Evaluation of an Isogenic Major Outer Membrane Protein-Deficient Mutant in the Human Model of
            <i>Haemophilus ducreyi</i>
            Infection

Self Cite

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 (35000HP⍀12) 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.

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Expression of the LspA1 and LspA2 Proteins by Haemophilus ducreyi Is Required for Virulence in Human Volunteers

Janowicz¹,

Fortney²,

Katz³

et al. 2004

Self Cite

“…Subjects gave informed consent for participation and for HIV serology, in accordance with the human experimentation guidelines of the U.S. Department of Health and Human Services and the Institutional Review Board of Indiana University-Purdue University of Indianapolis. The experimental challenge protocol, preparation and inoculation of the bacteria, calculation of the estimated delivered dose (EDD), surface cultures, and clinical observations were done exactly as described previously (4,5,54,55,58). Each subject was inoculated on one arm with three identical doses of the parent strain and on the other arm with three doses of twofold serial dilutions of the mutant.…”

Section: Methodsmentioning

confidence: 99%

Expression of Haemophilus ducreyi Collagen Binding Outer Membrane Protein NcaA Is Required for Virulence in Swine and Human Challenge Models of Chancroid

Fulcher

Cole

Janowicz³

et al. 2006

Self Cite

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.

“…Isogenic mutants of pal, hgbA, and dsrA were attenuated in their ability to form pustules compared to the parent (3,7,18). Seven mutants, including those with disruptions in hhdB, cdtC, hhdB and cdtC, momp, ftpA, lst, and losB, caused pustule formation rates that were similar to those caused by the parent (2,28,34,38,39). These results were surprising in that some of these candidate virulence factors (hhdB, cdtC, momp, losB) have roles in adherence, cell death, or serum resistance in vitro (10,11,(19)(20)(21)37).…”

mentioning

confidence: 93%

Transcription of Candidate Virulence Genes of Haemophilus ducreyi during Infection of Human Volunteers

Throm

Spinola

2001

Self Cite

Haemophilus ducreyi expresses several putative virulence factors in vitro. Isogenic mutant-to-parent comparisons have been performed in a human model of experimental infection to examine whether specific gene products are involved in pathogenesis. Several mutants (momp, ftpA, losB, lst, cdtC, and hhdB) were as virulent as the parent in the human model, suggesting that their gene products did not play a major role in pustule formation. However, we could not exclude the possibility that the gene of interest was not expressed during the initial stages of infection. Biopsies of pustules obtained from volunteers infected with H. ducreyi were subjected to reverse transcription-PCR. Transcripts corresponding to momp, ftpA, losB, lst, cdtB, and hhdA were expressed in vivo. In addition, transcripts for other putative virulence determinants such as ompA2, tdhA, lspA1, and lspA2 were detected in the biopsies. These results indicate that although several candidate virulence determinants are expressed during experimental infection, they do not have a major role in the initial stages of pathogenesis.Haemophilus ducreyi is a gram-negative bacterium that is the etiologic agent of chancroid, a genital ulcer disease that facilitates acquisition of human immunodeficiency virus type 1 (17, 35). During sexual intercourse, breaks in the epithelium may provide a portal of entry for H. ducreyi. After an incubation period of 1 to 7 days, a small erythematous papule develops. Pustules form 2 to 3 days later and eventually progress into a soft painful ulcer (25).A human model of H. ducreyi infection was developed by our laboratory to study H. ducreyi pathogenesis (4, 5, 27, 32). Volunteers are inoculated with bacteria at multiple sites on the skin of the upper arm via puncture wounds made with an allergy testing device. In the human model, 1 to 100 CFU are sufficient to initiate infection. Papules develop in 24 h, and pustules usually form 2 to 5 days later, consistent with the natural course of disease. The histopathology of pustules resembles that of naturally occurring ulcers. For subject safety, infection is limited to the pustular stage of disease, and subjects are typically infected for 7 to 14 days. Several putative virulence factors of H. ducreyi have been identified including pili, outer membrane proteins, toxins, and lipooligosaccharide (LOS) (8-10, 14, 15, 20, 22, 23, 26, 29, 33, 36). To study their role in pathogenesis, isogenic mutants were constructed and compared to the parent in the human model. To date, we have performed 10 mutant-to-parent comparison trials. Isogenic mutants of pal, hgbA, and dsrA were attenuated in their ability to form pustules compared to the parent (3, 7, 18). Seven mutants, including those with disruptions in hhdB, cdtC, hhdB and cdtC, momp, ftpA, lst, and losB, caused pustule formation rates that were similar to those caused by the parent (2,28,34,38,39). These results were surprising in that some of these candidate virulence factors (hhdB, cdtC, momp, losB) have roles in adherence, cell death,...