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PubMed2002

Development of a rapid immunodiagnostic test for Haemophilus ducreyi.

Haemophilus ducreyi is the etiologic agent of chancroid, a sexually transmitted disease that increases the rate of transmission of human immunodeficiency virus. Chancroid ulcerations are difficult to distinguish from those produced by syphilis and herpes. Diagnosis based solely on clinical grounds is inaccurate, and culture is insensitive. Highly sensitive PCR has largely superseded culture as the preferred method of laboratory diagnosis; however, neither culture nor PCR is feasible where chancroid is endemic. We developed a rapid (15-min) diagnostic test based on monoclonal antibodies (MAbs) to the hemoglobin receptor of H. ducreyi, HgbA. This outer membrane protein is conserved in all strains of H. ducreyi tested and is required for the establishment of experimental human infection. MAbs to HgbA were generated and tested for cross-reactivity against a panel of geographically diverse strains. Three MAbs were found to be unique and noncompetitive and bound to all strains of H. ducreyi tested. Using an immunochromatography format, we evaluated the sensitivity and specificity of the test using geographically diverse strains of H. ducreyi, other Haemophilus strains, and other bacteria known to superinfect genital ulcers. All H. ducreyi strains were positive, and all other bacteria were negative, resulting in a specificity of 100%. The minimum number of CFU of H. ducreyi detected was 2 x 10(6) CFU, and the minimum amount of purified HgbA protein detected was 8.5 ng. Although this level of sensitivity may not be sufficient to detect H. ducreyi in all clinical specimens, further work to increase the sensitivity could potentially make this a valuable bedside tool in areas where chancroid is endemic.

Antibodies, Monoclonal↗
PubMed1989

Chancroid and Haemophilus ducreyi.

Haemophilus ducreyi is the causative agent of chancroid, one of the genital ulcerative diseases. H. ducreyi is the major cause of genital ulcer disease in Africa and Southeast Asia and is of increasing concern in the United States. Definitive diagnosis of chancroid requires the isolation and identification of H. ducreyi, but isolation of this organism is difficult and the available medium is not optimal for all strains. Fluorescent antibody and serologic tests are of limited value. In general, our knowledge of this organism is rather limited, and indeed, recent studies have questioned the placement of H. ducreyi in the genus Haemophilus. H. ducreyi has relatively few biochemical activities, and epidemiologic studies are limited because there are limited phenotypic markers available for strain typing. Specific virulence factors of H. ducreyi have yet to be identified. Antimicrobial resistance in H. ducreyi is of special concern, as this organism has acquired both gram-negative and gram-positive resistance determinants. In addition, some of these determinants can be mobilized and transferred to other Haemophilus species or to Neisseria gonorrhoeae.

Amino Acid Sequence↗
PubMed1985

Haemophilus ducreyi.

Haemophilus ducreyi is the etiologic agent of chancroid, an infection which is endemic in tropical and subtropical areas of the world and which is becoming more prevalent in Europe and North America. Recently, several strains have demonstrated resistance to several antimicrobials by plasmid acquisition and cell envelope alteration making control of the spread of this organism a challenge. Equally challenging is the growth of this organism in the clinical microbiology laboratory because of its in vitro fastidious nature. Currently, the therapy of choice for Haemophilus ducreyi infection is trimethoprim sulfamethoxazole or erythromycin. Plasmid enumeration and restriction endonuclease fingerprinting in addition to outer membrane protein profiles may provide a basis for subtyping which would be useful in epidemiologic studies.

Animals↗
PubMed2001

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.

Cathepsin D↗
PubMed2004

A novel lectin, DltA, is required for expression of a full serum resistance phenotype in Haemophilus ducreyi.

Haemophilus ducreyi, the causative agent of chancroid, is highly resistant to the complement-mediated bactericidal activity of normal human serum (NHS). Previously, we identified DsrA (for ducreyi serum resistance A), a major factor required for expression of the serum resistance phenotype in H. ducreyi. We describe here a second outer membrane protein, DltA (for ducreyi lectin A), which also contributes to serum resistance in H. ducreyi. Isogenic dltA mutants, constructed in 35000HP wild-type and FX517 dsrA backgrounds, were more susceptible to the bactericidal effects of NHS than each respective parent, demonstrating the additive effect of the mutations. Furthermore, expression of dltA in H. influenzae strain Rd rendered this highly susceptible strain partially resistant to 5% NHS compared to a vector-control strain. Although primary basic local alignment search tool analysis of the dltA open reading frame revealed no close bacterial homologue, similarity to the beta-chain of the eukaryotic lectin ricin was noted. DltA shares highly conserved structural motifs with the ricin beta chain, such as cysteines and lectin-binding domains. To determine whether dltA was a lectin, ligand blots and affinity chromatography experiments were performed. DltA was affinity purified on immobilized lactose and N-acetylgalactosamine, and N-glycosylated but not glycosidase-treated model glycoproteins bound DltA. These data indicate that DltA is a lectin with specificity for lactose-related carbohydrates (CHO) and is important for H. ducreyi serum resistance.

Amino Acid Sequence↗
PubMed1997

Alterations in levels of DnaK and GroEL result in diminished survival and adherence of stressed Haemophilus ducreyi.

Haemophilus ducreyi is a hemin-requiring bacterium causing the genital ulcer disease chancroid. Previously we demonstrated that the heat shock protein GroEL was immunogenic and possibly highly expressed in a mammalian host. The present study was initiated to (i) determine the relative amounts of GroEL expressed by H. ducreyi during in vitro exposure to stresses and (ii) evaluate whether a high level of GroEL is directly or indirectly required for survival and adherence of stressed H. ducreyi. Using scanning densitometry of sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles, we found that H. ducreyi expressed high basal levels of GroEL, averaging fivefold greater than in Escherichia coli. These high GroEL levels increased up to twofold upon exposure of the organism to heat shock or high levels of hydrogen peroxide and during adherence to two human genital cell lines. Furthermore, when the gene for DnaK was present on a multicopy plasmid in H. ducreyi, a 1.8-fold increase in DnaK and a 2.3-fold reduction in GroEL were seen. These results suggest that DnaK serves as a negative modulator of H. ducreyi GroEL. Subsequently we found that H. ducreyi with lower GroEL had diminished ability to survive when challenged by heat and oxidative stresses. In addition, the long, parallel chains characteristic of virulent strains of H. ducreyi were absent when GroEL was lowered, so that fewer bacterial cells adhered to the human cells. These results suggest that the unusually high basal levels of GroEL are involved, either directly or indirectly, in the survival, chaining, and adherence of H. ducreyi in the presence of the combined stresses of the host environment.

Bacterial Adhesion↗
PubMed2006

Identification of genes involved in the expression of atypical lipooligosaccharide structures from a second class of Haemophilus ducreyi.

Haemophilus ducreyi is a gram-negative bacterium that is the causative agent of chancroid. Strain 35000HP has been well characterized and is representative of the majority of H. ducreyi strains. Strain 35000HP produces a lipooligosaccharide (LOS) that contains D-glycero-D-manno-heptose in the main oligosaccharide chain extension; the lbgB gene has been shown to encode the DD-heptosyltransferase. The lbgB gene is found in a gene cluster together with the lbgA gene, which encodes for the galactosyltransferase I. These two genes are flanked by two housekeeping genes, rpmE and xthA, encoding the ribosomal protein L31 and the exonuclease III, respectively. Recently, a second group of H. ducreyi strains have been identified. Strain 33921, a representative of the class II strains, produces an LOS that lacks DD-heptose in the oligosaccharide portion of its LOS. To better understand the biosynthesis of the DD-heptose-deficient 33921 LOS, we cloned and sequenced the corresponding lbgAB genomic region from strain 33921. Similar to strain 35000HP, the 33921 genome contains xthA and rpmE. However, between these two genes we identified genes encoding two putative glycosyltransferases that were not highly homologous to the 35000HP lbgAB genes. In this study, we demonstrate that the product of one of these genes encodes a galactosyltransferase. In addition, dot blot hybridization determined that 3 of 35 strains tested had the atypical transferases present, as did 4 strains characterized as class II strains by other criterion. These data indicate that the lbgAB genes can serve as one indicator of the classification of H. ducreyi strains.

Amino Acid Sequence↗
PubMed1999

Characterization of a WaaF (RfaF) homolog expressed by Haemophilus ducreyi.

Haemophilus ducreyi lipooligosaccharide (LOS) is capable of inducing an inflammatory response in skin (A. A. Campagnari, L. M. Wild, G. Griffiths, R. J. Karalus, M. A. Wirth, and S. M. Spinola, Infect. Immun. 59:2601-2608, 1991) and likely contributes to the virulence of this sexually transmitted pathogen (B. A. Bauer, M. K. Stevens, and E. J. Hansen, Infect. Immun. 68:4290-4298, 1998). An open reading frame in H. ducreyi 35000 was found to encode a predicted protein that was 59% identical to the protein product of the rfaF (waaF) gene of Salmonella typhimurium. The H. ducreyi waaF gene was able to complement an S. typhimurium rfaF (waaF) mutant, a result which confirmed the identity of this gene. In contrast to the rfaF (waaF) gene of enteric bacteria, the H. ducreyi waaF gene was not located adjacent to other genes involved in lipopolysaccharide expression. Inactivation of the H. ducreyi waaF gene by insertion mutagenesis resulted in expression of a LOS that migrated much faster than wild-type LOS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The LOS of this mutant also did not bind a monoclonal antibody directed against a cell surface-exposed epitope of wild-type H. ducreyi LOS. Testing of the wild-type H. ducreyi strain and its isogenic waaF mutant in the temperature-dependent rabbit model for dermal lesion production by H. ducreyi revealed that this waaF mutant was less virulent than the wild-type parent strain. Complementation of the H. ducreyi waaF mutant with the wild-type H. ducreyi waaF gene resulted in expression of both wild-type LOS and wild-type virulence by this mutant.

Amino Acid Sequence↗
PubMed1994

Identification of a hemolytic activity elaborated by Haemophilus ducreyi.

Haemophilus ducreyi is the causative agent of the sexually transmitted disease chancroid. We have identified a hemolytic activity expressed by H. ducreyi. This activity is most readily detected when horse erythrocytes are used as a target; however, low levels of activity can be detected with sheep, human, or rabbit erythrocyte targets. The activity is heat labile and protease sensitive.

Animals↗
PubMed1990

Use of the RapID-ANA system and sodium polyanetholesulfonate disk susceptibility testing in identifying Haemophilus ducreyi.

Haemophilus ducreyi has traditionally been difficult to identify. We have utilized simple test methods to identify 19 fresh isolates obtained during a recent outbreak of chancroid in Houston and six strains of H. ducreyi from other outbreaks. Tests were performed from growth on chocolate agar after 48 h of incubation at 35 degrees C with increased humidity and CO2. All isolates exhibited typical colonial morphology and Gram stain. Isolates were catalase negative and oxidase and nitrate positive (in enriched broth). The RapID NH system failed to identify these strains because of negative reactions with alkaline phosphatase and nitrate reductase. However, by using the RapID-ANA system, all strains were positive for alkaline phosphatase and arginine, glycine, and serine aminopeptidases. Their biochemical profiles were distinct from those obtained with 66 strains representing 13 species similar to H. ducreyi. We also investigated the use of sodium polyanetholesulfonate (SPS) disk susceptibility to identify and differentiate H. ducreyi from other species. All H. ducreyi isolates were susceptible, as evidenced by the presence of a zone of inhibition with an average size of 15 mm around the SPS disk. With the exceptions of Neisseria gonorrhoeae, Gardnerella vaginalis, and Capnocytophaga spp., no other strain showed any evidence of inhibition. The latter three organisms can be easily differentiated from H. ducreyi by various features including reactions in the RapID-ANA. We conclude that, by considering simple growth and biochemical characteristics, SPS susceptibilities, and reactions in RapID-ANA, it is possible for more clinical laboratories to definitively identify this organism.

Bacteriological Techniques↗
PubMed2002

Identification and characterization of the N-acetylglucosamine glycosyltransferase gene of Haemophilus ducreyi.

Haemophilus ducreyi is the causative agent of chancroid, a sexually transmitted ulcerative disease. In the present study, the Neisseria gonorrhoeae lgtA lipooligosaccharide glycosyltransferase gene was used to identify a homologue in the genome of H. ducreyi. The putative H. ducreyi glycosyltransferase gene (designated lgtA) was cloned and insertionally inactivated, and an isogenic mutant was constructed. Structural studies demonstrated that the lipooligosaccharide isolated from the mutant strain lacked N-acetylglucosamine and distal sugars found in the lipooligosaccharide produced by the parental strain. The isogenic mutant was transformed with a recombinant plasmid containing the putative glycosyltransferase gene. This strain produced the lipooligosaccharide glycoforms produced by the parental strain, confirming that the lgtA gene encodes the N-acetylglucosamine glycosyltransferase.

Bacterial Proteins↗
PubMed1995

Characterization of the hgbA locus encoding a hemoglobin receptor from Haemophilus ducreyi.

Haemophilus ducreyi can bind hemoglobin and use it as a source of heme, for which it has an obligate requirement. We previously identified and purified HgbA, a hemoglobin-binding outer membrane protein from H. ducreyi. In this report, we describe the molecular cloning, expression, DNA sequence, and mutagenesis of the structural gene for HgbA, hgbA. H. ducreyi and recombinant Escherichia coli expressing hgbA bound [125I]hemoglobin, establishing HgbA as a receptor. Insertions or deletions in the cloned hgbA gene abolished expression of HgbA and hemoglobin binding in E. coli. Mutagenesis of H. ducreyi by allelic exchange of insertions into hgbA abolished its ability to bind [125I]hemoglobin or utilize hemoglobin as a source of heme. The deduced protein sequence was similar to those of the TonB-dependent family of outer membrane receptors. The most similar member was HutA (heme receptor) from Vibrio cholerae. Tbp1 and Lbp1 (transferrin and lactoferrin receptors, respectively, from pathogenic Neisseria spp.) also showed very significant homology. Thus, by characterizing the hgbA locus, this work elucidates a potentially important role of HgbA in obtaining heme and/or iron from the host.

Amino Acid Sequence↗
PubMed2005

Identification of a novel sialic acid transporter in Haemophilus ducreyi.

Haemophilus ducreyi, the causative agent of chancroid, produces a lipooligosaccharide (LOS) which terminates in N-acetyllactosamine. This glycoform can be further extended by the addition of a single sialic acid residue to the terminal galactose moiety. H. ducreyi does not synthesize sialic acid, which must be acquired from the host during infection or from the culture medium when the bacteria are grown in vitro. However, H. ducreyi does not have genes that are highly homologous to the genes encoding known bacterial sialic acid transporters. In this study, we identified the sialic acid transporter by screening strains in a library of random transposon mutants for those mutants that were unable to add sialic acid to N-acetyllactosamine-containing LOS. Mutants that reacted with the monoclonal antibody 3F11, which recognizes the terminal lactosamine structure, and lacked reactivity with the lectin Maackia amurensis agglutinin, which recognizes alpha2,3-linked sialic acid, were further characterized to demonstrate that they produced a N-acetyllactosamine-containing LOS by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analyses. The genes interrupted in these mutants were mapped to a four-gene cluster with similarity to genes encoding bacterial ABC transporters. Uptake assays using radiolabeled sialic acid confirmed that the mutants were unable to transport sialic acid. This study is the first report of bacteria using an ABC transporter for sialic acid uptake.

Amino Sugars↗
PubMed2000

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.

Blood Bactericidal Activity↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed2004

Differential expression of porins OmpP2A and OmpP2B of Haemophilus ducreyi.

Haemophilus ducreyi is a strict human pathogen and the causative agent of the sexually transmitted disease chancroid. The genome of the human-passaged strain of H. ducreyi (35000HP) contains two homologous genes whose protein products have estimated molecular masses of 46 and 43 kDa. A comparative analysis of the deduced amino acid sequences revealed that these proteins share 27 to 33% identity to the outer membrane protein P2 (OmpP2), a major porin of Haemophilus influenzae. Therefore, these proteins have been designated OmpP2A and OmpP2B, respectively. The detection of ompP2A and ompP2B transcripts by reverse transcriptase PCR indicated that these genes were independently transcribed in H. ducreyi 35000HP. Western blot analysis of outer membrane proteins isolated from a geographically diverse collection of H. ducreyi clinical isolates revealed that OmpP2A and OmpP2B were differentially expressed among these strains. Although PCR analysis suggested that ompP2A and ompP2B were conserved among the strains tested, the differential expression observed was due to nucleotide additions and partial gene deletions. Purified OmpP2A and OmpP2B were isolated under nondenaturing conditions, and subsequent analysis demonstrated that these two proteins exhibited porin activity. OmpP2A and OmpP2B are the first porins described for H. ducreyi.

Amino Acid Sequence↗
PubMed1998

Cloning and characterization of tdhA, a locus encoding a TonB-dependent heme receptor from Haemophilus ducreyi.

Haemophilus ducreyi is unable to synthesize heme and must acquire it from its only known host, humans. We cloned and sequenced a gene encoding an outer membrane receptor for heme. It was designated tdhA (for TonB-dependent heme receptor A) since it was related by sequence homology to the family of TonB-dependent receptors. TdhA was strikingly similar to open reading frame HI0113 from the genome of Haemophilus influenzae Rd and also shared homology with five other heme receptors, including HxuC, HemR, HmuR, ChuA, and ShuA, from gram-negative bacteria. An Escherichia coli hemA tonB mutant strongly expressing H. ducreyi tdhA grew on low levels of heme as a source of heme only when an intact H. ducreyi Ton system plasmid was present, formally demonstrating functional TonB dependence. tdhA was expressed poorly in vitro by H. ducreyi and only under conditions of heme limitation. A survey of H. ducreyi revealed that all tested strains but one synthesized small amounts of TdhA in vitro under heme-limiting conditions. Surprisingly, an isogenic mutant of tdhA as well as its parent, 35000, both required the same high levels of heme for growth (50 microgram/ml [77 microM] on agar medium). This result, together with previous findings, suggests that in vitro, the uptake of heme by H. ducreyi is mediated by a TonB- and TdhA-independent mechanism, possibly diffusion.

Amino Acid Sequence↗
PubMed1989

DNA probes for the identification of Haemophilus ducreyi.

Haemophilus ducreyi ATCC 33922, a virulent, well-characterized strain, was used to construct a genomic library in a bacteriophage expression vector. Three DNA fragments were selected for use as probes on the basis of their ability to encode H. ducreyi-specific proteins, as demonstrated by reactivity with rabbit polyclonal antiserum. With DNA-DNA hybridization, the three probes, labeled with 32P, reacted strongly with 16 strains of H. ducreyi obtained from a variety of sources. Thirty-seven other bacterial isolates, representing 33 different species and including organisms likely to be encountered in the urogenital tract, were also tested with the three probes. Twenty-eight of these isolates, including the genital pathogen Neisseria gonorrhoeae, showed no hybridization with the probes. In addition, herpes simplex virus-infected tissue culture cells and Treponema pallidum-infected rabbit testicular fluid were also completely nonreactive. Nine isolates, six belonging to other Haemophilus species and three belonging to Pasteurella species, reacted weakly with the probes when approximately 3.0 x 10(7) to 6.0 x 10(7) CFU was tested. When 10(5) to 10(6) CFU of these organisms was tested, the weak reactions could no longer be seen. Yet this number of H. ducreyi still reacted strongly. In fact, the three probes consistently detected 10(4) CFU of H. ducreyi in pure and mixed cultures and even produced a weak signal when only 10(3) CFU was present. It is clear from our results that use of these probes will greatly facilitate the laboratory diagnosis of this genital pathogen.

Animals↗