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

Abstract

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,...

Characterization ofHaemophilus ducreyi cdtA, cdtB, andcdtCMutants in In Vitro and In Vivo Systems

“…RT-PCR analysis of RNA prepared from rabbit lesion material confirmed that all three genes of the H. ducreyi cdtABC cluster were transcribed in rabbit skin during infection. In vivo transcription of at least cdtB was also shown to occur in human volunteers infected with wild-type H. ducreyi (48). These findings suggest that the failure to observe diminished virulence with the H. ducreyi cdtA, cdtB, and cdtC mutants in rabbits and with the cdtC mutant in humans is unlikely to be due to a lack of expression of the relevant gene products in vivo.…”

A Superoxide Dismutase C Mutant of Haemophilus ducreyi Is Virulent in Human Volunteers

“…The larger EDD required for infection in the swine model suggests that H. ducreyi is a less efficient pathogen for pigs than for humans and that the absence of a candidate virulence determinant may have a more pronounced effect in swine than in humans. In the human model, H. ducreyi achieves a density of 10 5 CFU/lesion at the pustular stage, approximately 1 week after inoculation (38). In the swine model, the number of bacteria decreases from 1.7 ϫ 10 4 CFU to 1.7 ϫ 10 3 CFU 48 h after inoculation.…”

“…Neutrophils usually kill bacteria by ingesting them or by releasing toxic products such as superoxide radicals and antimicrobial proteins (10). H. ducreyi replicates during the initial stages of experimental infection in humans (3,38), which suggests that the organism escapes both phagocytosis and killing by toxic products of neutrophils. How H. ducreyi evades phagocytic defenses is unclear.…”

Development of a Rapid Immunodiagnostic Test for Haemophilus ducreyi

“…One study on pustules (the lesion just prior to the formation of ulcers) in an experimental human infection reported that there were approximately 10 5 CFU per lesion (56). Therefore, it is unclear what the minimum limit of detection is that is needed to accurately detect a reasonable amount of the HgbA released from H. ducreyi or what the number of organisms is that are heme stressed in clinical specimens.…”

“…A second immobilized MAb, directed against a separate antigenic epitope, allows the antigen-antibody complex to be concentrated and visually de- (30,56). Therefore, the generation of MAbs to the HgbA protein seemed to be a logical target.…”