these same human cells. CdtA appeared to be primarily associated with the H. ducreyi cell envelope, whereas both CdtB and CdtC were present primarily in the soluble fraction from sonicated cells. Both the cdtA mutant and the cdtB mutant were found to be fully virulent in the temperature-dependent rabbit model for experimental chancroid.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.69.9.5626-5634.2001
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2001
    detail.hit.zdb_id: 1483247-1
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  • 2
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    Online Resource
    Phenotypic Effect of Isogenic uspA1 and uspA2 Mutations on Moraxella catarrhalis 035E
    American Society for Microbiology ; 1998
    In:  Infection and Immunity Vol. 66, No. 7 ( 1998-07), p. 3113-3119
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 66, No. 7 ( 1998-07), p. 3113-3119
    Abstract: The UspA surface antigen of Moraxella catarrhalis was recently shown to be comprised of two different proteins (UspA1 and UspA2) which share an internal region containing 140 amino acids with 93% identity (C. Aebi, I. Maciver, J. L. Latimer, L. D. Cope, M. K. Stevens, S. E. Thomas, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 65:4367–4377, 1997). Isogenic uspA1 , uspA2 , and uspA1 uspA2 mutants were tested in a number of in vitro systems to determine what effect these mutations, either individually or together, might exert on the phenotype of M. catarrhalis 035E. Monoclonal antibodies specific for UspA1 or UspA2 were used in an indirect antibody accessibility assay to prove that both of these proteins were expressed on the surface of M. catarrhalis . All three mutants grew in vitro at the same rate and did not exhibit autoagglutination or hemagglutination properties that were detectably different from those of the wild-type parent strain. When tested for the ability to adhere to human epithelial cells, the wild-type parent strain and the uspA2 mutant readily attached to Chang conjunctival cells. In contrast, the uspA1 mutant and the uspA1 uspA2 double mutant both attached to these epithelial cells at a level nearly 2 orders of magnitude lower than that obtained with the wild-type parent strain, a result which suggested that expression of UspA1 by M. catarrhalis is essential for attachment to these epithelial cells. Both the wild-type parent strain and the uspA1 mutant were resistant to the bactericidal activity of normal human serum, whereas the uspA2 mutant and the uspA1 uspA2 double mutant were readily killed by this serum. This latter result indicated that the presence of UspA2 is essential for expression of serum resistance by M. catarrhalis .
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.66.7.3113-3119.1998
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 1998
    detail.hit.zdb_id: 1483247-1
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  • 3
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    Online Resource
    Expression of Cytolethal Distending Toxin and Hemolysin Is Not Required for Pustule Formation by Haemophilus ducreyi in Human Volunteers
    American Society for Microbiology ; 2001
    In:  Infection and Immunity Vol. 69, No. 3 ( 2001-03), p. 1938-1942
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 69, No. 3 ( 2001-03), p. 1938-1942
    Abstract: Haemophilus ducreyi makes cytolethal distending toxin (CDT) and hemolysin. In a previous human challenge trial, an isogenic hemolysin-deficient mutant caused pustules with a rate similar to that of its parent. To test whether CDT was required for pustule formation, six human subjects were inoculated with a CDT mutant and parent at multiple sites. The pustule formation rates were similar at both parent and mutant sites. A CDT and hemolysin double mutant was constructed and tested in five additional subjects. The pustule formation rates were similar for the parent and double mutant. These results indicate that neither the expression of CDT, nor that of hemolysin, nor both are required for pustule formation by H. ducreyi in humans.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.69.3.1938-1942.2001
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2001
    detail.hit.zdb_id: 1483247-1
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  • 4
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    Online Resource
    Haemophilus ducreyi Secretes a Filamentous Hemagglutinin-Like Protein
    American Society for Microbiology ; 1998
    In:  Journal of Bacteriology Vol. 180, No. 22 ( 1998-11-15), p. 6013-6022
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 180, No. 22 ( 1998-11-15), p. 6013-6022
    Abstract: We have identified two extremely large open reading frames (ORFs) in Haemophilus ducreyi 35000, lspA1 and lspA2 , each of which encodes a predicted protein product whose N-terminal half is approximately 43% similar to the N-terminal half of Bordetella pertussis filamentous hemagglutinin (FhaB). To the best of our knowledge, lspA1 (12,500 nucleotides [nt]) and lspA2 (14,800 nt) are among the largest prokaryotic ORFs identified to date. The predicted proteins, LspA1 and LspA2, are 86% identical overall to each other and also have limited amino acid sequence similarity at their N termini to other secreted bacterial proteins, including certain hemolysins. Southern blot analysis indicated that lspA1 and lspA2 sequences were present in 15 other geographically diverse H. ducreyi strains. Reverse transcriptase PCR analysis of total RNA isolated from H. ducreyi 35000 grown in liquid medium, grown on solid agar medium, and isolated from lesions of H. ducreyi -infected rabbits indicated that lspA1 and lspA2 were transcribed both in vitro and in vivo. A 260-kDa protein present in culture supernatant from eight virulent H. ducreyi strains reacted with both polyclonal serum from rabbits infected with H. ducreyi 35000 and a monoclonal antibody predicted to bind both LspA1 and LspA2. This 260-kDa protein in H. ducreyi 35000 culture supernatant was shown to be the protein product of the lspA1 ORF based on its reactivity with a monoclonal antibody specific for LspA1. Four H. ducreyi strains, previously shown to be avirulent in the temperature-dependent rabbit model for chancroid, did not produce either LspA1 or LspA2 in vitro. This finding raised the possibility that LspA1, LspA2, or both may be involved in the ability of H. ducreyi to cause lesions in this animal model.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.180.22.6013-6022.1998
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 1998
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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  • 5
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    Online Resource
    Haemophilus ducreyi Targets Src Family Protein Tyrosine Kinases To Inhibit Phagocytic Signaling
    American Society for Microbiology ; 2005
    In:  Infection and Immunity Vol. 73, No. 12 ( 2005-12), p. 7808-7816
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 73, No. 12 ( 2005-12), p. 7808-7816
    Abstract: Haemophilus ducreyi , the etiologic agent of the sexually transmitted disease chancroid, has been shown to inhibit phagocytosis of both itself and secondary targets in vitro. Immunodepletion of LspA proteins from H. ducreyi culture supernatant fluid abolished this inhibitory effect, indicating that the LspA proteins are necessary for the inhibition of phagocytosis by H. ducreyi . Fluorescence microscopy revealed that macrophages incubated with wild-type H. ducreyi , but not with a lspA1 lspA2 mutant, were unable to complete development of the phagocytic cup around immunoglobulin G-opsonized targets. Examination of the phosphotyrosine protein profiles of these two sets of macrophages showed that those incubated with wild-type H. ducreyi had greatly reduced phosphorylation levels of proteins in the 50-to-60-kDa range. Subsequent experiments revealed reductions in the catalytic activities of both Lyn and Hck, two members of the Src family of protein tyrosine kinases that are known to be involved in the proximal signaling steps of Fcγ receptor-mediated phagocytosis. Additional experiments confirmed reductions in the levels of both active Lyn and active Hck in three different immune cell lines, but not in HeLa cells, exposed to wild-type H. ducreyi . This is the first example of a bacte-rial pathogen that suppresses Src family protein tyrosine kinase activity to subvert phagocytic signaling in hostcells.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.73.12.7808-7816.2005
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2005
    detail.hit.zdb_id: 1483247-1
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  • 6
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    Identification of the znuA -Encoded Periplasmic Zinc Transport Protein of Haemophilus ducreyi
    American Society for Microbiology ; 1999
    In:  Infection and Immunity Vol. 67, No. 10 ( 1999-10), p. 5060-5068
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 67, No. 10 ( 1999-10), p. 5060-5068
    Abstract: The znuA gene of Haemophilus ducreyi encodes a 32-kDa (mature) protein that has homology to both the ZnuA protein of Escherichia coli and the Pzp1 protein of H. influenzae ; both of these latter proteins are members of a growing family of prokaryotic zinc transporters. Inactivation of the H. ducreyi 35000 znuA gene by insertional mutagenesis resulted in a mutant that grew more slowly than the wild-type parent strain in vitro unless ZnCl 2 was provided at a final concentration of 100 μM. Other cations tested did not restore growth of this H. ducreyi mutant to wild-type levels. The H. ducreyi ZnuA protein was localized to the periplasm, where it is believed to function as the binding component of a zinc transport system. Complementation of the znuA mutation with the wild-type H. ducreyi znuA gene provided in trans restored the ability of this H. ducreyi mutant to grow normally in the absence of exogenously added ZnCl 2 . The wild-type H. ducreyi znuA gene was also able to complement a H. influenzae pzp1 mutation. The H. ducreyi znuA isogenic mutant exhibited significantly decreased virulence ( P = 0.0001) when tested in the temperature-dependent rabbit model for experimental chancroid. This decreased virulence was not observed when the znuA mutant was complemented with the wild-type H. ducreyi znuA gene provided in trans .
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.67.10.5060-5068.1999
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 1999
    detail.hit.zdb_id: 1483247-1
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  • 7
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    Mutations in the lspA1 and lspA2 Genes of Haemophilus ducreyi Affect the Virulence of This Pathogen in an Animal Model System
    American Society for Microbiology ; 2004
    In:  Infection and Immunity Vol. 72, No. 2 ( 2004-02), p. 1221-1221
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 72, No. 2 ( 2004-02), p. 1221-1221
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.72.2.1221.2004
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2004
    detail.hit.zdb_id: 1483247-1
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  • 8
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    Online Resource
    Haemophilusducreyi Requires an Intact flp Gene Cluster for VirulenceinHumans
    American Society for Microbiology ; 2003
    In:  Infection and Immunity Vol. 71, No. 12 ( 2003-12), p. 7178-7182
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 71, No. 12 ( 2003-12), p. 7178-7182
    Abstract: An intact Haemophilus ducreyi flp operon is essential for microcolony formation in vitro. tadA is the 9th of 15 genes in the operon and has homology to NTPases of type IV secretion systems. Fifteen human volunteers were experimentally infected with both H. ducreyi 35000HP and the tadA mutant, 35000HP.400. Papules developed at similar rates at sites inoculated with the mutant and parent, while pustules formed at 36.4% of parent sites and at 0% of mutant sites ( P = 0.001). Compared to 35000HP, 35000HP.400 had only a modest but significant reduction in lesion scores in the temperature-dependent rabbit model of chancroid. These data suggest that proteins secreted by the flp locus are required for full expression of virulence by H. ducreyi in humans but have less of a role in virulence in an animal model of infection.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.71.12.7178-7182.2003
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2003
    detail.hit.zdb_id: 1483247-1
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  • 9
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    Online Resource
    Mapping of a Protective Epitope of the CopB Outer Membrane Protein of Moraxella catarrhalis
    American Society for Microbiology ; 1998
    In:  Infection and Immunity Vol. 66, No. 2 ( 1998-02), p. 540-548
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 66, No. 2 ( 1998-02), p. 540-548
    Abstract: A monoclonal antibody (MAb) (MAb 10F3) directed against the CopB outer membrane protein of Moraxella catarrhalis previously was found to enhance pulmonary clearance of M. catarrhalis in an animal model (M. Helminen, I. Maciver, J. L. Latimer, L. D. Cope, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 61:2003–2010, 1993). In the present study, this same MAb was shown to exert complement-dependent bactericidal activity against this pathogen in vitro. Nucleotide sequence analysis of the copB gene from two MAb 10F3-reactive and two MAb 10F3-unreactive strains of M. catarrhalis revealed that the deduced amino acid sequences of these four CopB proteins were at least 90% identical. Comparison of the amino acid sequences of these proteins allowed localization of possible MAb 10F3 binding sites to five relatively small regions of the CopB protein from M. catarrhalis O35E. When five synthetic peptides representing these regions were tested for their ability to bind MAb 10F3 in a direct enzyme-linked immunosorbent assay system, an oligopeptide containing 26 amino acids was shown to bind this MAb. The actual binding region for MAb 10F3 was localized further through the use of overlapping decapeptides that spanned this 26-mer. A fusion protein containing the same 26-mer readily bound MAb 10F3 and was used to immunize mice. The resultant antiserum contained antibodies that reacted with the CopB protein of the homologous M. catarrhalis strain in Western blot analysis and bound to the surface of both homologous and heterologous strains of M. catarrhalis.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.66.2.540-548.1998
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 1998
    detail.hit.zdb_id: 1483247-1
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  • 10
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    Expression of the LspA1 and LspA2 Proteins by Haemophilus ducreyi Is Required for Virulence in Human Volunteers
    American Society for Microbiology ; 2004
    In:  Infection and Immunity Vol. 72, No. 8 ( 2004-08), p. 4528-4533
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 72, No. 8 ( 2004-08), p. 4528-4533
    Abstract: Haemophilus ducreyi colocalizes with polymorphonuclear leukocytes and macrophages and evades phagocytosis during experimental infection of human volunteers. H. ducreyi contains two genes, lspA1 and lspA2 , which encode predicted proteins of 456 and 543 kDa, respectively. Compared to its wild-type parent, an lspA1 lspA2 double mutant does not inhibit phagocytosis by macrophage and myelocytic cell lines in vitro and is attenuated in an experimental rabbit model of chancroid. To test whether expression of LspA1 and LspA2 was necessary for virulence in humans, six volunteers were experimentally infected. Each volunteer was inoculated with three doses (ranging from 85 to 112 CFU) of the parent (35000HP) in one arm and three doses (ranging from 60 to 822 CFU) of the mutant (35000HPΩ12) in the other arm. The papule formation rates were 88% (95% confidence interval [95% CI], 76.8 to 99.9%) at 18 parent sites and 72% (95% CI, 44.4 to 99.9%) at 18 mutant sites ( P = 0.19). However, papules were significantly smaller at mutant sites (mean size, 24.8 mm 2 ) than at parent sites (mean size, 39.1 mm 2 ) 24 h after inoculation ( P = 0.0002). The pustule formation rates were 44% (95% CI, 5.8 to 77.6%) at parent sites and 0% (95% CI, 0 to 39.4%) at mutant sites ( P = 0.009). With the caveat that biosafety regulations preclude testing of a complemented mutant in human subjects, these results indicate that expression of LspA1 and LspA2 facilitates the ability of H. ducreyi to initiate disease and to progress to pustule formation in humans.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.72.8.4528-4533.2004
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2004
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