Acid-induced disassembly of the Haemophilus ducreyi cytolethal distending toxin.

The cytolethal distending toxins (CDTs) produced by many Gram-negative pathogens are tripartite genotoxins with a single catalytic subunit (CdtB) and two cell-binding subunits (CdtA + CdtC). CDT moves by vesicle carriers from the cell surface to the endosomes and through the Golgi apparatus en route to the endoplasmic reticulum (ER). CdtA dissociates from the rest of the toxin before reaching the Golgi apparatus, and CdtB separates from CdtC in the ER. The free CdtB subunit, which is only active after holotoxin disassembly, then crosses the ER membrane and enters the nucleus where it generates DNA breaks. We hypothesized that the acidified lumen of the endosomes is responsible for separating CdtA from the CdtB/CdtC heterodimer. To test this prediction, possible acid-induced disruptions to the CDT holotoxin were monitored by size exclusion chromatography and surface plasmon resonance. We found that CDT could not efficiently assemble from its individual subunits at the early endosome pH of 6.3. Partial disassembly of the CDT holotoxin also occurred at pH 6.3, with complete separation of CdtA from an intact CdtB/CdtC heterodimer occurring at both pH 6.0 and the late endosome pH of 5.6. Acidification caused the precipitation of CdtA at pH 6.5 and below, but neither CdtB nor CdtC were affected by a pH as low as 5.2. Circular dichroism further showed that the individual CdtB subunit adopts a different secondary structure as compared to its structure in the holotoxin. We conclude the first stage of CDT disassembly occurs in the early endosomes, where an acid-induced alteration to CdtA releases it from the CdtB/CdtC heterodimer.

Genes Differentially Expressed by Haemophilus ducreyi during Anaerobic Growth Significantly Overlap Those Differentially Expressed during Experimental Infection of Human Volunteers.

Haemophilus ducreyi causes cutaneous ulcers in children and the genital ulcer disease chancroid in adults. In humans, H. ducreyi is found in the anaerobic environment of an abscess; previous studies comparing bacterial gene expression levels in pustules with the inocula (∼4-h aerobic mid-log-phase cultures) identified several upregulated differentially expressed genes (DEGs) that are associated with anaerobic metabolism. To determine how H. ducreyi alters its gene expression in response to anaerobiosis, we performed RNA sequencing (RNA-seq) on both aerobic and anaerobic broth cultures harvested after 4, 8, and 18 h of growth. Principal-coordinate analysis (PCoA) plots showed that anaerobic growth resulted in distinct transcriptional profiles compared to aerobic growth. During anaerobic growth, early-time-point comparisons (4 versus 8 h) identified few DEGs at a 2-fold change in expression and a false discovery rate (FDR) of <0.01. By 18 h, we observed 18 upregulated and 16 downregulated DEGs. DEGs involved in purine metabolism, the uptake and use of alternative carbon sources, toxin production, nitrate reduction, glycine metabolism, and tetrahydrofolate synthesis were upregulated; DEGs involved in electron transport, thiamine biosynthesis, DNA recombination, peptidoglycan synthesis, and riboflavin synthesis or modification were downregulated. To examine whether transcriptional changes that occur during anaerobiosis overlap those that occur during infection of human volunteers, we compared the overlap of DEGs obtained from 4 h of aerobic growth to 18 h of anaerobic growth to those found between the inocula and pustules in previous studies; the DEGs significantly overlapped. Thus, a major component of H. ducreyi gene regulation in vivo involves adaptation to anaerobiosis. IMPORTANCE In humans, H. ducreyi resides in the anaerobic environment of an abscess and appears to upregulate genes involved in anaerobic metabolism. How anaerobiosis alone affects gene transcription in H. ducreyi is unknown. Using RNA-seq, we investigated how anaerobiosis affects gene transcription over time compared to aerobic growth. Our results suggest that a substantial component of H. ducreyi gene regulation in vivo overlaps the organism's response to anaerobiosis in vitro. Our data identify potential therapeutic targets that could be inhibited during in vivo growth.

Cell transfection of purified cytolethal distending toxin B subunits allows comparing their nuclease activity while plasmid degradation assay does not.

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.

Molecular characterization of the interaction of sialic acid with the periplasmic binding protein from Haemophilus ducreyi.

The primary role of bacterial periplasmic binding proteins is sequestration of essential metabolites present at a low concentration in the periplasm and making them available for active transporters that transfer these ligands into the bacterial cell. The periplasmic binding proteins (SiaPs) from the tripartite ATP-independent periplasmic (TRAP) transport system that transports mammalian host-derived sialic acids have been well studied from different pathogenic bacteria, including Haemophilus influenzae, Fusobacterium nucleatum, Pasteurella multocida, and Vibrio cholerae SiaPs bind the sialic acid N-acetylneuraminic acid (Neu5Ac) with nanomolar affinity by forming electrostatic and hydrogen-bonding interactions. Here, we report the crystal structure of a periplasmic binding protein (SatA) of the ATP-binding cassette (ABC) transport system from the pathogenic bacterium Haemophilus ducreyi The structure of Hd-SatA in the native form and sialic acid-bound forms (with Neu5Ac and N-glycolylneuraminic acid (Neu5Gc)), determined to 2.2, 1.5, and 2.5 Å resolutions, respectively, revealed a ligand-binding site that is very different from those of the SiaPs of the TRAP transport system. A structural comparison along with thermodynamic studies suggested that similar affinities are achieved in the two classes of proteins through distinct mechanisms, one enthalpically driven and the other entropically driven. In summary, our structural and thermodynamic characterization of Hd-SatA reveals that it binds sialic acids with nanomolar affinity and that this binding is an entropically driven process. This information is important for future structure-based drug design against this pathogen and related bacteria.

Putative vaccine candidates and drug targets identified by reverse vaccinology and subtractive genomics approaches to control Haemophilus ducreyi, the causative agent of chancroid.

Chancroid is a sexually transmitted infection (STI) caused by the Gram-negative bacterium Haemophilus ducreyi The control of chancroid is difficult and the only current available treatment is antibiotic therapy; however, antibiotic resistance has been reported in endemic areas. Owing to recent outbreaks of STIs worldwide, it is important to keep searching for new treatment strategies and preventive measures. Here, we applied reverse vaccinology and subtractive genomic approaches for the in silico prediction of potential vaccine and drug targets against 28 strains of H. ducreyi We identified 847 non-host homologous proteins, being 332 exposed/secreted/membrane and 515 cytoplasmic proteins. We also checked their essentiality, functionality and virulence. Altogether, we predicted 13 candidate vaccine targets and three drug targets, where two vaccines (A01_1275, ABC transporter substrate-binding protein; and A01_0690, Probable transmembrane protein) and three drug targets (A01_0698, Purine nucleoside phosphorylase; A01_0702, Transcription termination factor; and A01_0677, Fructose-bisphosphate aldolase class II) are harboured by pathogenicity islands. Finally, we applied a molecular docking approach to analyse each drug target and selected ZINC77257029, ZINC43552589 and ZINC67912117 as promising molecules with favourable interactions with the target active site residues. Altogether, the targets identified here may be used in future strategies to control chancroid worldwide.

Haemophilus ducreyi Seeks Alternative Carbon Sources and Adapts to Nutrient Stress and Anaerobiosis during Experimental Infection of Human Volunteers.

Haemophilus ducreyi causes the sexually transmitted disease chancroid in adults and cutaneous ulcers in children. In humans, H. ducreyi resides in an abscess and must adapt to a variety of stresses. Previous studies (D. Gangaiah, M. Labandeira-Rey, X. Zhang, K. R. Fortney, S. Ellinger, B. Zwickl, B. Baker, Y. Liu, D. M. Janowicz, B. P. Katz, C. A. Brautigam, R. S. MunsonJr, E. J. Hansen, and S. M. Spinola, mBio 5:e01081-13, 2014, http://dx.doi.org/10.1128/mBio.01081-13) suggested that H. ducreyi encounters growth conditions in human lesions resembling those found in stationary phase. However, how H. ducreyi transcriptionally responds to stress during human infection is unknown. Here, we determined the H. ducreyi transcriptome in biopsy specimens of human lesions and compared it to the transcriptomes of bacteria grown to mid-log, transition, and stationary phases. Multidimensional scaling showed that the in vivo transcriptome is distinct from those of in vitro growth. Compared to the inoculum (mid-log-phase bacteria), H. ducreyi harvested from pustules differentially expressed ∼93 genes, of which 62 were upregulated. The upregulated genes encode homologs of proteins involved in nutrient transport, alternative carbon pathways (l-ascorbate utilization and metabolism), growth arrest response, heat shock response, DNA recombination, and anaerobiosis. H. ducreyi upregulated few genes (hgbA, flp-tad, and lspB-lspA2) encoding virulence determinants required for human infection. Most genes regulated by CpxRA, RpoE, Hfq, (p)ppGpp, and DksA, which control the expression of virulence determinants and adaptation to a variety of stresses, were not differentially expressed in vivo, suggesting that these systems are cycling on and off during infection. Taken together, these data suggest that the in vivo transcriptome is distinct from those of in vitro growth and that adaptation to nutrient stress and anaerobiosis is crucial for H. ducreyi survival in humans.

Distinct Roles for CdtA and CdtC during Intoxication by Cytolethal Distending Toxins.

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.

DksA and (p)ppGpp have unique and overlapping contributions to Haemophilus ducreyi pathogenesis in humans.

The (p)ppGpp-mediated stringent response is important for bacterial survival in nutrient limiting conditions. For maximal effect, (p)ppGpp interacts with the cofactor DksA, which stabilizes (p)ppGpp's interaction with RNA polymerase. We previously demonstrated that (p)ppGpp was required for the virulence of Haemophilus ducreyi in humans. Here, we constructed an H. ducreyi dksA mutant and showed it was also partially attenuated for pustule formation in human volunteers. To understand the roles of (p)ppGpp and DksA in gene regulation in H. ducreyi, we defined genes potentially altered by (p)ppGpp and DksA deficiency using transcriptome sequencing (RNA-seq). In bacteria collected at stationary phase, lack of (p)ppGpp and DksA altered expression of 28% and 17% of H. ducreyi open reading frames, respectively, including genes involved in transcription, translation, and metabolism. There was significant overlap in genes differentially expressed in the (p)ppGpp mutant relative to the dksA mutant. Loss of (p)ppGpp or DksA resulted in the dysregulation of several known virulence determinants. Deletion of dksA downregulated lspB and rendered the organism less resistant to phagocytosis and increased its sensitivity to oxidative stress. Both mutants had reduced ability to attach to human foreskin fibroblasts; the defect correlated with reduced expression of the Flp adhesin proteins in the (p)ppGpp mutant but not in the dksA mutant, suggesting that DksA regulates the expression of an unknown cofactor(s) required for Flp-mediated adherence. We conclude that both (p)ppGpp and DksA serve as major regulators of H. ducreyi gene expression in stationary phase and have both overlapping and unique contributions to pathogenesis.

Phosphoethanolamine Transferase LptA in Haemophilus ducreyi Modifies Lipid A and Contributes to Human Defensin Resistance In Vitro.

Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, ß-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 ß-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.

Development and validation of a high-throughput cell-based screen to identify activators of a bacterial two-component signal transduction system.

CpxRA is a two-component signal transduction system (2CSTS) found in many drug-resistant Gram-negative bacteria. In response to periplasmic stress, CpxA autophosphorylates and donates a phosphoryl group to its cognate response regulator, CpxR. Phosphorylated CpxR (CpxR-P) upregulates genes involved in membrane repair and downregulates multiple genes that encode virulence factors, which are trafficked across the cell membrane. Mutants that constitutively activate CpxRA in Salmonella enterica serovar Typhimurium and Haemophilus ducreyi are avirulent in mice and humans, respectively. Thus, the activation of CpxRA has high potential as a novel antimicrobial/antivirulence strategy. Using a series of Escherichia coli strains containing a CpxR-P-responsive lacZ reporter and deletions in genes encoding CpxRA system components, we developed and validated a novel cell-based high-throughput screen (HTS) for CpxRA activators. A screen of 36,000 compounds yielded one hit compound that increased reporter activity in wild-type cells. This is the first report of a compound that activates, rather than inhibits, a 2CSTS. The activity profile of the compound against CpxRA pathway mutants in the presence of glucose suggested that the compound inhibits CpxA phosphatase activity. We confirmed that the compound induced the accumulation of CpxR-P in treated cells. Although the hit compound contained a nitro group, a derivative lacking this group retained activity in serum and had lower cytotoxicity than that of the initial hit. This HTS is amenable for the screening of larger libraries to find compounds that activate CpxRA by other mechanisms, and it could be adapted to find activators of other two-component systems.

Trimeric autotransporter DsrA is a major mediator of fibrinogen binding in Haemophilus ducreyi.

Haemophilus ducreyi is the etiologic agent of the sexually transmitted genital ulcer disease chancroid. In both natural and experimental chancroid, H. ducreyi colocalizes with fibrin at the base of the ulcer. Fibrin is obtained by cleavage of the serum glycoprotein fibrinogen (Fg) by thrombin to initiate formation of the blood clot. Fg binding proteins are critical virulence factors in medically important Gram-positive bacteria. H. ducreyi has previously been shown to bind Fg in an agglutination assay, and the H. ducreyi Fg binding protein FgbA was identified in ligand blotting with denatured proteins. To better characterize the interaction of H. ducreyi with Fg, we examined Fg binding to intact, viable H. ducreyi bacteria and identified a novel Fg binding protein. H. ducreyi bound unlabeled Fg in a dose-dependent manner, as measured by two different methods. In ligand blotting with total denatured cellular proteins, digoxigenin (DIG)-Fg bound only two H. ducreyi proteins, the trimeric autotransporter DsrA and the lectin DltA; however, only the isogenic dsrA mutant had significantly less cell-associated Fg than parental strains in Fg binding assays with intact bacteria. Furthermore, expression of DsrA, but not DltA or an empty vector, rendered the non-Fg-binding H. influenzae strain Rd capable of binding Fg. A 13-amino-acid sequence in the C-terminal section of the passenger domain of DsrA appears to be involved in Fg binding by H. ducreyi. Taken together, these data suggest that the trimeric autotransporter DsrA is a major determinant of Fg binding at the surface of H. ducreyi.

Mutational analysis of hemoglobin binding and heme utilization by a bacterial hemoglobin receptor.

Iron is an essential nutrient for most living organisms. To acquire iron from their environment, Gram-negative bacteria use TonB-dependent transporters that bind host proteins at the bacterial surface and transport iron or heme to the periplasm via the Ton machinery. TonB-dependent transporters are barrel-shaped outer membrane proteins with 22 transmembrane domains, 11 surface-exposed loops, and a plug domain that occludes the pore. To identify key residues of TonB-dependent transporters involved in hemoglobin binding and heme transport and thereby locate putative protective epitopes, the hemoglobin receptor of Haemophilus ducreyi HgbA was used as a model of iron/heme acquisition from hemoglobin. Although all extracellular loops of HgbA are required by H. ducreyi to use hemoglobin as a source of iron/heme, we previously demonstrated that hemoglobin binding by HgbA only involves loops 5 and 7. Using deletion, substitution, and site-directed mutagenesis, we were able to differentiate hemoglobin binding and heme acquisition by HgbA. Deletion or substitution of the GYEAYNRQWWA region of loop 5 and alanine replacement of selected histidines affected hemoglobin binding by HgbA. Conversely, mutation of the phenylalanine in the loop 7 FRAP domain or substitution of the NRQWWA motif of loop 5 significantly abrogated utilization of heme from hemoglobin. Our findings show that hemoglobin binding and heme utilization by a bacterial hemoglobin receptor involve specific motifs of HgbA.

Cellular interactions of the cytolethal distending toxins from Escherichia coli and Haemophilus ducreyi.

The cytolethal distending toxins (CDTs) compose a subclass of intracellularly acting genotoxins produced by many Gram-negative pathogenic bacteria that disrupt the normal progression of the eukaryotic cell cycle. Here, the intoxication mechanisms of CDTs from Escherichia coli (Ec-CDT) and Haemophilus ducreyi (Hd-CDT), which share limited amino acid sequence homology, were directly compared. Ec-CDT and Hd-CDT shared comparable in vitro DNase activities of the CdtB subunits, saturable cell surface binding with comparable affinities, and the requirement for an intact Golgi complex to induce cell cycle arrest. In contrast, disruption of endosome acidification blocked Hd-CDT-mediated cell cycle arrest and toxin transport to the endoplasmic reticulum and nucleus, while having no effects on Ec-CDT. Phosphorylation of the histone protein H2AX, as well as nuclear localization, was inhibited for Hd-CdtB, but not Ec-CdtB, in cells expressing dominant negative Rab7 (T22N), suggesting that Hd-CDT, but not Ec-CDT, is trafficked through late endosomal vesicles. In support of this idea, significantly more Hd-CdtB than Ec-CdtB co-localized with Rab9, which is enriched in late endosomal compartments. Competitive binding studies suggested that Ec-CDT and Hd-CDT bind to discrete cell surface determinants. These results suggest that Ec-CDT and Hd-CDT are transported within cells by distinct pathways, possibly mediated by their interaction with different receptors at the cell surface.

Carbon storage regulator A contributes to the virulence of Haemophilus ducreyi in humans by multiple mechanisms.

The carbon storage regulator A (CsrA) controls a wide variety of bacterial processes, including metabolism, adherence, stress responses, and virulence. Haemophilus ducreyi, the causative agent of chancroid, harbors a homolog of csrA. Here, we generated an unmarked, in-frame deletion mutant of csrA to assess its contribution to H. ducreyi pathogenesis. In human inoculation experiments, the csrA mutant was partially attenuated for pustule formation compared to its parent. Deletion of csrA resulted in decreased adherence of H. ducreyi to human foreskin fibroblasts (HFF); Flp1 and Flp2, the determinants of H. ducreyi adherence to HFF cells, were downregulated in the csrA mutant. Compared to its parent, the csrA mutant had a significantly reduced ability to tolerate oxidative stress and heat shock. The enhanced sensitivity of the mutant to oxidative stress was more pronounced in bacteria grown to stationary phase compared to that in bacteria grown to mid-log phase. The csrA mutant also had a significant survival defect within human macrophages when the bacteria were grown to stationary phase but not to mid-log phase. Complementation in trans partially or fully restored the mutant phenotypes. These data suggest that CsrA contributes to virulence by multiple mechanisms and that these contributions may be more profound in bacterial cell populations that are not rapidly dividing in the human host.

Sialylation of lipooligosaccharides is dispensable for the virulence of Haemophilus ducreyi in humans.

Sialylated glycoconjugates on the surfaces of mammalian cells play important roles in intercellular communication and self-recognition. The sialic acid preferentially expressed in human tissues is N-acetylneuraminic acid (Neu5Ac). In a process called molecular mimicry, many bacterial pathogens decorate their cell surface glycolipids with Neu5Ac. Incorporation of Neu5Ac into bacterial glycolipids promotes bacterial interactions with host cell receptors called Siglecs. These interactions affect bacterial adherence, resistance to serum killing and phagocytosis, and innate immune responses. Haemophilus ducreyi, the etiologic agent of chancroid, expresses lipooligosaccharides (LOS) that are highly sialylated. However, an H. ducreyi sialyltransferase (lst) mutant, whose LOS contain reduced levels of Neu5Ac, is fully virulent in human volunteers. Recently, a second sialyltransferase gene (Hd0053) was discovered in H. ducreyi, raising the possibility that Hd0053 compensated for the loss of lst during human infection. CMP-Neu5Ac is the obligate nucleotide sugar donor for all bacterial sialyltransferases; LOS derived from an H. ducreyi CMP-Neu5Ac synthetase (neuA) mutant has no detectable Neu5Ac. Here, we compared an H. ducreyi neuA mutant to its wild-type parent in several models of pathogenesis. In human inoculation experiments, the neuA mutant formed papules and pustules at rates that were no different than those of its parent. When grown in media with and without Neu5Ac supplementation, the neuA mutant and its parent had similar phenotypes in bactericidal, macrophage uptake, and dendritic cell activation assays. Although we cannot preclude a contribution of LOS sialylation to ulcerative disease, these data strongly suggest that sialylation of LOS is dispensable for H. ducreyi pathogenesis in humans.

Genetics and molecular specificity of sialylation of Histophilus somni lipooligosaccharide (LOS) and the effect of LOS sialylation on Toll-like receptor-4 signaling.

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-ß-[1-4]-GlcNAc-R), which is expressed on strain 2336, whereas HssT-II preferentially sialylated lacto-N-biose (LNB, Gal-ß-[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 ß-(1-3)-(LNB) to ß-(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.

Characterization of the CpxRA regulon in Haemophilus ducreyi.

The Haemophilus ducreyi 35000HP genome encodes a homolog of the CpxRA two-component cell envelope stress response system originally characterized in Escherichia coli. CpxR, the cytoplasmic response regulator, was shown previously to be involved in repression of the expression of the lspB-lspA2 operon (M. Labandeira-Rey, J. R. Mock, and E. J. Hansen, Infect. Immun. 77:3402-3411, 2009). In the present study, the H. ducreyi CpxR and CpxA proteins were shown to closely resemble those of other well-studied bacterial species. A cpxA deletion mutant and a CpxR-overexpressing strain were used to explore the extent of the CpxRA regulon. DNA microarray and real-time reverse transcriptase (RT) PCR analyses indicated several potential regulatory targets for the H. ducreyi CpxRA two-component regulatory system. Electrophoretic mobility shift assays (EMSAs) were used to prove that H. ducreyi CpxR interacted with the promoter regions of genes encoding both known and putative virulence factors of H. ducreyi, including the lspB-lspA2 operon, the flp operon, and dsrA. Interestingly, the use of EMSAs also indicated that H. ducreyi CpxR did not bind to the promoter regions of several genes predicted to encode factors involved in the cell envelope stress response. Taken together, these data suggest that the CpxRA system in H. ducreyi, in contrast to that in E. coli, may be involved primarily in controlling expression of genes not involved in the cell envelope stress response.

Cytolethal distending toxin family members are differentially affected by alterations in host glycans and membrane cholesterol.

Cytolethal distending toxins (CDTs) are tripartite protein exotoxins produced by a diverse group of pathogenic Gram-negative bacteria. Based on their ability to induce DNA damage, cell cycle arrest, and apoptosis of cultured cells, CDTs are proposed to enhance virulence by blocking cellular division and/or directly killing epithelial and immune cells. Despite the widespread distribution of CDTs among several important human pathogens, our understanding of how these toxins interact with host cells is limited. Here we demonstrate that CDTs from Haemophilus ducreyi, Aggregatibacter actinomycetemcomitans, Escherichia coli, and Campylobacter jejuni differ in their abilities to intoxicate host cells with defined defects in host factors previously implicated in CDT binding, including glycoproteins, and glycosphingolipids. The absence of cell surface sialic acid sensitized cells to intoxication by three of the four CDTs tested. Surprisingly, fucosylated N-linked glycans and glycolipids, previously implicated in CDT-host interactions, were not required for intoxication by any of the CDTs tested. Finally, altering host-cellular cholesterol, also previously implicated in CDT binding, affected intoxication by only a subset of CDTs tested. The findings presented here provide insight into the molecular and cellular basis of CDT-host interactions.

Bacterial intoxication evokes cellular senescence with persistent DNA damage and cytokine signalling.

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/gammaH2AX(+) foci), enhanced senescence-associated beta-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.

An immunogenic, surface-exposed domain of Haemophilus ducreyi outer membrane protein HgbA is involved in hemoglobin binding.

HgbA is the sole TonB-dependent receptor for hemoglobin (Hb) acquisition of Haemophilus ducreyi. Binding of Hb to HgbA is the initial step in heme acquisition from Hb. To better understand this step, we mutagenized hgbA by deletion of each of the 11 putative surface-exposed loops and expressed each of the mutant proteins in trans in host strain H. ducreyi FX547 hgbA. All mutant proteins were expressed, exported, and detected on the surface by anti-HgbA immunoglobulin G (IgG). Deletion of sequences in loops 5 and 7 of HgbA abolished Hb binding in two different formats. In contrast, HgbA proteins containing deletions in the other nine loops retained the ability to bind Hb. None of the clones expressing mutant proteins were able to grow on plates containing low concentrations of Hb. Previously we demonstrated in a swine model of chancroid infection that an HgbA vaccine conferred complete protection from a challenge infection. Using anti-HgbA IgG from this study and the above deletion mutants, we show that loops 4, 5, and 7 of HgbA were immunogenic and surface exposed and that IgG directed against loops 4 and 5 blocked Hb binding. Furthermore, loop 6 was cleaved by protease on intact H. ducreyi, suggesting surface exposure. These data implicate a central domain of HgbA (in respect to the primary amino acid sequence) as important in Hb binding and suggest that this region of the molecule might have potential as a subunit vaccine.