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Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. The triple mutant was significantly more susceptible to both α- and β-defensins; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin LL-37 may be more important than defensin resistance to H. ducreyi pathogenesis.

Cytolethal distending toxins (CDTs) are heterotrimeric protein exotoxins produced by a diverse array of Gram-negative pathogens. The enzymatic subunit, CdtB, possesses DNase and phosphatidylinositol 3-4-5 trisphosphate phosphatase activities that induce host cell cycle arrest, cellular distension and apoptosis. To exert cyclomodulatory and cytotoxic effects CDTs must be taken up from the host cell surface and transported intracellularly in a manner that ultimately results in localization of CdtB to the nucleus. However, the molecular details and mechanism by which CDTs bind to host cells and exploit existing uptake and transport pathways to gain access to the nucleus are poorly understood. Here, we report that CdtA and CdtC subunits of CDTs derived from Haemophilus ducreyi (Hd-CDT) and enteropathogenic E. coli (Ec-CDT) are independently sufficient to support intoxication by their respective CdtB subunits. CdtA supported CdtB-mediated killing of T-cells and epithelial cells that was nearly as efficient as that observed with holotoxin. In contrast, the efficiency by which CdtC supported intoxication was dependent on the source of the toxin as well as the target cell type. Further, CdtC was found to alter the subcellular trafficking of Ec-CDT as determined by sensitivity to EGA, an inhibitor of endosomal trafficking, colocalization with markers of early and late endosomes, and the kinetics of DNA damage response. Finally, host cellular cholesterol was found to influence sensitivity to intoxication mediated by Ec-CdtA, revealing a role for cholesterol or cholesterol-rich membrane domains in intoxication mediated by this subunit. In summary, data presented here support a model in which CdtA and CdtC each bind distinct receptors on host cell surfaces that direct alternate intracellular uptake and/or trafficking pathways.

The Cytolethal Distending Toxin (CDT) is produced by many pathogenic bacteria. CDT is known to induce genomic DNA damage to host eukaryotic cells through its catalytic subunit, CdtB. CdtB is structurally homologous to DNase I and has a nuclease activity, dependent on several key residues. Yet some differences between various CdtB subunit activities, and discrepancies between biochemical and cellular data, have been observed. To better characterise the role of CdtB in the induction of DNA damage, we affinity-purified wild-type and mutants of CdtB, issued from E. coli and H. ducreyi, under native and denaturing conditions. We then compared their nuclease activity by a classic in vitro assay using plasmid DNA, and two different eukaryotic assays-the first assay where host cells were transfected with a plasmid encoding CdtB, the second assay where host cells were directly transfected with purified CdtB. We show here that in vitro nuclease activities are difficult to quantify, whereas CdtB activities in host cells can be easily interpreted and confirmed the loss of function of the catalytic mutant. Our results highlight the importance of performing multiple assays while studying the effects of bacterial genotoxins, and indicate that the classic in vitro assay should be complemented with cellular assays.

Cytolethal distending toxins (CDTs) are proteins produced and secreted by facultative pathogenic strains of Gram‐negative bacteria with potentially genotoxic effects. Mammalian cells exposed to CDTs undergo cell type‐dependent cell‐cycle arrest or apoptosis; however, the cell fate responses to such intoxication are mechanistically incompletely understood. Here we show that both normal and cancer cells (BJ, IMR‐90 and WI‐38 fibroblasts, HeLa and U2‐OS cell lines) that survive the acute phase of intoxication by Haemophilus ducreyi CDT possess the hallmarks of cellular senescence. This characteristic phenotype included persistently activated DNA damage signalling (detected as 53BP1/γH2AX+ foci), enhanced senescence‐associated β‐galactosidase activity, expansion of promyelocytic leukaemia nuclear compartments and induced expression of several cytokines (especially interleukins IL‐6, IL‐8 and IL‐24), overall features shared by cells undergoing replicative or premature cellular senescence. We conclude that analogous to oncogenic, oxidative and replicative stresses, bacterial intoxication represents another pathophysiological stimulus that induces premature senescence, an intrinsic cellular response that may mechanistically underlie the ‘distended’ morphology evoked by CDTs. Finally, the activation of the two anticancer barriers, apoptosis and cellular senescence, together with evidence of chromosomal aberrations (micronucleation) reported here, support the emerging genotoxic and potentially oncogenic effects of this group of bacterial toxins, and warrant further investigation of their role(s) in human disease.

Haemophilus ducreyi contains 3 TonB-dependent receptors: the hemoglobin receptor HgbA, which is required for virulence in humans; the heme receptor TdhA; and an uncharacterized conserved hypothetical protein TdX (HD0646). A double tdX/tdhA mutant (FX527) was constructed on the background of a human-passaged variant of strain 35000 (35000HP). Six volunteers were infected with 35000HP at 3 sites on one arm and with FX527 at 3 sites on the other. The pustule formation rate was 55.6% (95% confidence interval [CI], 35.7%–75.4%) at 18 parent-strain sites and 44.4% (95% CI, 15.0%–73.9%) at 18 mutant-strain sites (P=.51). Similar amounts of 35000HP and FX527 were recovered from pustules in semiquantitative culture. Thus, TdX and TdhA are not necessary for virulence, whereas HgbA is both necessary and sufficient for virulence in humans. The data suggest that hemoglobin is the sole source of heme/iron used by H. ducreyi in vivo and has implications for the potential of HgbA as a vaccine

The lipooligosaccharides (LOS) of Haemophilus ducreyi are highly sialylated, a modification that has been implicated in resistance to host defense and in virulence. In previous work, we demonstrated that H. ducreyi scavenges sialic acid from the extracellular milieu and incorporates those residues into LOS. Here we report that H. ducreyi can use unnatural sialic acids bearing elongated N-acyl groups from three to seven carbon atoms in length, resulting in outer membrane presentation of unnatural sialyl-LOS. The unnatural variant comprises ≈90% of cell surface sialosides when exogenous substrates are added to the media at micromolar concentrations, despite the availability of natural sialic acid in the growth media. Although they represent the majority of cell surface sialosides, analogs with longer N-acyl groups diminish the overall level of LOS sialylation, culminating in complete inhibition of LOS sialylation by N-octanoyl sialic acid. Thus, sialylation of H. ducreyi LOS can be modulated with respect to the structure of the terminal sialic acid residue and the extent to which the LOS acceptor is modified by supplying the bacteria with various sialic acid analogs.

Cytolethal distending toxins (CDTs) are unique among bacterial protein toxins in their ability to cause DNA damage, due to their functional similarity to the mammalian deoxyribonuclease I (DNase I). The cellular response to CDT intoxication is characterised by activation of DNA damage-induced checkpoint responses, and the final outcome is cell type dependent. Cells of epithelial origin and normal keratinocytes are arrested in the G2 phase of the cell cycle, normal fibroblasts are also arrested in G1, while B cells die of apoptosis. CDTs are encoded by three linked genes ( cdtA, cdtB and cdtC), and CdtB is the toxin subunit which possesses the DNase I-like activity. All the three genes have to be present in the bacterium in order to produce an active cytotoxin, however cytotoxic Haemophilus ducreyi CDT, purified from a CdtABC recombinant E. coli strain, contains the CdtB and CdtC subunits, suggesting that they constitute the holotoxin and that CdtC may be required for CdtB internalization. The role of the CdtA subunit is currently unknown, but it might modify and therefore activate CdtC. This review will focus on the cellular responses induced by CDTs in mammalian cells.

Objective: To screen a collection of isolates of Haemophilus ducreyi for expression of the cytolethal distending toxin (CDT). Methods: 45 clinical isolates of H ducreyi were screened for cytotoxic activity by examining the effect of culture supernatants on Hela cells. Expression was confirmed using immunoblotting with CDT specific monoclonal antibodies and the presence of the cdt genes determined by amplification of the cdt genes in a multiplex polymerase chain assay. Results: Of the 45 clinical isolates, six isolates from differing geographical origins did not demonstrate cytotoxic activity. Expression of CDT was also not detected in these six isolates using immunoblotting and the genes cdtA, cdtB, and cdtC were not amplified using PCR. The remaining isolates demonstrated cytotoxic activity, expressed the CDT proteins, and the presence of the cdt genes was confirmed. Conclusions: CDT is considered a virulence factor of H ducreyi but was found to be absent in 13% of isolates from different geographical origins.

In a study of 13 local and four reference strains of Haemophilus ducreyi all grew well on a selective medium consisting of Bacto proteose No 3 agar (Difco), soluble starch, IsoVitalex, human blood, and vancomycin. All the strains reduced nitrate, were alkaline-phosphatase-positive, and (with one exception) used glucose, fructose, and mannose, beta-lactamase was produced by 12 local strains. Erythromycin was the the most effective antibiotic tested, followed by streptomycin, co-trimoxazole, and spectinomycin.

The tripartite cytolethal distending toxin (CDT) induces cell cycle arrest and apoptosis in eukaryotic cells 1 , 2 . 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 3 , 4 , 5 , 6 , 7 . 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.