INTRODUCTION

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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, or serum resistance in vitro (10, 11, 19-21, 37). Although the results of human challenge trials suggested that these gene products were not required for pustule formation, we could not exclude the possibility that the gene of interest was simply not transcribed in the initial stages of infection. If a gene was transcribed and its isogenic mutant was still virulent, then it would be very unlikely that the gene product played a major role in virulence.

Previous studies indicated that sera from patients with chancroid contained elevated levels of antibodies to HgbA, TdhA, D15, Hlp, hemolysin, cytolethal distending toxin (CDT), and LOS of H. ducreyi when compared to control sera, suggesting that these factors were expressed during natural infection (1, 12, 16, 22, 24). Confocal microscopy performed on lesions from experimentally infected subjects indicated that ftpA, pal, hlp, momp, and/or ompA2 was expressed in vivo (6). However, detection of gene expression by serology and confocal microscopy is limited by the availability of appropriate antigens or antibodies.

In this study, we examined whether transcripts of candidate virulence genes of H. ducreyi were present in lesions of experimentally infected human subjects. We also estimated the number of CFU in an entire lesion and the number of CFU that allowed detection of bacterial transcripts in human tissue.

	MATERIALS AND METHODS

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Bacteria and culture conditions. H. ducreyi 35000HP is a human-passaged variant of 35000 described previously (5, 31). H. ducreyi 35000HP was grown on chocolate agar plates supplemented with 1% IsoVitaleX and incubated at 33°C with 5% CO₂ or in brain heart infusion broth containing 50 µg of hemin per ml, 1% IsoVitaleX, and 5% heat-inactivated fetal calf serum and incubated with aeration at 33°C.

Human subjects and biopsies. Tissue was collected from eight adults (two female and six male; mean age ± standard deviation [SD], 33.6 ± 10.4 years) who participated in several mutant-to-parent comparison trials (7, 18, 39) or who were specifically infected for this study. Informed consent was obtained from the subjects for participation and for human immunodeficiency virus 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 at Indianapolis. Enrollment procedures and exclusion criteria are described elsewhere in detail (5, 32). Six volunteers were inoculated on the upper arm with estimated delivered doses (EDD) of 60 to 100 CFU of H. ducreyi 35000, 35000HP, or 35000.304, an isogenic cdtC and hhdB double mutant. Subjects were observed until they achieved clinical endpoint, defined as resolution of disease at all sites, 14 days of infection, or the development of a painful pustule. At clinical endpoint, a total of eight pustules were biopsied with a punch forceps.

RNA isolation and reverse transcription (RT). The remaining homogenate was recovered and usually 600 to 700 µl was centrifuged at 16,000 × g for 5 min at 4°C. Pellets were suspended in 1 ml of Ultraspec RNA (Biotecx Laboratories, Inc., Houston, Tex.), and RNA was isolated according to the manufacturer's instructions. RNA was suspended in diethyl pyrocarbonate-treated H₂O and incubated with 15 U of DNase I (GenHunter Corp, Nashville, Tenn.) at 37°C for 45 min. DNase I was heat inactivated by incubating the reaction mixture at 75°C for 6 min. For in vitro experiments, RNA was isolated as described above from approximately 10⁸ CFU of 35000HP grown to mid-logarithmic phase.

PCR. Amplification of most target cDNAs was performed with MgCl₂ (5 mM) and the appropriate primers (30 pM) in 100-µl reaction mixtures using the PCR Core Kit Plus (Roche Molecular Biochemicals, Indianapolis, Ind.) as per the manufacturer's instructions. ompA2 cDNA was amplified in the presence of MgCl₂ (2.5 mM). The synthetic primers used to amplify target cDNA were based on published sequences (9, 22, 27, 30, 36) (Table 1). Robert Munson, Jr. (Ohio State University, Columbus), kindly provided the primers Cdt3 and Cdt4 (Table 1). Reaction mixtures were denatured at 95°C for 5 min, then cycled 45 times through 95°C for 1 min, 60°C for 25 s, and 72°C for 2 min, with a final extension at 72°C for 7 min and then held at 4°C using a thermal cycler (Perkin-Elmer PCR System 2400). The annealing temperature for most primers was 60°C except for ompA2 and lspA1 primers, which annealed at 63°C, and ftpA and -actin primers, which annealed at 50 and 55°C, respectively. Genomic DNA from 35000HP and H₂O were used as templates for positive and negative controls, respectively. Ten microliters of cDNA or RNA was used as template for PCRs. Amplicons were analyzed by electrophoresis of 10 µl of each PCR on 1.2% agarose gels stained with ethidium bromide.

Nucleotide sequence analysis. Amplicons from PCRs using 35000HP as template and each primer set were sequenced on an ABI Prism 310 (Applied Biosystems) automated sequencing system employing dye terminators. Sequencing data were compared to published sequences for each gene of interest.

Limit of detection. To determine the limit of detection for each H. ducreyi primer set, three samples of RNA prepared from 35000HP grown to mid-logarithmic phase were used to make cDNA that was subsequently serially diluted and used as template in PCR. Amplicons were analyzed as described above. The number of CFU that gave rise to each RT-PCR sample loaded on the gel was calculated. The limit of detection was defined as the lowest number of CFU whose cDNA yielded a visible RT-PCR product on an ethidium bromide-stained gel.

Analysis of amplicons prepared from biopsies. All RT-PCR products obtained from biopsies were subjected to agarose gel electrophoresis and Southern blot analysis. DNA was transferred to a nylon membrane by a standard capillary method. Probes were prepared from amplicons that were obtained using 35000HP genomic DNA as template and the appropriate primer set. Probe labeling, hybridization, and detection were performed as described previously (34). If a band of the expected size was detected by agarose gel and Southern blotting or Southern blotting alone, the gene of interest was considered to have been transcribed in vivo. For -actin, RT-PCR products were only analyzed by agarose gel electrophoresis and stained with ethidium bromide. The number of CFU corresponding to the RT-PCR sample loaded on the gel was calculated based on the quantitative culture for each biopsy and the portion of each biopsy that was converted to cDNA.

	RESULTS AND DISCUSSION

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Specimens. Eight pustules were biopsied from six volunteers who were infected for 7.0 ± 1.1 days (mean ± SD). Two additional biopsies were obtained from two uninfected volunteers. Biopsies were homogenized, and a portion was cultured on selective and nonselective media. While H. ducreyi was the only bacterium recovered from pustules, no bacteria were recovered from uninfected skin.

RT-PCR of H. ducreyi mRNA in vitro. To verify that the target sequences were amplified, PCR products obtained from 35000HP genomic DNA and each of the H. ducreyi primer sets were sequenced. The sequence of each amplicon matched that of the expected product (data not shown).

Detection of H. ducreyi mRNA in control samples. To detect potential inhibitors or false-positive signals, we performed RT-PCR on homogenates prepared from two biopsies of uninfected skin with and without H. ducreyi. No products were detected in samples from uninfected skin, but the expected PCR products were amplified from uninfected skin homogenized with H. ducreyi. To verify that cDNA was present in the PCR, -actin was amplified from uninfected skin with and without H. ducreyi (data not shown). Although uninfected skin did not contain the same cells or genes expressed as inflamed tissue, the data suggested that the RT-PCR products were H. ducreyi specific and not eukaryotic in origin.

Detection of transcripts of candidate virulence genes in vivo. Five biopsies obtained from sites inoculated with the parent strain were analyzed for the presence of H. ducreyi cDNAs. Since the products of pal, hlp, and ftpA were detected by confocal microscopy in vivo (6), these genes served as positive controls for the specimens. pal, hlp, and ftpA cDNAs were detected in all the specimens. Thus, the biopsies were also analyzed for the presence of other cDNAs.

View larger version (73K): [in this window] [in a new window]   FIG. 1.    Composite agarose gels stained with ethidium bromide (A and C) and Southern blot (B and D) of PCR and RT-PCR products. PCR was performed with either losB primers (panels A and B) or ftpA primers (panels C and D). Lanes 1 and 2 contain 35000HP genomic DNA and no template, respectively. cDNA (lanes 3, 5, 7, and 9) and RNA that was not reverse transcribed (lanes 4, 6, 8, and 10) were prepared from biopsy 165 (lanes 3 and 4), biopsy 164 (lanes 5 and 6), uninfected skin homogenized with H. ducreyi (lanes 7 and 8), and uninfected skin (lanes 9 and 10).