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Background: Haemophilus ducreyi encounters several classes of antimicrobial peptides (APs) in vivo and utilizes the sensitive-to-antimicrobial-peptides (Sap) transporter as one mechanism of AP resistance. A mutant lacking the periplasmic solute-binding component, SapA, was somewhat more sensitive to the cathelicidin LL-37 than the parent strain and was partially attenuated for virulence. The partial attenuation led us to question whether the transporter is fully abrogated in the sapA mutant., Methods: We generated a nonpolar sapBC mutant, which lacks both inner membrane permeases of the Sap transporter, and tested the mutant for virulence in human volunteers. In vitro, we compared LL-37 resistance phenotypes of the sapBC and sapA mutants., Results: Unlike the sapA mutant, the sapBC mutant was fully attenuated for virulence in human volunteers. In vitro, the sapBC mutant exhibited significantly greater sensitivity than the sapA mutant to killing by LL-37. Similar to the sapA mutant, the sapBC mutant did not affect H. ducreyi's resistance to human defensins., Conclusions: Compared with the sapA mutant, the sapBC mutant exhibited greater attenuation in vivo, which directly correlated with increased sensitivity to LL-37 in vitro. These results strongly suggest that the SapBC channel retains activity when SapA is removed.

Cytidine 5'-monophosphate (CMP)-sialic acid synthetases (CSSs) catalyze the formation of CMP-sialic acid from CTP and sialic acid, a key step for sialyltransferase-catalyzed biosynthesis of sialic acid-containing oligosaccharides and glycoconjugates. More than 50 different sialic acid forms have been identified in nature. To facilitate the enzymatic synthesis of sialosides with diverse naturally occurring sialic acid forms and their non-natural derivatives, CMP-sialic acid synthetases with promiscuous substrate specificity are needed. Herein we report the cloning, characterization, and substrate specificity studies of a new CSS from Pasteurella multocida strain P-1059 (PmCSS) and a CSS from Haemophillus ducreyi (HdCSS). Based on protein sequence alignment and substrate specificity studies of these two CSSs and a Neisseria meningitidis CSS (NmCSS), as well as crystal structure modeling and analysis of NmCSS, NmCSS mutants (NmCSS_S81R and NmCSS_Q163A) with improved substrate promiscuity were generated. The strategy of combining substrate specificity studies of enzymes from different sources and protein crystal structure studies can be a general approach for designing enzyme mutants with improved activity and substrate promiscuity.

The Cu,Zn superoxide dismutases (Cu,Zn SOD) isolated from some Gram-negative bacteria possess a His-rich N-terminal metal binding extension. The N-terminal domain of Haemophilus ducreyi Cu,Zn SOD has been previously proposed to play a copper(II)-, and may be a zinc(II)-chaperoning role under metal ion starvation, and to behave as a temporary (low activity) superoxide dismutating center if copper(II) is available. The N-terminal extension of Cu,Zn SOD from Actinobacillus pleuropneumoniae starts with an analogous sequence (HxDHxH), but contains considerably fewer metal binding sites. In order to study the possibility of the generalization of the above mentioned functions over all Gram-negative bacteria possessing His-rich N-terminal extension, here we report thermodynamic and solution structural analysis of the copper(II) and zinc(II) complexes of a peptide corresponding to the first eight amino acids (HADHDHKK-NH(2), L) of the enzyme isolated from A. pleuropneumoniae. In equimolar solutions of Cu(II)/Zn(II) and the peptide the MH(2)L complexes are dominant in the neutral pH-range. L has extraordinary copper(II) sequestering capacity (K(D,Cu)=7.4×10(-13)M at pH 7.4), which is provided only by non-amide (side chain) donors. The central ion in CuH(2)L is coordinated by four nitrogens {NH(2),3N(im)} in the equatorial plane. In ZnH(2)L the peptide binds to zinc(II) through a {NH(2),2N(im),COO(-)} donor set, and its zinc binding affinity is relatively modest (K(D,Zn)=4.8×10(-7)M at pH 7.4). Consequently, the presented data do support a general chaperoning role of the N-terminal His-rich region of Gram-negative bacteria in copper(II) uptake, but do not confirm similar function for zinc(II). Interestingly, the complex CuH(2)L has very high SOD-like activity, which may further support the multifunctional role of the copper(II)-bound N-terminal His-rich domain of Cu,Zn SODs of Gram-negative bacteria. The proposed structure for the MH(2)L complexes has been verified by semiempirical quantum chemical calculations (PM6), too., (Copyright © 2011 Elsevier Inc. All rights reserved.)

X-ray absorption near-edge structure (XANES) spectroscopy and molecular dynamics (MD) simulations have been jointly applied to the study of the Cu,Zn superoxide dismutase from Haemophilus ducreyi (HdSOD) in interaction with the carbon monoxide molecule. The configurational flexibility of the Fe(II)-heme group, intercalated between the two subunits, has been sampled by MD simulations and included in the XANES data analysis without optimization in the structural parameter space. Our results provide an interpretation of the observed discrepancy in the Fe-heme distances as detected by extended X-ray absorption fine structure (EXAFS) spectroscopy and the classical XANES analysis, in which the structural parameters are optimized in a unique structure. Moreover, binding of the CO molecule to the heme induces a long range effect on the Cu,Zn active site, as evidenced by both MD simulations and in vitro experiments. MD simulation of the CO bound system, in fact, highlighted a structural rearrangement of the protein-protein hydrogen bond network in the region of the Cu,Zn active site, correlated with an increase in water accessibility at short distance from the copper atom. In line, in vitro experiments evidenced an increase of copper accessibility to a chelating agent when the CO molecule binds to the heme group, as compared to a heme deprived HdSOD. Altogether, our results support the hypothesis that the HdSOD is a heme-sensor protein, in which binding to small gaseous molecules modulates the enzyme superoxide activity as an adaptive response to the bacterial environment.

Histophilus somni is an etiologic agent of bovine respiratory and systemic diseases. Most pathogenic strains of H. somni that have been tested (36 of 42) are able to utilize N-acetyl-5-neuraminic acid (Neu5Ac) to sialylate their lipooligosaccharide (LOS). Homologs of all the genes required for transport, metabolism, and regulation of Neu5Ac in Haemophilus influenzae were identified in the sequenced genomes of H. somni. Three open reading frames (ORFs) in H. somni strain 2336 were identified that contained homology to genes required for LOS sialylation in related bacteria. ORF-1 (hssT-I), ORF-2 (hssT-II), and ORF-3 (neuA(Hs)) were predicted to encode for putative proteins with 37% amino acid homology to an α-(2-3)-sialyltransferase in H. influenzae, 43% amino acid homology to an Haemophilus ducreyi sialyltransferase, and 72% amino acid homology to an H. influenzae CMP-Neu5Ac synthetase, respectively. The specific enzyme activity of each ORF was determined using synthetic acceptor substrates. The HssT-I sialyltransferase primarily sialylated N-acetyllactosamine (LacNAc, Gal-β-[1-4]-GlcNAc-R), which is expressed on strain 2336, whereas HssT-II preferentially sialylated lacto-N-biose (LNB, Gal-β-[1-3]-GlcNAc-R), which is expressed on a phase variant of strain 2336: strain 738. Phase variation of the terminal galactose linkage in strain 738 from β-(1-3)-(LNB) to β-(1-4)-(LacNAc) was confirmed using monoclonal antibody reactivity and nuclear magnetic resonance spectroscopy. Sialylated LOS induced significantly less chemokine response from macrophages derived from Toll-like receptor (TLR)-4 knockout mice than from de-sialylated LOS. Furthermore, sialylated LOS induced significantly less NF-κB activity from mouse-derived bone marrow macrophages than de-sialylated LOS. Therefore, sialylation inhibited LOS signaling through TLR-4. In conclusion, H. somni utilizes linkage-specific sialyltransferases to sialylate its LOS to avoid innate host defense mechanisms despite simultaneous epitope phase variation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We have carried out an X-ray Absorption Spectroscopy (XAS) study of ferric, ferrous, CO- and NO-bound Haemophilus ducreyi Cu,ZnSOD (HdSOD) in solution to investigate the structural modifications induced by the binding of small gaseous ligands to heme in this enzyme. The combined analysis of EXAFS and XANES data has allowed us to characterize the local structure around the Fe-heme with 0.02A accuracy, revealing a heterogeneity in the distances between iron and the two histidine ligands which was not evident in the X-ray crystal structure. In addition, we have shown that the metal oxidation state does not influence the Fe-heme coordination environment, whereas the presence of the CO and NO ligands induces local structural rearrangements in the enzyme which are very similar to those already observed in other hexa-coordinated heme proteins, such as neuroglobin., (2010 Elsevier Inc. All rights reserved.)

The Cu,Zn superoxide dismutase (Cu,ZnSOD) isolated from Haemophilus ducreyi possesses a His-rich N-terminal metal binding domain, which has been previously proposed to play a copper(II) chaperoning role. To analyze the metal binding ability and selectivity of the histidine-rich domain we have carried out thermodynamic and solution structural analysis of the copper(II) and zinc(II) complexes of a peptide corresponding to the first 11 amino acids of the enzyme (H(2)N-HGDHMHNHDTK-OH, L). This peptide has highly versatile metal binding ability and provides one and three high affinity binding sites for zinc(II) and copper(II), respectively. In equimolar solutions the MHL complexes are dominant in the neutral pH-range with protonated lysine epsilon-amino group. As a consequence of its multidentate nature, L binds zinc and copper with extraordinary high affinity (K(D,Zn)=1.6x10(-9)M and K(D,Cu)=5.0x10(-12)M at pH 7.4) and appears as the strongest zinc(II) and copper(II) chelator between the His-rich peptides so far investigated. These K(D) values support the already proposed role of the N-terminal His-rich region of H. ducreyi Cu,ZnSOD in copper recruitment under metal starvation, and indicate a similar function in the zinc(II) uptake, too. The kinetics of copper(II) transfer from L to the active site of Cu-free N-deleted H. ducreyi Cu,ZnSOD showed significant pH and copper-to-peptide ratio dependence, indicating specific structural requirements during the metal ion transfer to the active site. Interestingly, the complex CuHL has significant superoxide dismutase like activity, which may suggest multifunctional role of the copper(II)-bound N-terminal His-rich domain of H. ducreyi Cu,ZnSOD.

The Cu,Zn superoxide dismutase (Cu,ZnSOD) from Haemophilus ducreyi is the only enzyme of this class which binds a heme molecule at its dimer interface. To explore the role of the enzyme in this heme-obligate bacterium, a sodC mutant was created by insertional inactivation. No difference in growth rate was observed during heme limitation. In contrast, under heme rich conditions growth of the sodC mutant was impaired compared to the wild type strain. This growth defect was abolished by supplementation of exogenous catalase. Genetic complementation of the sodC mutant in trans demonstrated that the enzymatic property or the heme-binding activity of the protein could repair the growth defect of the sodC mutant. These results indicate that Cu,ZnSOD protects Haemophilus ducreyi from heme toxicity.

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.

The N-terminal metal binding extension of the Cu,Zn superoxide dismutase from Haemophilus ducreyi is constituted by a histidine-rich region followed by a methione-rich sequence which shows high similarity with protein motifs involved in the binding of Cu(I). X-ray absorption spectroscopy experiments selectively carried out with peptides corresponding to the two metal binding regions indicate that both sequences can bind either Cu(II) or Cu(I). However, competition experiments demonstrate that Cu(II) is preferred by histidine residues belonging to the first half of the motif, while the methionine-rich region preferentially binds Cu(I) via the interaction with three methionine sulfur atoms. Moreover, we have observed that the rate of copper transfer from the peptides to the active site of a copper-free form of the Cu,Zn superoxide dismutase mutant lacking the N-terminal extension depends on the copper oxidation state and on the residues involved in metal binding, histidine residues being critically important for the efficient transfer. Differences in the enzyme reactivation rates in the presence of mixtures of the two peptides when compared to those obtained with the single peptides suggest that the two halves of the N-terminal domain functionally interact during the process of copper transfer, possibly through subtle modifications of the copper coordination environment.

The tripartite cytolethal distending toxin (CDT) induces cell cycle arrest and apoptosis in eukaryotic cells. The subunits CdtA and CdtC associate with the nuclease CdtB to form a holotoxin that translocates CdtB into the host cell, where it acts as a genotoxin by creating DNA lesions. Here we show that the crystal structure of the holotoxin from Haemophilus ducreyi reveals that CDT consists of an enzyme of the DNase-I family, bound to two ricin-like lectin domains. CdtA, CdtB and CdtC form a ternary complex with three interdependent molecular interfaces, characterized by globular, as well as extensive non-globular, interactions. The lectin subunits form a deeply grooved, highly aromatic surface that we show to be critical for toxicity. The holotoxin possesses a steric block of the CdtB active site by means of a non-globular extension of the CdtC subunit, and we identify putative DNA binding residues in CdtB that are essential for toxin activity.

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.

Streptococcus agalactiae (GBS) is a major cause of serious newborn bacterial infections. Crucial to GBS evasion of host immunity is the production of a capsular polysaccharide (CPS) decorated with sialic acid, which inactivates the alternative complement pathway. The CPS operons of serotypes Ia and III GBS have been described, but the CPS sialyltransferase gene was not identified. We identified cpsK, an open reading frame in the CPS operon of most serotypes, which was homologous to the lipooligosaccharide (LOS) sialyltransferase gene, lst, of Haemophilus ducreyi. To determine if cpsK might encode a sialyltransferase, we complemented a H. ducreyi lst mutant with cpsK. CpsK was expressed in H. ducreyi and LOS was isolated and analysed for sialic acid content by SDS-PAGE and high-performance liquid chromatography (HPLC). Sialo-LOS was seen in the wild-type, cpsK- or lst-complemented mutant strains, but not in the mutant without cpsK. Addition of Neu5Ac to the LOS was confirmed by mass spectroscopy. Lectin binding studies detected terminal Neu5Ac(alpha 2-->3)Gal(beta 1- on LOS produced by the wild-type, cpsK or lst-complemented mutant strain LOS, compared with the mutant alone. Our data characterize the first sialyltransferase gene from a Gram- positive bacterium and provide compelling evidence that its product catalyses the alpha2,3 addition of Neu5Ac to H. ducreyi LOS and therefore the terminal side-chain of GBS CPS. Phylogenetic studies further indicated that lst and cpsK are related but distinct from sialyltransferases of most other bacteria and, along with their similar codon usage bias and G + C content, suggests acquisition by lateral transfer from an ancestral low G + C organism.

All Haemophilus ducreyi strains examined contain a lipooligosaccharide (LOS) consisting of a single but variable branch oligosaccharide that emanates off the first heptose (Hep-I) of a conserved Hep(3)-phosphorylated 3-deoxy-D-manno-octulosonic acid-lipid A core. In a previous report, identification of tandem genes, lbgA and lbgB, that are involved in LOS biosynthesis was described (Stevens et al., Infect. Immun. 65:651-660, 1997). In a separate study, the same gene cluster was identified and the lbgB (losB) gene was found to be required for transfer of the second sugar, D-glycero-D-manno-heptose (DD-Hep), of the major branch structure (Gibson et al., J. Bacteriol. 179:5062-5071, 1997). In this study, we identified the function of the neighboring upstream gene, lbgA, and found that it is necessary for addition of the third sugar in the dominant oligosaccharide branch, a galactose-linked beta1-->4, to the DD-Hep. LOS from an lbgA mutant and an lbgAB double mutant were isolated and were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, carbohydrate analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy. The results showed that the mutant strains synthesize truncated LOS glycoforms that terminate after addition of the first glucose (lbgAB) or the disaccharide DD-Hepalpha1-->6Glcbeta1 (lbgA) that is attached to the heptose core. Both mutants show a significant reduction in the ability to adhere to human keratinocytes. Although minor differences were observed after two-dimensional gel electrophoresis of total proteins from the wild-type and mutant strains, the expression levels of the vast majority of proteins were unchanged, suggesting that the differences in adherence and invasion are due to differences in LOS. These studies add to the mounting evidence for a role of full-length LOS structures in the pathophysiology of H. ducreyi infection.

A pCb plasmid encoding a beta-lactamase from Haemophilus ducreyi was transferred to Escherichia coli, purified, and characterized. The beta-lactamase could be isolated from a culture filtrate and further purified by ammonium sulfate and chelating Sepharose fast flow loaded with Zn(2+). The purified enzyme resulted in a major band at approximately 30-kDa on SDS-PAGE and its pI was determined to be 5.4. The beta-lactamase could hydrolyze both penicillin antibiotics including ampicillin, benzylpenicillin, and carbenicillin as well as cephalosporin antibiotics including nitrocefin, cephalothin, cephaloridine, and cefoperazone. However, benzylpenicillin was the best substrate. The enzyme activity was inhibited by clavulanic acid but not by boric acid, cefotaxime, ethylenediaminetetraacetic acid, or phenylmethylsulfonyl fluoride. The sequence of the beta-lactamase gene was also determined. It confirmed that the enzyme belonged to a class A beta-lactamase which had 99% identity to the ampicillin resistance transposon Tn3 of pBR322. Two nucleotides were different between the E. coli (Tn3) and H. ducreyi (pCb) genes that affected the amino-acid sequence. The valine at position 82 (ABL 84) was changed to isoleucine and the alanine at position 182 (ABL 184) was changed to valine. Genetic homogeneity among beta-lactamases is remarkable. Amino acid sequencing of some beta-lactamases has shown that substitution of only a few amino acids in the bla gene leads to high-level resistance against specific cephalosporins., (Copyright 2001 Academic Press.)

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.

A group of Cu,Zn-superoxide dismutases from pathogenic bacteria is characterized by histidine-rich N-terminal extensions that are in a highly exposed and mobile conformation. This feature allows these proteins to be readily purified in a single step by immobilized metal affinity chromatography. The Cu,Zn-superoxide dismutases from both Haemophilus ducreyi and Haemophilus parainfluenzae display anomalous absorption spectra in the visible region due to copper binding at the N-terminal region. Reconstitution experiments of copper-free enzymes demonstrate that, under conditions of limited copper availability, this metal ion is initially bound at the N-terminal region and subsequently transferred to an active site. Evidence is provided for intermolecular pathways of copper transfer from the N-terminal domain of an enzyme subunit to an active site located on a distinct dimeric molecule. Incubation with EDTA rapidly removes copper bound at the N terminus but is much less effective on the copper ion bound at the active site. This indicates that metal binding by the N-terminal histidines is kinetically favored, but the catalytic site binds copper with higher affinity. We suggest that the histidine-rich N-terminal region constitutes a metal binding domain involved in metal uptake under conditions of metal starvation in vivo. Particular biological importance for this domain is inferred by the observation that its presence enhances the protection offered by periplasmic Cu,Zn-superoxide dismutase toward phagocytic killing.

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 causes chancroid, a sexually transmitted cutaneous genital ulcer disease associated with increased heterosexual transmission of human immunodeficiency virus. H. ducreyi expresses a periplasmic copper-zinc superoxide dismutase (Cu, Zn SOD) that protects the bacterium from killing by exogenous superoxide in vitro. We hypothesized that the Cu,Zn SOD would protect H. ducreyi from immune cell killing, enhance survival, and affect ulcer development in vivo. In order to test this hypothesis and study the role of the Cu,Zn SOD in H. ducreyi pathogenesis, we compared a Cu,Zn SOD-deficient H. ducreyi strain to its isogenic wild-type parent with respect to survival and ulcer development in immunocompetent and immunosuppressed pigs. The Cu,Zn SOD-deficient strain was recovered from significantly fewer inoculated sites and in significantly lower numbers than the wild-type parent strain or a merodiploid (sodC+ sodC) strain after infection of immunocompetent pigs. In contrast, survival of the wild-type and Cu,Zn SOD-deficient strains was not significantly different in pigs that were rendered neutropenic by treatment with cyclophosphamide. Ulcer severity in pigs was not significantly different between sites inoculated with wild type and sites inoculated with Cu,Zn SOD-deficient H. ducreyi. Our data suggest that the periplasmic Cu,Zn SOD is an important virulence determinant in H. ducreyi, protecting the bacterium from host immune cell killing and contributing to survival and persistence in the host.

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.

Haemophilus ducreyi, the cause of the sexually transmitted disease chancroid produces a lipooligosaccharide (LOS) containing a terminal sialyl N-acetyllactosamine trisaccharide. Previously, we reported the identification and characterization of the N-acetylneuraminic acid cytidylsynthetase gene (neuA). Forty-nine base pairs downstream of the synthetase gene is an open reading frame (ORF) encoding a protein with a predicted molecular weight of 34,646. This protein has weak homology to the polysialyltransferase of Escherichia coli K92. Downstream of this ORF is the gene encoding the H. ducreyi homologue of the Salmonella typhimurium rmlB gene. Mutations were constructed in the neuA gene and the gene encoding the second ORF by insertion of an Omega kanamycin cassette, and isogenic strains were constructed. LOS was isolated from each strain and characterized by SDS-polyacrylamide gel electrophoresis, carbohydrate, and mass spectrometric analysis. LOS isolated from strains containing a mutation in neuA or in the second ORF, designated lst, lacked the sialic acid-containing glycoform. Complementation studies were performed. The neuA gene and the lst gene were each cloned into the shuttle vector pLS88 after polymerase chain reaction amplification. Complementation of the mutation in the lst gene was observed, but we were unable to complement the neuA mutation. Since it is possible that transcription of the neuA gene and the lst gene were coupled, we constructed a nonpolar mutation in the neuA gene. In this construct, the neuA mutation was complemented, suggesting transcriptional coupling of the neuA gene and the lst gene. Sialyltransferase activity was detected by incorporation of 14C-labeled NeuAc from CMP-NeuAc into trichloroacetic acid-precipitable material when the lst gene was overexpressed in the nonpolar neuA mutant. We conclude that the lst gene encodes the H. ducreyi sialyltransferase. Since the lst gene product has little, if any, structural relationship to other sialyltransferases, this protein represents a new class of sialyltransferase.

Haemophilus ducreyi is the etiologic agent of the sexually transmitted disease chancroid, an ulcerative condition implicated in increased HIV transmission. There is increasing evidence for the roles of oxidative stress proteins including superoxide dismutase enzymes in the survival and persistence of pathogenic organisms within the host. The sodA gene of Haemophilus ducreyi was isolated from a genomic plasmid library on the basis of its ability to rescue the hydrogen peroxide hypersensitivity of an Escherichia coli sodA sodB strain. The H. ducreyi SodA protein also complemented the aerobic growth defect of the E. coli sodA sodB strain in minimal medium. The deduced amino-acid sequence of the H. ducreyi sodA gene product is 74 and 70% identical to the Mn-SODs of Haemophilus influenzae and E. coli, respectively. However, unlike Mn-SODs, the H ducreyi SodA protein was inhibited by hydrogen peroxide in native gels stained for SOD activity.

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 TnphoA insertion mutations in an H. ducreyi 35000 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.

The sodC gene of Haemophilus ducreyi was cloned and sequenced. The deduced amino acid sequence of this protein exhibited 71.6% identity and 81.8% similarity to the H. influenzae and H. parainfluenzae copper (Cu), zinc (Zn)-superoxide dismutase (SOD) enzymes. This gene was localized to a 2.2-kb H. ducreyi chromosomal DNA insert in plasmid pHdSOD. SOD activity was expressed in cell-free extracts of Escherichia coli containing the recombinant plasmid pHdSOD and was localized to the periplasmic space. The Cu,Zn-SOD produced by the H. ducreyi sodC gene did not complement the aerobic growth defect of an E. coli SOD-deficient mutant.

An N-acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-NeuAc synthetase) was isolated from a Haemophilus ducreyi strain 35000 cell lysate and partially characterized. The enzyme catalyzes the reaction of CTP and NeuAc to form CMP-NeuAc, which is the nucleotide sugar donor used by sialyltransferases. Previous studies have shown that the outer membrane lipooligosaccharides of H. ducreyi contain terminal sialic acid attached to N-acetyllactosamine and that this modification is likely important to its pathogenesis. Therefore, to investigate the role of sialic acid in H. ducreyi pathogenesis, the gene encoding the CMP-NeuAc synthetase was cloned using degenerate oligonucleotide probes derived from NH2-terminal sequence data, and the nucleotide sequence was determined. The derived amino acid sequence of the CMP-NeuAc synthetase gene has homology to other CMP-NeuAc synthetases and to a lesser extent to CMP-2-keto-3-deoxy-D-manno-octulosonic acid synthetases. The gene was cloned into a T7 expression vector, the protein expressed in Escherichia coli, and purified to apparent homogeneity by anion exchange, Green 19 dye, and hydrophobic interaction chromatography. The final step yielded 20 mg of pure protein/liter of culture. The protein has a predicted molecular mass of 25440.6 Da, which was confirmed by electrospray mass spectrometry (Mexpt = 25439.9 +/- 1.4 Da). The enzyme appears to exist as a dimer by size exclusion chromatography. In contrast to other bacterial CMP-NeuAc synthetases, the H. ducreyi enzyme exhibited a different substrate specificity, being capable of also using N-glycolylneuraminic acid as a substrate.

The in vitro susceptibilities to various antimicrobial agents of 100 strains of beta-lactamase-producing Haemophilus ducreyi recently isolated from patients in New Orleans, La., were determined by an agar dilution method. All strains were highly susceptible to ceftizoxime, ceftriaxone, ceftazidime, azithromycin, erythromycin, ciprofloxacin, and sparfloxacin. beta-Lactam-beta-lactamase inhibitor combinations were less active, but all strains were susceptible to them. Doxycycline exhibited the poorest activity, and the rate of resistance to doxycycline varied depending on the time after inoculation that the MIC was determined.

We determined the susceptibilities of 94 strains of Haemophilus ducreyi isolated in various municipalities in the United States between 1982 and 1989 to the following antimicrobial agents: amoxicillin-clavulanic acid, ceftriaxone, erythromycin, azithromycin, ciprofloxacin, ofloxacin, trimethoprim, and spectinomycin. Ceftriaxone (MIC, less than or equal to 0.008 micrograms/ml), azithromycin (MIC, less than or equal to 0.125 micrograms/ml), erythromycin (MIC, less than or equal to 0.125 micrograms/ml), ciprofloxacin (MIC, less than or equal to 0.25 micrograms/ml), and ofloxacin (MIC, less than or equal to 0.25 micrograms/ml) were highly active against all isolates. Amoxicillin-clavulanic acid (MICs, 0.25 to 8.0 micrograms/ml), trimethoprim (MICs, 0.06 to 16.0 micrograms/ml), and spectinomycin (MICs, 2.0 to greater than or equal to 32.0 micrograms/ml) were less active against these isolates. Isolates possessing the 5.7-MDa beta-lactamase plasmid were less susceptible to erythromycin, trimethoprim, and spectinomycin than were isolates possessing the 3.2-MDa beta-lactamase plasmid. The susceptibilities of plasmidless isolates to erythromycin, trimethoprim, and spectinomycin were distributed bimodally; the median MIC for the more susceptible plasmidless isolates corresponded to that for isolates with the 3.2-MDa plasmid, and the median MIC for the less susceptible plasmidless isolates corresponded to that for isolates with the 5.7-MDa plasmid. Thus, plasmid profiles may be valuable markers for geographical variations in antimicrobial susceptibilities of H. ducreyi strains that may indicate the relative efficacy of regimens for the treatment of chancroid. Of the regimens recommended by the U.S. Public Health Service for the treatment of chancroid, our results support the use of erythromycin, ceftriaxone, and ciprofloxacin, and perhaps ofloxacin, but suggest that amoxicillin-clavulanic acid and sulfamethoxazole-trimethoprim should be used with caution.

A collection of 20 strains of Haemophilus ducreyi including freshly isolated, low-passage and multi-passage reference strains was examined for immunoglobulin A1 protease production by SDS-PAGE and immunoblotting. None of the strains demonstrated IgA1 protease activity despite the fact that different culture media were used. By direct immunofluorescence testing, binding of IgA to Haemophilus ducreyi organisms could be demonstrated.

A collection of 100 clinical isolates of Haemophilus ducreyi from Thailand were all found to harbor a 5.4-kb plasmid, designated pTH126, which was shown to contain the bla ROB-1 gene. Restriction enzyme analysis and DNA-DNA hybridization studies confirmed that pTH126 was similar to the ROB-1 beta-lactamase plasmid pVM105 from Actinobacillus pleuropneumoniae. In approximately one-half of the isolates, pTH126 was found together with pHD131, which mediates TEM-1 beta-lactamase production.

Ten isolates of Haemophilus ducreyi, including seven clinical isolates and three laboratory reference strains, were assessed for their ability to produce extracellular enzymes which might contribute to their pathogenicity. Protease, elastase, lecithinase, lipase or collagenase enzyme activity were not detected in culture filtrates from any of the isolates tested using either plate or spectrophotometrical assays. Furthermore, cell-free culture filtrates did not exhibit cytotoxic activity in vitro when tested using either an established tissue culture cell line (Vero) or a primary cell culture of human keratinocytes. These results suggest that extracellular products do not play a role in host invasion or production of tissue damage by H. ducreyi.

Chancroid, the disease caused by H. ducreyi is one of the common sexually transmitted diseases (STD) in Thailand and other tropical countries. In Thailand, the diagnosis of chancroid is still based on clinical appearance which may be confused with other STD manifested by genital ulcers. In recent years the increasing resistance strains of H. ducreyi to these antimicrobial agents has been reported so that cultivation and antimicrobial susceptibility tests of this organism have become more important. This study showed that MBV is the best medium for isolation with a success rate of 48%. All strains tested from isolates of this study were resistant to ampicillin, due to production of beta-lactamase. Approximately 99% of the strains were resistant to tetracycline 92% of strains were resistant to sulfamethoxazole and 32% were resistant to trimethoprim. All isolates were susceptible to chloramphenicol, ceftriaxone, erythromycin and the fluorinated quinolones ciprofloxacin, norfloxacin, ofloxacin and pefloxacin. Beta-lactamase enzymes produced by 37 strains of H. ducreyi were determined for their isoelectric point (pI). All had pI of 5.4, indicative of plasmid-mediated beta-lactamase type TEM-1.

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.

We have studied the in-vitro susceptibility of 30 strains of Haemophilus ducreyi to 22 antibacterial agents. Twenty-seven strains produced a beta-lactamase that had an isoelectric point similar to the isoelectric point of the TEM 1 type beta-lactamase produced by pSF 2124. Except for cefamandole, the 30 strains had similar susceptibilities to the different cephalosporins, cefotaxime being the most active compound. Twenty-two isolates were resistant to tetracycline and doxycycline; minimal inhibitory concentration (MIC) of minocycline was 4 mg/l for all the strains. One isolate was resistant to chloramphenicol (MIC = 16 mg/l). All strains were relatively resistant to colistin and nalidixic acid, and susceptible to streptomycin, kanamycin, gentamicin, erythromycin, rifampicin and vancomycin.

We have studied the genetic basis of beta-lactamase production in eight strains of Haemophilus ducreyi isolated in diverse areas of the world. Beta-lactamase production in all strains was mediated by plasmids having a molecular mass of either 5.7 or 7.0 megadaltons. Plasmids of 5.7 megadaltons were shown to carry the entire sequence of pFA7, the beta-lactamase specifying plasmid found in isolates of Neisseria gonorrhoeae epidemiologically linked to West Africa. Plasmids of 7.0 megadaltons were shown to carry the entire sequence of pFA3, the beta-lactamase specifying plasmid found in Far Eastern isolates of N. gonorrhoeae. Both groups of H. ducreyi plasmids were shown to carry physically complete and functional TnA sequences. Thus we have identified two types of H. ducreyi beta-lactamase plasmid which are identical to the two types of N. gonorrhoeae beta-lactamase plasmid, except that they carry complete TnA sequences.

Eighty-nine strains of Haemophilus ducreyi from a chancroid epidemic in Orange County, California, were examined for plasmid content. Seventy-eight (88%) of these isolates were found to contain a plasmid of 3.2 megadaltons which conferred beta-lactamase production. Restriction endonuclease digests indicated that this was the same plasmid that was found in some strains of beta-lactamase-producing Neisseria gonorrhoeae.

The enzymatic activities of two reference strains of Haemophilus ducreyi and thirty clinical isolates were investigated by conventional biochemical tests and the API-ZYM test kit system which included 97 synthetic substrates. No strains converted delta-aminolevulinic acid to porphyrins, but they all reduced nitrates to nitrites. All strains possessed aminopeptidase activity against beta-naphthylamide derivatives of L-alanine, L-arginine, L-glutamine, glycine, L-leucine, L-lysine and L-serine. No trypsinor chymotrypsin-like activities were detected. All strains had phosphatase activity with broad pH range, and phosphoamidase activity. No glycosidase was detected by the substrates tested.

Multiresistant strains of Haemophilus ducreyi, the aetiological agent of chancroid, are prevalent in Nairobi, Kenya, where tetracyclines and sulphonamides are no longer very effective in the treatment of chancroid. The following regimens (given three times daily for seven days) were compared in a double-blind randomised trial--amoxycillin 500 mg, amoxycillin 500 mg and clavulanic acid 125 mg, and amoxycillin 500 mg and clavulanic acid 250 mg. 68 of 100 ulcers were culture-positive for H. ducreyi. All strains of H. ducreyi produced beta-lactamase. At day 7 none of the amoxycillin-treated patients had responded clinically or bacteriologically, whereas all but 2 of 56 patients treated with an amoxycillin/clavulanic-acid regimen had responded clinically and H. ducreyi had been eradicated from their ulcers. The combination of amoxycillin-clavulanic acid appears to be very effective for the treatment of chancroid. The results of this study accord with H. ducreyi as the primary pathogen of chancroid.

We characterized pJB1, the plasmid previously reported to mediate beta-lactamase production in Haemophilus ducreyi. We studied its relationship to pMR0360 and RSF0885, the plasmids responsible for beta-lactamase production in Neisseria gonorrhoeae and Haemophilus parainfluenzae, respectively. Although pJB1 was maintained as a multicopy pool in Escherichia coli, it was not stably maintained in the absence of antibiotic selection. Electron microscope heteroduplex studies showed that it carried 100% of the transposable ampicillin resistance sequence TnA. This sequence was transposed to plasmid pUB307 at a low rate. Heteroduplexes between pMR0360 and pJB1 showed that they contained 3.3 megadaltons of homologous sequences. Two sets of nonhomologous sequences, one a TnA sequence and the other a non-TnA sequence, took the form of insertion loops. For plasmids pMR0360 and RSF0885, previously shown to be highly related, the nonhomologous sequences took the form of a substitution loop. We concluded that all three plasmids shared major portions of their sequences but differed in discrete segments. pJB1 was the first such plasmid to have a physically and functionally intact TnA sequence.

Two hundred strains of Haemophilus ducreyi, isolated in different parts of the world, were investigated using the API-ZYM system, which included 95 different substrates. All strains produced aminopeptidase against beta-naphthylamide derivatives of L-lysine, glycine, L-arginine, L-alanine, D-L-methionine, glycyl-glycine, glycyl-L-alanine and L-leucine. All strains also produced alkaline and acid phosphatase and phosphohydrolase. Nearly all strains showed esterase activity against butyrate, valerate, caproate and caprylate, but the reactions were very weak. No glycosidase activity could be detected. Of the 47 aminopeptidase tests showing variable reactions, only the results for S-benzyl-L-cystine, L-ornithine, L-alanyl-L-phenyl-alanyl-L-proline, L-hystidyl-L-leucyl-L-histidine and L-histidyl-L-serine arylamidase obtained on strains from Asia, Africa and Europe were significantly different (p less than 0.05). On the basis of test results for L-ornithine arylamidase and L-alanyl-L-phenyl-alanyl-L-proline arylamidase, the distribution of three biovars found among the isolates of the different continents was significantly different (p less than 0.0001), whereas African strains isolated in Kenya and South Africa yielded the same enzymatic pattern. Thus, these enzymes may constitute a marker system for the epidemiological study of Haemophilus ducreyi.

A TnA-containing 6.0-megadalton plasmid (pJB1) isolated from Haemophilus ducreyi was shown to code for a beta-lactamase similar to the TEM-1-type beta-lactamase originating from the ampicillin transposon Tn2.

The susceptibility of 19 isolates of Haemophilus ducreyi from a recent chancroid outbreak and four reference strains was determined in vitro to 13 antimicrobial agents. The rabbit intradermal test for virulence was positive for all of the local isolates, but not for the reference strains. The "nonvirulent" reference strains were inhibited by lower minimum inhibitory concentrations (MICs) of most agents tested. For the virulent isolates, the range of MICs (in micrograms per milliliter) of the following were: of vancomycin, 8 to 128; of polymyxin, 32 to 128; of cloxacillin, 32 to 64; of tetracycline, 0.5 to 32; of cephalothin, 4 to 8; of doxycycline, 0.25 to 8; and of kanamycin, 1 to 8. Three strains were resistant to penicillin and ampicillin (MIC >/= 128 mug/ml), and these three strains produced beta-lactamase. The remainder were susceptible to 4 mug/ml. All strains were susceptible to rifampin (MIC

The minimal inhibitory concentration (MIC) of chloramphenicol for a clinical isolate of Haemophilus ducreyi, strain CEB-10, was 16 micrograms/ml. This strain was also resistant to tetracycline (MIC = 64 micrograms/ml) and ampicillin. The presence of a chloramphenicol acetyltransferase (CAT) activity was demonstrated.

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.

Three clinical isolates of Haemophilus ducreyi, representing at least two subtypes, were shown to be resistant to streptomycin and kanamycin. They also produced a beta-lactamase and chloramphenicol acetyltransferase and were resistant to tetracycline. In the three strains the resistance to both aminoglycoside antibiotics was encoded by a plasmid of ca. 4.7 kilobases which apparently did not carry ampicillin, chloramphenicol, or tetracycline resistance genes, as determined after transfer to Escherichia coli by transformation. Resistance to streptomycin and kanamycin was due to the presence of two aminoglycoside phosphotransferases (APH). The enzyme modifying kanamycin was a 3',5"-APH of type I [APH(3',5")-I], as inferred from its substrate profile and immunological cross-reactivity with the APH(3',5")-I encoded by the transposable element Tn903. However, the APH(3',5")-I gene in H. ducreyi did not appear to be carried by Tn903.