Localization of Haemophilus ducreyi in naturally acquired chancroidal ulcers

Haemophilus ducreyi causes the sexually transmitted genital ulcer disease chancroid. In human inoculation experiments, bacteria colocalize with neutrophils and macrophages but remain extracellular. The organism also colocalizes with collagen and fibrin but not with keratinocytes, fibroblasts, laminin, or fibronectin. These relationships are established by 48 h postinoculation and persist through the pustular stage of disease. To extend these observations to the ulcerative stage of disease, and to compare results in the human model with those of natural disease, we obtained biopsies from patients with naturally acquired chancroid. All ulcers were culture positive for H. ducreyi and histologically very similar to pustules from the human model. Staining with H. ducreyi-specific monoclonal antibodies demonstrated H. ducreyi within 5 biopsies. The organism was chiefly found within the granulocytic infiltrate of the ulcer. Dual staining for H. ducreyi and eukaryotic tissue components showed that H. ducreyi colocalized with neutrophils and fibrin at the ulcerative stage of disease. No bacteria were associated with keratinocytes, fibroblasts, or collagen. Overall, these findings are consistent with results from the human model. This is the first reported study to localize bacteria specifically identified as H. ducreyi within naturally acquired chancroid.     

Distribution, cloning, characterisation and mutagenesis of sodC, the gene encoding copper/zinc superoxide dismutase, a potential determinant of virulence, in Haemophilus ducreyi

The sodC gene encoding the periplasmic enzyme copper/zinc superoxide dismutase (CuZnSOD) has been cloned from Haemophilus ducreyi, the causative agent of the genital ulcer disease, chancroid. Examination of a collection of diverse strains indicates that it is present throughout the species. Cloned sodC has been expressed in E. coli and shown to encode active enzyme. Insertional mutagenesis was used to construct a non-functional version of the gene. This has been transferred into the chromosome of the parent H. ducreyi strain by electroporation and homologous recombination, in preparation for studies of the role of this enzyme in the interactive biology of the organism with its host, perhaps in protecting bacteria from superoxide radicals and their reactive progeny generated by neutrophils in the context of host defence.     

The Hd0053 gene of Haemophilus ducreyi encodes an alpha2,3-sialyltransferase

Haemophilus ducreyi is a Gram-negative bacterium that causes chancroid, a sexually transmitted genital ulcer disease. Different lipooligosaccharide (LOS) structures have been identified from H. ducreyi strain 35000, including those sialylated glycoforms. Surface LOS of H. ducreyi is considered an important virulence factor that is involved in ulcer formation, cell adhesion, and invasion of host tissue. Gene Hd0686 of H. ducreyi, designated lst (for lipooligosaccharide sialyltransferase), was identified to encode an alpha2,3-sialyltransferase that is important for the formation of sialylated LOS. Here, we show that Hd0053 of H. ducreyi genomic strain 35000HP, the third member of the glycosyltransferase family 80 (GT80), also encodes an alpha2,3-sialyltransferase that may be important for LOS sialylation.     

A novel heme protein, the Cu,Zn-superoxide dismutase from Haemophilus ducreyi

Haemophilus ducreyi, the causative agent of the genital ulcerative disease known as chancroid, is unable to synthesize heme, which it acquires from humans, its only known host. Here we provide evidence that the periplasmic Cu,Zn-superoxide dismutase from this organism is a heme-binding protein, unlike all the other known Cu,Zn-superoxide dismutases from bacterial and eukaryotic species. When the H. ducreyi enzyme was expressed in Escherichia coli cells grown in standard LB medium, it contained only limited amounts of heme covalently bound to the polypeptide but was able efficiently to bind exogenously added hemin. Resonance Raman and electronic spectra at neutral pH indicate that H. ducreyi Cu,Zn-superoxide dismutase contains a 6-coordinated low spin heme, with two histidines as the most likely axial ligands. By site-directed mutagenesis and analysis of a structural model of the enzyme, we identified as a putative axial ligand a histidine residue (His-64) that is present only in the H. ducreyi enzyme and that was located at the bottom of the dimer interface. The introduction of a histidine residue in the corresponding position of the Cu,Zn-superoxide dismutase from Haemophilus parainfluenzae was not sufficient to confer the ability to bind heme, indicating that other residues neighboring His-64 are involved in the formation of the heme-binding pocket. Our results suggest that periplasmic Cu,Zn-superoxide dismutase plays a role in heme metabolism of H. ducreyi and provide further evidence for the structural flexibility of bacterial enzymes of this class.     

Use of electroporation to construct isogenic mutants of Haemophilus ducreyi.

Little is known about the genetics of Haemophilus ducreyi, the etiologic agent of chancroid. To develop a method for constructing isogenic mutants of this organism that could be utilized in pathogenesis-related studies, electroporation techniques were evaluated as a means of introducing DNA into this organism. Electroporation of the plasmid shuttle vector pLS88 into H. ducreyi yielded approximately 10(6) antibiotic-resistant transformants per microgram of plasmid DNA. Studies of the feasibility of moving mutated genes into H. ducreyi were initiated by using NotI linker insertion and mini-Tn10kan mutagenesis techniques to introduce insertion mutations into cloned H. ducreyi genes encoding cell envelope antigens. In the former case, a gene encoding chloramphenicol acetyltransferase was then inserted into the NotI linker site created in the cloned H. ducreyi gene. The recombinant Escherichia coli strains containing these mutated plasmids no longer expressed the homologous H. ducreyi cell envelope antigens, as evidenced by their lack of reactivity with monoclonal antibody probes for these H. ducreyi proteins. Subsequent electroporation of both circular and linearized forms of plasmids carrying these mutated H. ducreyi genes into the homologous wild-type strain of H. ducreyi yielded antibiotic-resistant transformants which also lacked reactivity with the cell envelope antigen-specific monoclonal antibodies. Southern blot analysis confirmed that homologous recombination had occurred in these monoclonal antibody-unreactive transformants, resulting in the replacement of the wild-type allele with the mutated allele. Allelic exchange was most efficient when linear DNA molecules were used for electroporation. These results indicate that electroporation methods can be utilized to construct isogenic mutants of H. ducreyi.     

Neutrophil degranulation induced by Haemophilus ducreyi

Haemophilus ducreyi is the causative bacterium of genital ulcers, which are collectively known as chancroid. Little is known about the cytotoxicity of H. ducreyi. The virulent strains are relatively resistant to phagocytosis and apoptosis by neutrophils. Therefore, experiments were designed to examine whether neutrophil degranulation caused by H. ducrey would provide insights into the virulence mechanisms through which cellular damage is affected by the organism. Clinical isolates of eight strains of H. ducreyi and the culture strain type CIP542 (Collection Institute Pasteur) were incubated with neutrophils harvested from human donor blood. The release by the organism of lysosomal enzymes from intracellular granules of neutrophils was indicative of degranulation. The results showed that H. ducreyi triggered the release of lysosomal enzymes from human neutrophils, and that the magnitude of the release was dependent both on the ratio of bacteria to neutrophils and the duration of incubation. In vitro experiments involving HeLa cells were designed to determine the manner in which H. ducreyi initiated the process of degranulation. The morphological changes associated with degranulation were visualized by confocal and transmission electron microscopy. This is the first report that describes degranulation of neutrophils induced by H. ducreyi which causes chancroid infection.     

Cloning and characterization of the lipooligosaccharide galactosyltransferase II gene of Haemophilus ducreyi

Haemophilus ducreyi is the etiologic agent of chancroid, a genital ulcer disease. The lipooligosaccharide (LOS) is considered to be a major virulence determinant and has been implicated in the adherence of H. ducreyi to keratinocytes. Strain A77, an isolate from the Paris collection, is serum sensitive, poorly adherent to fibroblasts, and deficient in microcolony formation. Structural analysis indicates that the LOS of strain A77 lacks the galactose residue found in the N-acetyllactosamine portion of the strain 35000HP LOS as well as the sialic acid substitution. From an H. ducreyi 35000HP genomic DNA library, a clone complementing the defect in A77 was identified by immunologic screening with monoclonal antibody (MAb) 3F11, a MAb which recognizes the N-acetyllactosamine portion of strain 35000HP LOS. The clone contained a 4-kb insert that was sequenced. One open reading frame which encodes a protein with a molecular weight of 33,400 was identified. This protein has homology to glycosyltransferases of Haemophilus influenzae, Haemophilus somnus, Neisseria species, and Pasteurella haemolytica. The putative H. ducreyi glycosyltransferase gene was insertionally inactivated, and an isogenic mutant of strain 35000HP was constructed. The most complex LOS glycoform produced by the mutant has a mobility on sodium dodecyl sulfate-polyacrylamide gel identical to that of the LOS of strain A77 and lacks the 3F11-binding epitope. Structural studies confirm that the most complex glycoform of the LOS isolated from the mutant lacks the galactose residue found in the N-acetyllactosamine portion of the strain 35000HP LOS. Although previously published data suggested that the serum-sensitive phenotype of A77 was due to the LOS mutation, we observed that the complemented A77 strain retained its serum-sensitive phenotype and that the galactosyltransferase mutant retained its serum-resistant phenotype. Thus, the serum sensitivity of strain A77 cannot be attributed to the galactosyltransferase mutation in strain A77.     

Haemophilus ducreyi: clinical features,epidemiology, and prospects for disease control

Haemophilus ducreyi is the causative agent of the genital ulcer disease chancroid. Chancroid is common in developing countries and facilitates human immunodeficiency virus transmission. In this review, the clinical features, epidemiology, and prospects for disease control are discussed in the context of experimental and natural infection of humans.     

The cytolethal distending toxin of Haemophilus ducreyi aggravates dermal lesions in a rabbit model of chancroid

Haemophilus ducreyi, the etiologic agent of the sexually transmitted disease chancroid, produces a cytolethal distending toxin (HdCDT) that inhibits cultured cell proliferation, leading to cell death. A rabbit model of dermal infection was used to investigate the roles of H. ducreyi bacteria and HdCDT in the development, clinical appearance, and persistence of infection. A non-toxin producing H. ducreyi strain, and for comparison purposes a non-capsulated Haemophilus influenzae strain, were inoculated intradermally, with and without co-administration of purified HdCDT. Co-administration of HdCDT resulted in significant aggravation of H. ducreyi-induced inflammatory lesions, and development of ulcers in rabbit skin. Less pronounced inflammatory lesions and lack of epithelial eruption were observed after inoculation with H. influenzae. Histopathological sections of the H. ducreyi-induced lesions, in both the presence and absence of HdCDT, showed dense infiltrates of the same type inflammatory cells, with the exception of a prominent endothelial cell proliferation noted in sections from lesions caused by H. ducreyi and toxin. Signs of chronic inflammation with involvement of T cells, macrophages, eosinophils, and granuloma formation were observed after H. ducreyi inoculation both with and without toxin. In conclusion, H. ducreyi causes a pronounced, chronic inflammation with involvement of T cells and macrophages, and in combination with HdCDT production of ulcers in the rabbit model. These pathogenic mechanisms may promote the development and persistence of chancroid ulcers.     

Proposed second class of Haemophilus ducreyi strains show altered protein and lipooligosaccharide profiles

Haemophilus ducreyi is the etiologic agent of chancroid, a sexually transmitted genital ulcer disease. Previously we have shown that the protein profiles and lipooligosaccharide (LOS) structures from various strains of H. ducreyi are generally well conserved. Previous studies have demonstrated that at least one strain, 33921, has a variant protein profile and LOS structure. In this study, both the whole cell lysate and the membrane proteins from strain 33921 were further examined and compared to the prototypical strain 35000HP by 2-DE and by the 16-BAC (benzyldimethyl-n-hexadecylammonium chloride)/SDS-PAGE two-detergent system, respectively. These comparisons demonstrated that a number of the proteins that could be identified from both strains had altered positions on the gels, both in their apparent molecular weight and pI values. Strain 33921 has been suggested to be a member of a second class of strains, termed class II strains. In this study, the proteomic profiles and the LOS structures from the five potential class II strains were examined and found to be similar to strain 33921.     

Periplasmic copper–zinc superoxide dismutase protects Haemophilus ducreyi from exogenous superoxide

Haemophilus ducreyi causes chancroid, a sexually transmitted genital ulcer disease implicated in increased heterosexual transmission of HIV. As part of an effort to identify H . ducreyi gene products involved in virulence and pathogenesis, we created random Tn phoA insertion mutations in an H . ducreyi 35 000 library cloned in Escherichia coli . Inserts encoding exported or secreted PhoA fusion proteins were characterized by DNA sequencing. One such clone encoded a Cu–Zn superoxide dismutase (SOD) enzyme. The Cu–Zn SOD was periplasmic in H . ducreyi and accounted for most of the detectable SOD activity in whole-cell lysates of H . ducreyi grown in vitro . To investigate the function of the Cu–Zn SOD, we created a Cu–Zn SOD-deficient H . ducreyi strain by inserting a cat cassette into the sodC gene. The wild-type and Cu–Zn SOD null mutant strains were equally resistant to excess cytoplasmic superoxide induced by paraquat, demonstrating that the Cu–Zn SOD did not function in the detoxification of cytoplasmic superoxide. However, the Cu–Zn SOD null strain was significantly more susceptible to killing by extracellular superoxide than the wild type. This result suggests that the H . ducreyi Cu–Zn SOD may play a role in bacterial defence against oxidative killing by host immune cells during infection.     

Rapid divergence of two classes of Haemophilus ducreyi

Haemophilus ducreyi, the etiologic agent of chancroid, expresses variants of several key virulence factors. While previous reports suggested that H. ducreyi strains formed two clonal populations, the differences between, and diversity within, these populations were unclear. To assess their variability, we examined sequence diversity at 11 H. ducreyi loci, including virulence and housekeeping genes, augmenting published data sets with PCR-amplified genes to acquire data for at least 10 strains at each locus. While sequences from all 11 loci place strains into two distinct groups, there was very little variation within each group. The difference between alleles of the two groups was variable and large at 3 loci encoding surface-exposed proteins (0.4 < K(S) < 1.3, where K(S) is divergence at synonymous sites) but consistently small at genes encoding cytoplasmic or periplasmic proteins (K(S) < 0.09). The data suggest that the two classes have recently diverged, that recombination has introduced variant alleles into at least 3 distinct loci, and that these alleles have been confined to one of the two classes. In addition, recombination is evident among alleles within, but not between, classes. Rather than clones of the same species, these properties indicate that the two classes may form distinct species.     

A mouse model of Haemophilus ducreyi infection (chancroid)

Abstract Six- to 8-week-old CBA mice were inoculated intradermally and/or subcutaneously on each flank with one of 3 strains of Haemophilus ducreyi , 2 of these being recent isolates and the other a reference strain. Pustular nodules developed at all sites the day after 10⁷ organisms had been given. Severe ulceration similar to chancroid occurred in about half of the lesions which took at least 13 days to heal. All three strains of H. ducreyi were isolated from the mice during the first week. The model is invaluable in helping to understand the pathogenicity of H. ducreyi as well as the immunopathogenesis and other aspects of chancroid.     

Binding of Haemophilus ducreyi to carbohydrate receptors is mediated by the 58.5-kDa GroEL heat shock protein

The bacterium Haemophilus ducreyi causes the sexually transmitted disease chancroid, which is characterized by the appearance of mucocutaneous, persistent ulcers on the external genitals. To identify carbohydrate receptors that mediate the attachment of this pathogen to host cells, we investigated the binding of 35S-methionine-labeled H. ducreyi strains to a panel of defined glycosphingolipids that were separated on thin layer chromatography plates. H. ducreyi bound to lactosylceramide, gangliotriaosylceramide, gangliotetraosylceramide, neolactotetraosylceramide, the GM3 ganglioside, and sulfatide. To elucidate the role of the surface-located 58.5-kDa GroEL heat shock protein (HSP) of H. ducreyi in attachment, we investigated the binding of purified HSP to the same panel of glycosphingolipids. Our results suggest that the 58.5-kDa GroEL HSP of H. ducreyi is responsible for the attachment of this bacterium to the majority of the tested glycosphingolipids, and thus represents a potential bacterial adhesin.     

Biosynthesis of sialylated lipooligosaccharides in Haemophilus ducreyi is dependent on exogenous sialic acid and not mannosamine. Incorporation studies using N-acylmannosamine analogues, N-glycolylneuraminic acid, and 13C-labeled N-acetylneuraminic acid

Haemophilus ducreyi is a Gram-negative bacterium that causes chancroid, a sexually transmitted disease. Cell surface lipooligosaccharides (LOS) of H. ducreyi are thought to play important biological roles in host infection. The vast majority of H. ducreyi strains contain high levels of sialic acid (N-acetylneuraminic acid, NeuAc) in their LOS. Here we investigate the biosynthetic origin of H. ducreyi sialosides by metabolic incorporation studies using a panel of N-acylmannosamine and sialic acid analogues. Incorporation of sialosides into LOS was assessed by matrix-assisted laser desorption and electrospray ionization mass spectrometry. A Fourier transform ion cyclotron resonance mass spectrometer provided accurate mass measurements, and a quadrupole time-of-flight instrument was used to obtain characteristic fragment ions and partial carbohydrate sequences. Exogenously supplied N-acetylmannosamine analogues were not converted to LOS-associated sialosides at a detectable level. In contrast, exogenous (13)C-labeled N-acetylneuraminic acid ([(13)C]NeuAc) and N-glycolylneuraminic acid (NeuGc) were efficiently incorporated into LOS in a dose-dependent fashion. Moreover, approximately 1.3 microM total exogenous sialic acid was sufficient to obtain about 50% of the maximum production of sialic acid-containing glycoforms observed under in vitro growth conditions. Together, these data suggest that the expressed levels of sialylated LOS glycoforms observed in H. ducreyi are in large part controlled by the exogenous concentrations of sialic acid and at levels one might expect in vivo. Moreover, these studies show that to properly exploit the sialic acid biosynthetic pathway for metabolic oligosaccharide engineering in H. ducreyi and possibly other prokaryotes that share similar pathways, precursors based on sialic acid and not mannosamine must be used.     

Rapid control of a chancroid outbreak: implications for Canada.

From June to November 1987 an outbreak of chancroid occurred in Winnipeg, the first in more than 10 years; 14 people (9 men, 5 women) were involved. Nine of the cases were confirmed through culture. A control strategy was implemented in November 1987 that included presumptive treatment of genital ulcer disease with single-dose antimicrobial therapy, intensive tracing of contacts and treatment of asymptomatic sexual contacts. The origin of the outbreak was not determined, and an epidemiologic link between all the patients could not be demonstrated. The isolates were found to contain the same plasmid; this suggested that a single clone of Haemophilus ducreyi was responsible for the outbreak.     

Facile construction of mutations in Haemophilus ducreyi using lacZ as a counter-selectable marker

Haemophilus ducreyi is a Gram-negative bacterium which is the causative agent of chancroid, an ulcerative sexually transmitted disease. In order to understand the pathogenesis of H. ducreyi disease, studies designed to identify potential virulence determinants and construct mutants deficient in the elaboration of these determinants have been undertaken in several laboratories. At the present time, construction of isogenic mutants is accomplished by electroporation of linearized DNA containing insertionally inactivated H. ducreyi genes followed by selection for the resistance marker encoded on the inactivated gene. In our experience, certain mutants are difficult to construct using this procedure. In the construction of strains containing lacZ as a reporter gene, we observed that the growth of lacZ expressing H. ducreyi was inhibited in the presence of X-gal. We have exploited this observation to develop a new strategy for the construction of isogenic H. ducreyi mutants.     

Evolution of the cutaneous immune response to experimental Haemophilus ducreyi infection and its relevance to HIV-1 acquisition

Haemophilus ducreyi causes the sexually transmitted disease chancroid, which facilitates HIV-1 transmission. Skin biopsies were obtained from subjects experimentally infected with H. ducreyi to study the evolution of the immune response and immunophenotypes relevant to transmission of HIV-1. Compared with peripheral blood, there was an enrichment of T cells and macrophages after 48 h of infection in the skin. Neutrophils became the predominant cell type by 7-9 days. By immunohistochemistry, macrophage-inflammatory protein-1alpha was not present early in infection, but was abundant at later stages. RANTES was present throughout the papular and pustular stages of experimental infection, but not present in uninfected control skin. Stromal cell-derived factor-1 was present at low levels in all samples examined. Macrophages in lesions had significantly increased expression of CCR5 and CXCR4 compared with peripheral blood cells, and CD4 T cells had significant up-regulation of CCR5. The magnitude of increased expression of these receptors was not replicated when PBMCs were incubated with H. ducreyi or H. ducreyi lipooligosaccharide in vitro. Together with the disruption of mucosal and skin barriers, the presence of cells with up-regulated HIV-1 coreceptors in H. ducreyi-infected lesions may provide an environment that facilitates the acquisition of R5 (CCR5), X4 (CXCR4), and dual-tropic HIV-1 strains.     

Investigation of the kinetic mechanism of cytidine 5'-monophosphate N-acetylneuraminic acid synthetase from Haemophilus ducreyi with new insights on rate-limiting steps from product inhibition analysis.

The presence of sialic acid as a component of cell surface lipooligosaccharides or capsular polysaccharides has been shown to be correlated with the virulence of a number of Gram-negative mucosal pathogens, including several Haemophilus and Neisseria spp. As part of our efforts to evaluate the role of sialic acid in the pathobiology of these organisms, we have initiated studies of the enzymes from Haemophilus ducreyi (the infectious agent of chancroid) responsible for the activation and attachment of sialic acid to the lipooligosaccharide. In this report, we describe results of an investigation of the steady-state kinetic mechanism of the activating enzyme, cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase. Using a combination of initial velocity, product inhibition, and dead-end inhibition studies, the reaction is shown to be freely reversible and to proceed through an ordered bi-bi kinetic mechanism in which CTP binds first and CMP-NeuAc dissociates last. In addition, a detailed analysis of the kinetic expressions for the observable constants is presented showing how the variation in apparent product inhibition constants (Kii) can be used to predict the rate-limiting step in kcat, which appears to be dissociation of CMP-NeuAc in this enzyme. To our knowledge, this relationship has not been previously recognized.