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  • American Society for Microbiology  (13)
  • Janowicz, Diane M.  (13)
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  • American Society for Microbiology  (13)
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  • 1
    Online Resource
    Online Resource
    A Class I Haemophilus ducreyi Strain Containing a Class II hgbA Allele Is Partially Attenuated in Humans: Implications for HgbA Vaccine Efficacy Trials
    American Society for Microbiology ; 2019
    In:  Infection and Immunity Vol. 87, No. 7 ( 2019-07)
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 87, No. 7 ( 2019-07)
    Abstract: Haemophilus ducreyi causes chancroid and is a major cause of cutaneous ulcers in children. Due to environmental reservoirs, both class I and class II H. ducreyi strains persist in cutaneous ulcer regions of endemicity following mass drug administration of azithromycin, suggesting the need for a vaccine. The hemoglobin receptor (HgbA) is a leading vaccine candidate, but its efficacy in animal models is class specific. Controlled human infection models can be used to evaluate vaccines, but only a class I strain (35000HP) has been characterized in this model. As a prelude to evaluating HgbA vaccines in the human model, we tested here whether a derivative of 35000HP containing a class II hgbA allele (FX548) is as virulent as 35000HP in humans. In eight volunteers infected at three sites with each strain, the papule formation rate was 95.8% for 35000HP versus 62.5% for FX548 ( P  = 0.021). Excluding doses of FX548 that were ≥2-fold higher than those of 35000HP, the pustule formation rate was 25% for 35000HP versus 11.7% for FX548 ( P  = 0.0053). By Western blot analysis, FX548 and 35000HP expressed equivalent amounts of HgbA in whole-cell lysates and outer membranes. The growth of FX548 and 35000HP was similar in media containing hemoglobin or hemin. By whole-genome sequencing and single-nucleotide polymorphism analysis, FX548 contained no mutations in open reading frames other than hgbA . We conclude that by an unknown mechanism, FX548 is partially attenuated in humans and is not a suitable strain for HgbA vaccine efficacy trials in the model.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.00112-19
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2019
    detail.hit.zdb_id: 1483247-1
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  • 2
    Online Resource
    Online Resource
    Determination of an Interaction Network between an Extracellular Bacterial Pathogen and the Human Host
    American Society for Microbiology ; 2019
    In:  mBio Vol. 10, No. 3 ( 2019-06-25)
    Details
    In: mBio, American Society for Microbiology, Vol. 10, No. 3 ( 2019-06-25)
    Abstract: A major gap in understanding infectious diseases is the lack of information about molecular interaction networks between pathogens and the human host. Haemophilus ducreyi causes the genital ulcer disease chancroid in adults and is a leading cause of cutaneous ulcers in children in the tropics. We developed a model in which human volunteers are infected on the upper arm with H. ducreyi until they develop pustules. To define the H. ducreyi and human interactome, we determined bacterial and host transcriptomic and host metabolomic changes in pustules. We found that in vivo H. ducreyi transcripts were distinct from those in the inocula, as were host transcripts in pustule and wounded control sites. Many of the upregulated H. ducreyi genes were found to be involved in ascorbic acid and anaerobic metabolism and inorganic ion/nutrient transport. The top 20 significantly expressed human pathways showed that all were involved in immune responses. We generated a bipartite network for interactions between host and bacterial gene transcription; multiple positively correlated networks contained H. ducreyi genes involved in anaerobic metabolism and host genes involved with the immune response. Metabolomic studies showed that pustule and wounded samples had different metabolite compositions; the top ion pathway involved ascorbate and aldarate metabolism, which correlated with the H. ducreyi transcriptional response and upregulation of host genes involved in ascorbic acid recycling. These data show that an interactome exists between H. ducreyi and the human host and suggest that H. ducreyi exploits the metabolic niche created by the host immune response. IMPORTANCE Dual RNA sequencing (RNA-seq) offers the promise of determining an interactome at a transcriptional level between a bacterium and the host but has yet to be done on any bacterial infection in human tissue. We performed dual RNA-seq and metabolomics analyses on wounded and infected sites following experimental infection of the arm with H. ducreyi . Our results suggest that H. ducreyi survives in an abscess by utilizing l -ascorbate as an alternative carbon source, possibly taking advantage of host ascorbic acid recycling, and that H. ducreyi also adapts by upregulating genes involved in anaerobic metabolism and inorganic ion and nutrient transport. To our knowledge, this is the first description of an interaction network between a bacterium and the human host at a site of infection.
    Type of Medium: Online Resource
    ISSN: 2161-2129 , 2150-7511
    URL: Article
    DOI: 10.1128/mBio.01193-19
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2019
    detail.hit.zdb_id: 2557172-2
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  • 3
    Online Resource
    Online Resource
    Carbon Storage Regulator A Contributes to the Virulence of Haemophilus ducreyi in Humans by Multiple Mechanisms
    American Society for Microbiology ; 2013
    In:  Infection and Immunity Vol. 81, No. 2 ( 2013-02), p. 608-617
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 81, No. 2 ( 2013-02), p. 608-617
    Abstract: The carbon storage regulator A (CsrA) controls a wide variety of bacterial processes, including metabolism, adherence, stress responses, and virulence. Haemophilus ducreyi , the causative agent of chancroid, harbors a homolog of csrA . Here, we generated an unmarked, in-frame deletion mutant of csrA to assess its contribution to H. ducreyi pathogenesis. In human inoculation experiments, the csrA mutant was partially attenuated for pustule formation compared to its parent. Deletion of csrA resulted in decreased adherence of H. ducreyi to human foreskin fibroblasts (HFF); Flp1 and Flp2, the determinants of H. ducreyi adherence to HFF cells, were downregulated in the csrA mutant. Compared to its parent, the csrA mutant had a significantly reduced ability to tolerate oxidative stress and heat shock. The enhanced sensitivity of the mutant to oxidative stress was more pronounced in bacteria grown to stationary phase compared to that in bacteria grown to mid-log phase. The csrA mutant also had a significant survival defect within human macrophages when the bacteria were grown to stationary phase but not to mid-log phase. Complementation in trans partially or fully restored the mutant phenotypes. These data suggest that CsrA contributes to virulence by multiple mechanisms and that these contributions may be more profound in bacterial cell populations that are not rapidly dividing in the human host.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.01239-12
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2013
    detail.hit.zdb_id: 1483247-1
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  • 4
    Online Resource
    Online Resource
    Haemophilus ducreyi SapA Contributes to Cathelicidin Resistance and Virulence in Humans
    American Society for Microbiology ; 2010
    In:  Infection and Immunity Vol. 78, No. 3 ( 2010-03), p. 1176-1184
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 78, No. 3 ( 2010-03), p. 1176-1184
    Abstract: Haemophilus ducreyi is an extracellular pathogen of human epithelial surfaces that resists human antimicrobial peptides (APs). The organism's genome contains homologs of genes s ensitive to a ntimicrobial p eptides ( sap operon) in nontypeable Haemophilus influenzae . In this study, we characterized the sap -containing loci of H. ducreyi 35000HP and demonstrated that sapA is expressed in broth cultures and H. ducreyi -infected tissue; sapA is also conserved among both class I and class II H. ducreyi strains. We constructed a nonpolar sapA mutant of H. ducreyi 35000HP, designated 35000HP sapA , and compared the percent survival of wild-type 35000HP and 35000HP sapA exposed to several human APs, including α-defensins, β-defensins, and the cathelicidin LL-37. Unlike an H. influenzae sapA mutant, strain 35000HP sapA was not more susceptible to defensins than strain 35000HP was. However, we observed a significant decrease in the survival of strain 35000HP sapA after exposure to LL-37, which was complemented by introducing sapA in trans . Thus, the Sap transporter plays a role in resistance of H. ducreyi to LL-37. We next compared mutant strain 35000HP sapA with strain 35000HP for their ability to cause disease in human volunteers. Although both strains caused papules to form at similar rates, the pustule formation rate at sites inoculated with 35000HP sapA was significantly lower than that of sites inoculated with 35000HP (33.3% versus 66.7%; P = 0.007). Together, these data establish that SapA acts as a virulence factor and as one mechanism for H. ducreyi to resist killing by antimicrobial peptides. To our knowledge, this is the first demonstration that an antimicrobial peptide resistance mechanism contributes to bacterial virulence in humans.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.01014-09
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2010
    detail.hit.zdb_id: 1483247-1
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  • 5
    Online Resource
    Online Resource
    Activation of the CpxRA System by Deletion of cpxA Impairs the Ability of Haemophilus ducreyi To Infect Humans
    American Society for Microbiology ; 2010
    In:  Infection and Immunity Vol. 78, No. 9 ( 2010-09), p. 3898-3904
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 78, No. 9 ( 2010-09), p. 3898-3904
    Abstract: Haemophilus ducreyi must adapt to the environment of the human host to establish and maintain infection in the skin. Bacteria generally utilize stress response systems, such as the CpxRA two-component system, to adapt to hostile environments. CpxRA is the only obvious two-component system contained in the H. ducreyi genome and negatively regulates the lspB-lspA2 operon, which encodes proteins that enable the organism to resist phagocytosis. We constructed an unmarked, in-frame H. ducreyi cpxA deletion mutant, 35000HPΔ cpxA . In human inoculation experiments, 35000HPΔ cpxA formed papules at a rate and size that were significantly less than its parent and was unable to form pustules compared to the parent. CpxA usually has kinase and phosphatase activities for CpxR, and the deletion of CpxA leads to the accumulation of activated CpxR due to the loss of phosphatase activity and the ability of CpxR to accept phosphate groups from other donors. Using a reporter construct, the lspB-lspA2 promoter was downregulated in 35000HPΔ cpxA , confirming that CpxR was activated. Deletion of cpxA downregulated DsrA, the major determinant of serum resistance in the organism, causing the mutant to become serum susceptible. Complementation in trans restored parental phenotypes. 35000HPΔ cpxA is the first H. ducreyi mutant that is impaired in its ability to form both papules and pustules in humans. Since a major function of CpxRA is to control the flow of protein traffic across the periplasm, uncontrolled activation of this system likely causes dysregulated expression of multiple virulence determinants and cripples the ability of the organism to adapt to the host.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.00432-10
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2010
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  • 6
    Online Resource
    Online Resource
    Expression of Haemophilus ducreyi Collagen Binding Outer Membrane Protein NcaA Is Required for Virulence in Swine and Human Challenge Models of Chancroid
    American Society for Microbiology ; 2006
    In:  Infection and Immunity Vol. 74, No. 5 ( 2006-05), p. 2651-2658
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 74, No. 5 ( 2006-05), p. 2651-2658
    Abstract: Haemophilus ducreyi , the etiologic agent of the sexually transmitted genital ulcer disease chancroid, has been shown to associate with dermal collagen fibers within infected skin lesions. Here we describe NcaA, a previously uncharacterized outer membrane protein that is important for H. ducreyi collagen binding and host colonization. An H. ducreyi strain lacking the ncaA gene was impaired in adherence to type I collagen but not fibronectin (plasma or cellular form) or heparin. The mutation had no effect on serum resistance or binding to HaCaT keratinocytes or human foreskin fibroblasts in vitro. Escherichia coli expressing H. ducreyi NcaA bound to type I collagen, demonstrating that NcaA is sufficient to confer collagen attachment. The importance of NcaA in H. ducreyi pathogenesis was assessed using both swine and human experimental models of chancroid. In the swine model, 20% of lesions from sites inoculated with the ncaA mutant were culture positive for H. ducreyi 7 days after inoculation, compared to 73% of wild-type-inoculated sites. The average number of CFU recovered from mutant-inoculated lesions was also significantly reduced compared to that recovered from wild-type-inoculated sites at both 2 and 7 days after inoculation. In the human challenge model, 8 of 30 sites inoculated with wild-type H. ducreyi progressed to the pustular stage, compared to 0 of 30 sites inoculated with the ncaA mutant. Together these results demonstrate that the collagen binding protein NcaA is required for H. ducreyi infection.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.74.5.2651-2658.2006
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2006
    detail.hit.zdb_id: 1483247-1
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  • 7
    Online Resource
    Online Resource
    DksA and (p)ppGpp Have Unique and Overlapping Contributions to Haemophilus ducreyi Pathogenesis in Humans
    American Society for Microbiology ; 2015
    In:  Infection and Immunity Vol. 83, No. 8 ( 2015-08), p. 3281-3292
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 83, No. 8 ( 2015-08), p. 3281-3292
    Abstract: The (p)ppGpp-mediated stringent response is important for bacterial survival in nutrient limiting conditions. For maximal effect, (p)ppGpp interacts with the cofactor DksA, which stabilizes (p)ppGpp's interaction with RNA polymerase. We previously demonstrated that (p)ppGpp was required for the virulence of Haemophilus ducreyi in humans. Here, we constructed an H. ducreyi dksA mutant and showed it was also partially attenuated for pustule formation in human volunteers. To understand the roles of (p)ppGpp and DksA in gene regulation in H. ducreyi , we defined genes potentially altered by (p)ppGpp and DksA deficiency using transcriptome sequencing (RNA-seq). In bacteria collected at stationary phase, lack of (p)ppGpp and DksA altered expression of 28% and 17% of H. ducreyi open reading frames, respectively, including genes involved in transcription, translation, and metabolism. There was significant overlap in genes differentially expressed in the (p)ppGpp mutant relative to the dksA mutant. Loss of (p)ppGpp or DksA resulted in the dysregulation of several known virulence determinants. Deletion of dksA downregulated lspB and rendered the organism less resistant to phagocytosis and increased its sensitivity to oxidative stress. Both mutants had reduced ability to attach to human foreskin fibroblasts; the defect correlated with reduced expression of the Flp adhesin proteins in the (p)ppGpp mutant but not in the dksA mutant, suggesting that DksA regulates the expression of an unknown cofactor(s) required for Flp-mediated adherence. We conclude that both (p)ppGpp and DksA serve as major regulators of H. ducreyi gene expression in stationary phase and have both overlapping and unique contributions to pathogenesis.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.00692-15
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2015
    detail.hit.zdb_id: 1483247-1
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  • 8
    Online Resource
    Online Resource
    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
    detail.hit.zdb_id: 1483247-1
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  • 9
    Online Resource
    Online Resource
    A (p)ppGpp-Null Mutant of Haemophilus ducreyi Is Partially Attenuated in Humans Due to Multiple Conflicting Phenotypes
    American Society for Microbiology ; 2014
    In:  Infection and Immunity Vol. 82, No. 8 ( 2014-08), p. 3492-3502
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 82, No. 8 ( 2014-08), p. 3492-3502
    Abstract: (p)ppGpp responds to nutrient limitation through a global change in gene regulation patterns to increase survival. The stringent response has been implicated in the virulence of several pathogenic bacterial species. Haemophilus ducreyi , the causative agent of chancroid, has homologs of both relA and spoT , which primarily synthesize and hydrolyze (p)ppGpp in Escherichia coli . We constructed relA and relA spoT deletion mutants to assess the contribution of (p)ppGpp to H. ducreyi pathogenesis. Both the relA single mutant and the relA spoT double mutant failed to synthesize (p)ppGpp, suggesting that relA is the primary synthetase of (p)ppGpp in H. ducreyi . Compared to the parent strain, the double mutant was partially attenuated for pustule formation in human volunteers. The double mutant had several phenotypes that favored attenuation, including increased sensitivity to oxidative stress. The increased sensitivity to oxidative stress could be complemented in trans . However, the double mutant also exhibited phenotypes that favored virulence. When grown to the mid-log phase, the double mutant was significantly more resistant than its parent to being taken up by human macrophages and exhibited increased transcription of lspB , which is involved in resistance to phagocytosis. Additionally, compared to the parent, the double mutant also exhibited prolonged survival in the stationary phase. In E. coli , overexpression of DksA compensates for the loss of (p)ppGpp; the H. ducreyi double mutant expressed higher transcript levels of dksA than the parent strain. These data suggest that the partial attenuation of the double mutant is likely the net result of multiple conflicting phenotypes.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.01994-14
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2014
    detail.hit.zdb_id: 1483247-1
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  • 10
    Online Resource
    Online Resource
    Dysregulated Immune Profiles for Skin and Dendritic Cells Are Associated with Increased Host Susceptibility to Haemophilus ducreyi Infection in Human Volunteers
    American Society for Microbiology ; 2007
    In:  Infection and Immunity Vol. 75, No. 12 ( 2007-12), p. 5686-5697
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 75, No. 12 ( 2007-12), p. 5686-5697
    Abstract: In experimentally infected human volunteers, the cutaneous immune response to Haemophilus ducreyi is orchestrated by serum, polymorphonuclear leukocytes, macrophages, T cells, and myeloid dendritic cells (DC). This response either leads to spontaneous resolution of infection or progresses to pustule formation, which is associated with the failure of phagocytes to ingest the organism and the presence of Th1 and regulatory T cells. In volunteers who are challenged twice, some subjects form at least one pustule twice (PP group), while others have all inoculated sites resolve twice (RR group). Here, we infected PP and RR subjects with H. ducreyi and used microarrays to profile gene expression in infected and wounded skin. The PP and RR groups shared a core response to H. ducreyi . Additional transcripts that signified effective immune function were differentially expressed in RR infected sites, while those that signified a hyperinflammatory, dysregulated response were differentially expressed in PP infected sites. To examine whether DC drove these responses, we profiled gene expression in H. ducreyi -infected and uninfected monocyte-derived DC. Both groups had a common response that was typical of a type 1 DC (DC1) response. RR DC exclusively expressed many additional transcripts indicative of DC1. PP DC exclusively expressed differentially regulated transcripts characteristic of DC1 and regulatory DC. The data suggest that DC from the PP and RR groups respond differentially to H. ducreyi. PP DC may promote a dysregulated T-cell response that contributes to phagocytic failure, while RR DC may promote a Th1 response that facilitates bacterial clearance.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.00777-07
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2007
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