A DltA mutant of Haemophilus ducreyi Is partially attenuated in its ability to cause pustules in human volunteers

Haemophilus ducreyi produces two outer membrane proteins, called DltA (H. ducreyi lectin A) and DsrA (H. ducreyi serum resistance A), that contribute to the ability of the organism to evade complement-mediated serum killing. In contrast to their isogenic parent strain, 35000HP, the DsrA mutant FX517 exhibits 0% survival in 50% normal human serum and the DltA mutant FX533 exhibits 23% survival. Compared to 35000HP, FX517 does not cause pustule formation in human volunteers. To test whether DltA was required for virulence in humans, seven volunteers were experimentally infected with 35000HP and FX533. Four subjects were inoculated with fixed doses of 35000HP (101 CFU or 130 CFU) at three sites on one arm and escalating doses of FX533 (range, 46 CFU to 915 CFU) at three sites on the other arm. Pustules only developed at mutant-injected sites at doses nearly twofold higher than that of the parent, suggesting that FX533 was partially attenuated. Three subjects were inoculated with similar doses of the parent (67 CFU) and mutant (104 CFU) at three sites. Pustules formed at five of nine parent sites and one of nine mutant sites. Overall, the papule and pustule formation rates for 35000HP and FX533 were similar for the trial. However, for the five subjects who received similar doses of the parent and mutant, pustules developed at 7 of 15 sites (46.7%; 95% confidence interval [CI], 16.9% to 76.5%) inoculated with the parent and at 1 of 15 (6.7%; 95% CI, 0.1% to 18.4%) sites inoculated with the mutant (P = 0.043). We concluded that the DltA mutant was attenuated in its ability to cause disease at doses similar to that of the parent.     

A superoxide dismutase C mutant of Haemophilus ducreyi is virulent in human volunteers

Haemophilus ducreyi produces a periplasmic copper-zinc superoxide dismutase (Cu-Zn SOD), which is thought to protect the organism from exogenous reactive oxygen species generated by neutrophils during an inflammatory response. We had previously identified the gene, sodC, responsible for the production and secretion of Cu-Zn SOD and constructed an isogenic H. ducreyi strain with a mutation in the sodC gene (35000HP-sodC-cat). Compared to the parent, the mutant does not survive in the presence of exogenous superoxide (L. R. San Mateo, M. Hobbs, and T. H. Kawula, Mol. Microbiol. 27:391-404, 1998) and is impaired in the swine model of H. ducreyi infection (L. R. San Mateo, K. L. Toffer, P. E. Orndorff, and T. H. Kawula, Infect. Immun. 67:5345-5351, 1999). To test whether Cu-Zn SOD is important for bacterial survival in vivo, six human volunteers were experimentally infected with 35000HP and 35000HP-sodC-cat and observed for papule and pustule formation. Papules developed at similar rates at sites inoculated with the mutant or parent. The pustule formation rates were 75% (95% confidence intervals [CI], 43 to 95%) at 12 parent-inoculated sites and 67% (95% CI, 41 to 88%) at 18 mutant-inoculated sites (P = 0.47). There was no significant difference in levels of H. ducreyi recovery from mutant- and parent-inoculated biopsy sites. These results suggest that expression of Cu-Zn SOD does not play a major role in the survival of this pathogen in the initial stages of experimental infection of humans.     

Haemophilus ducreyi adheres to human keratinocytes

Haemophilus ducreyi, Moraxella catarrhalis and a non-piliated Escherichia coli K-12 strain were studied for their ability to bind to human keratinocytes in vitro. Epidermal cells isolated from neonatal foreskins were grown to confluence in serum-free keratinocyte media. Probing of the monolayers with anti-cytokeratin antibody showed that 97% of cells were keratinocytes. Bacteria were grown to mid-log phase and seeded onto the monolayers. At various timepoints monolayers were washed with PBS to remove non-adherent bacteria, and the monolayers were quantitatively cultured. After 120 min, 15 to 23% of the H. ducreyi inocula bound to the monolayer, while less than 1% of the M. catarrhalis or E. coli controls bound. Wet mounts of fixed monolayers observed with differential interference contrast microscopy confirmed the quantitative data. We conclude that H. ducreyi binds to keratinocytes and that this process may play a role in the initiation of chancroid.     

Expression of the LspA1 and LspA2 proteins by Haemophilus ducreyi is required for virulence in human volunteers

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 Omega 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.     

Expression of cytolethal distending toxin and hemolysin is not required for pustule formation by Haemophilus ducreyi in human volunteers

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.     

Haemophilus ducreyi lipooligosaccharide mutant defective in expression of beta-1,4-glucosyltransferase is virulent in humans

The lipooligosaccharide (LOS) of Haemophilus ducreyi contains a major glycoform that is immunochemically identical to paragloboside, a glycosphingolipid precursor of major human blood group antigens. We recently identified the gene responsible for the glucosyltransferase activity and constructed an isogenic mutant (35000glu-) deficient in this activity. 35000glu- makes an LOS that consists only of the heptose trisaccharide core and 2-keto-deoxyoctulosonic acid (KDO). For this study, the mutant was reconstructed in the 35000HP (human passaged [HP]) background. Five human subjects were inoculated with 35000HP and 35000HPglu- in a dose-response trial. The pustule formation rates were 40% (95% confidence interval [CI], 13.7 to 72.6%) at 10 sites for 35000HP and 46.7% (95% CI, 24.8 to 69.9%) at 15 sites for 35000HPglu-. The histopathology and recovery rates of H. ducreyi from surface cultures and biopsies obtained from mutant and parent sites were similar. These results indicate that the expression of glycoforms with sugar moieties extending beyond the heptose trisaccharide core is not required for pustule formation by H. ducreyi in humans.     

Expression of OmpP2A and OmpP2B is not required for pustule formation by Haemophilus ducreyi in human volunteers

Haemophilus ducreyi express two porin proteins, termed OmpP2A and OmpP2B. To test whether expression of OmpP2A and OmpP2B was necessary for virulence in humans, eight volunteers were experimentally infected with the parent (35000HP) in one arm and a double OmpP2A OmpP2B mutant (35000HP::P2AB) in the other arm. The pustule formation rates were 58.3% (95% CI, 33.2-83.5%) for the parent and 41.7% (95% CI, 19.3-64.0%) for the mutant (P=0.25). Biopsy of 35000HP and 35000HP::P2AB-infected sites yielded similar amounts of bacteria in quantitative culture. These results indicate that expression of OmpP2A and OmpP2B is not necessary to initiate disease or to progress to pustule formation in humans.     

A P60 mutant of Listeria monocytogenes is impaired in its ability to cause infection in intragastrically inoculated mice

A spontaneous P60 mutant of Listeria monocytogenes was less able to cause systemic infection in A/J mice, following intragastric inoculation, than the parental wild type strain (SLCC 5764, serotype 1/2a). Significantly fewer CFU were recovered from internal organs (spleen, liver, gall bladder) and from the cecum of mice inoculated intragastrically with the P60 mutant than mice inoculated with wild type L. monocytogenes. The P60 mutant also exhibited a diminished ability to invade and multiply within Caco-2 intestinal epithelial cells. These findings indicate that P60 is required for maximal virulence of L. monocytogenes in the gastrointestinal tract of mice.     

Expression of sialylated or paragloboside-like lipooligosaccharides are not required for pustule formation by Haemophilus ducreyi in human volunteers

The lipooligosaccharide (LOS) of Haemophilus ducreyi, the etiologic agent of chancroid, chemically and immunologically resembles human glycosphingolipid antigens. To test whether LOS that contains paragloboside-like structures was required for pustule formation, an isogenic mutant (35000HP-RSM2) was constructed in losB, which encodes D-glycero-D-manno-heptosyltransferase. 35000HP-RSM2 produces a truncated LOS whose major glycoform terminates in a single glucose attached to a heptose trisaccharide core and 2-keto-3-deoxyoctulosonic acid. Five human subjects were inoculated with 35000HP and 35000HP-RSM2 in a dose-response trial. For estimated delivered doses (EDDs) of >/=25 CFU, the pustule formation rates were 80% for 35000HP and 58% for 35000HP-RSM2. Preliminary data indicated that a previously described Tn916 losB mutant made a minor glycoform that does not require DD-heptose to form the terminal N-acetyllactosamine. If 35000HP-RSM2 made this glycoform, then 35000HP-RSM2 could theoretically make a sialylated glycoform. To test whether sialylated LOS was required for pustule formation, a second trial comparing an isogenic sialyltransferase mutant (35000HP-RSM203) to 35000HP was performed in five additional subjects. For EDDs of >/=25 CFU, the pustule formation rates were 30% for both 35000HP and 35000HP-RSM203. The histopathology and recovery rates of H. ducreyi from surface cultures and biopsies obtained from mutant and parent sites in both trials were similar. These results indicate that neither the expression of a major glycoform resembling paragloboside nor sialylated LOS is required for pustule formation by H. ducreyi in humans.     

Haemophilus ducreyi partially activates human myeloid dendritic cells

Dendritic cells (DC) orchestrate innate and adaptive immune responses to bacteria. How Haemophilus ducreyi, which causes genital ulcers and regional lymphadenitis, interacts with DC is unknown. H. ducreyi evades uptake by polymorphonuclear leukocyte and macrophage-like cell lines by secreting LspA1 and LspA2. Many H. ducreyi strains express cytolethal distending toxin (CDT), and recombinant CDT causes apoptosis of DC in vitro. Here, we examined interactions between DC and H. ducreyi 35000HP, which produces LspA1, LspA2, and CDT. In human volunteers infected with 35000HP, the ratio of myeloid DC to plasmacytoid DC was 2.8:1 in lesions, compared to a ratio of 1:1 in peripheral blood. Using myeloid DC derived from monocytes as surrogates for lesional DC, we found that DC infected with 35000HP remained as viable as uninfected DC for up to 48 h. Gentamicin protection and confocal microscopy assays demonstrated that DC ingested and killed 35000HP, but killing was incomplete at 48 h. The expression of LspA1 and LspA2 did not inhibit the uptake of H. ducreyi, despite inactivating Src kinases. Infection of DC with live 35000HP caused less cell surface marker activation than infection with heat-killed 35000HP and lipopolysaccharide (LPS) and inhibited maturation by LPS. However, infection of DC with live bacteria caused the secretion of significantly higher levels of interleukin-6 and tumor necrosis factor alpha than infection with heat-killed bacteria and LPS. The survival of H. ducreyi in DC may provide a mechanism by which the organism traffics to lymph nodes. Partial activation of DC may abrogate the establishment of a full Th1 response and an environment that promotes phagocytosis.     

Haemophilus ducreyi attaches to and invades human epithelial cells in vitro.

Haemophilus ducreyi is a sexually transmitted pathogen that causes genital ulcers and inguinal adenopathy. Because chancroidal ulcers are most commonly located on the foreskins of uncircumcised males, we utilized human foreskin epithelial cells (HFECs) to investigate the initial interaction of H. ducreyi with its host. The eight different strains of H. ducreyi that were studied varied in their abilities to attach to these epithelial cells, with six strains consistently attaching to > or = 90% of HFECs and two strains attaching to < 25% of HFECs. The strains with low levels of adherence also failed to exhibit chaining in broth culture and were avirulent in the rabbit model, suggesting that virulence in this model and attachment may be linked. The most adherent strain, LA228R, was further evaluated for its ability to invade HFECs and HEp-2 cells. Scanning electron microscopy and transmission electron microscopy of HFECs after interaction with LA228R produced images consistent with attachment, ingestion into vesicles, and escape from the vesicles into the cytoplasm. In addition, the gentamicin protection assay and inhibition of invasion by cytochalasin B and D indicated that LA228R was able to invade both HFECs and HEp-2 cells. Further examination of the mechanisms involved in the adherence and invasion of H. ducreyi into epithelial cells and their correlation with virulence will provide a better understanding of the pathogenesis of the disease caused by this important pathogen.     

Role of lipopolysaccharide and complement in susceptibility of Haemophilus ducreyi to human serum

The role of lipopolysaccharide (LPS) in the susceptibility of Haemophilus ducreyi to human serum and the mechanism of complement activation by serum-susceptible (Sers) strains were investigated. Serum treated with 2 mM Mg2+ and 20 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was nonbactericidal, but inulin-treated serum remained bactericidal. Absorption of serum with heat-killed whole cells of an Sers strain removed its bactericidal activity against the absorbing strain and also against other Sers strains. LPS obtained from Sers strains inhibited the bactericidal activity of serum against all Sers strains, whereas LPS from serum-resistant (Serr) strains and an Serr isogenic strain did not. However, high concentrations of LPS from the Serr strain inhibited the bactericidal activity of serum, an indication that part of the structural site involved in serum susceptibility is retained in the LPS of this strain. The LPS of Sers strains exhibited higher anticomplement activity than the LPS of Serr strains. These findings suggest that the classical pathway of complement activation is involved in the serum killing of H. ducreyi and that LPS composition may contribute to their susceptibility to complement-mediated serum bactericidal activity.     

Standardization of an enzyme immunoassay for human antibody to Haemophilus ducreyi.

We standardized a serologic enzyme immunoassay (EIA) for human immunoglobulin G and M antibodies against Haemophilus ducreyi. We evaluated the performance of this test with respect to the time from acute chancroid and coinfection with human immunodeficiency virus (HIV). Antibody to a crude, soluble bacterial antigen of one H. ducreyi strain was detected in a panel of serum samples from clinically and microbiologically confirmed cases of chancroid and from controls. Test interpretation was standardized for optimal sensitivity and specificity. Performance of the EIA was enhanced in the period of early convalescence from acute primary chancroid and was not diminished in the presence of HIV coinfection. The EIA performed adequately as a serologic screening test for field evaluation and epidemiologic application in conjunction with sexually transmitted disease and HIV detection and control efforts.     

Haemophilus ducreyi associates with phagocytes, collagen, and fibrin and remains extracellular throughout infection of human volunteers

In a previous study, Haemophilus ducreyi was found in the pustule and dermis of samples obtained at the clinical end point in the human model of infection. To understand the kinetics of localization, we examined infected sites at 0, 24, and 48 h after inoculation and at the clinical end point. Immediately after inoculation, bacteria were found predominantly in the dermis but also in the epidermis. Few bacteria were detectable at 24 h; however, by 48 h, bacteria were readily seen in the pustule and dermis. H. ducreyi was associated with polymorphonuclear leukocytes and macrophages in the pustule and at its base, but was not associated with T cells, Langerhans' cells, or fibroblasts. H. ducreyi colocalized with collagen and fibrin but not laminin or fibronectin. Association with phagocytes, collagen, and fibrin was seen as early as 48 h and persisted at the pustular stage of disease. Optical sectioning by confocal microscopy and transmission electron microscopy both failed to demonstrate intracellular H. ducreyi. These data identify collagen and fibrin as potentially important targets of adherence in vivo and strongly suggest that H. ducreyi remains extracellular throughout infection and survives by resisting phagocytic killing in vivo.     

Expression of peptidoglycan-associated lipoprotein is required for virulence in the human model of Haemophilus ducreyi infection

Haemophilus ducreyi expresses a peptidoglycan-associated lipoprotein (PAL) that exhibits extensive homology to Haemophilus influenzae protein 6. We constructed an isogenic PAL mutant (35000HP-SMS4) by the use of a suicide vector that contains lacZ as a counterselectable marker. H. ducreyi 35000HP-SMS4 and its parent, 35000HP, had similar growth rates in broth and similar lipooligosaccharide profiles. 35000HP-SMS4 formed smaller, more transparent colonies than 35000HP and, unlike its parent, was hypersensitive to antibiotics. Complementation of the mutant in trans restored the parental phenotypes. To test whether expression of PAL is required for virulence, nine human volunteers were experimentally infected. Each subject was inoculated with two doses (41 to 89 CFU) of live 35000HP and one dose of heat-killed bacteria on one arm and with three doses (ranging from 28 to 800 CFU) of live 35000HP-SMS4 on the other arm. Papules developed at similar rates at sites inoculated with the mutant or parent but were significantly smaller at mutant-inoculated sites than at parent-inoculated sites. The pustule formation rate was 72% (95% confidence interval [CI], 46.5 to 90.3%) at 18 parent sites and 11% (95% CI, 2.4 to 29.2%) at 27 mutant sites (P < 0.0001). The rates of recovery of H. ducreyi from surface cultures were 8% (n = 130; 95% CI, 4.3 to 14.6%) for parent-inoculated sites and 0% (n = 120; 95% CI, 0.0 to 2.5%) for mutant-inoculated sites (P < 0.001). H. ducreyi was recovered from six of seven biopsied parent-inoculated sites and from one of three biopsied mutant-inoculated sites. Confocal microscopy confirmed that the bacteria present in a mutant inoculation site pustule lacked a PAL-specific epitope. Although biosafety regulations precluded our testing the complemented mutant in humans, these results suggest that expression of PAL facilitates the ability of H. ducreyi to progress to the pustular stage of disease.     

Enzymic activity of Haemophilus ducreyi

The enzymic activity of 29 Haemophilus ducreyi strains on 28 substrates is described. The results are compared with those of seven other authors. There is agreement only about the presence of alkaline phosphatase and arginine aminopeptidase and the lack of glycosidases. Possible reasons for the contradictions in the eight reports are discussed.     

Specificity of the immune response to Haemophilus ducreyi

The specificity of the antibody response toHaemophilus ducreyiin sera from patients attending a sexually transmitted disease clinic in South Africa has been studied using immunoblotting. Patients with chancroid were shown to have higher levels of IgG (mean 0.74, SD 0.34) toH. ducreyithan those with no history of chancroid (mean 0.34, SD 0.19). The pattern of the antibody specificity was highly variable between patients with culture proven chancroid but there was no observed strain specificity. In comparison, the patterns obtained using sera from patients without known exposure toH. ducreyishowed less variation between patients and were of less intensity at the dilution used. Sera from patients with chancroid recognised epitopes on proteins that varied in molecular weight between strains, particularly of 60–66kDa (10 of 36 patients) and 25–27kDa (8 of 36 patients). In addition epitopes were recognised on the GroEL and/or DnaK heat shock proteins in 13 of 36 sera tested. There was no apparent change in the epitopes recognised on proteins between the homologous and heterologous strains. Patterns of antibody specificity in sequential sera only varied in one of six patients tested.