A Haemophilus ducreyi strain lacking the yfeABCD iron transport system is virulent in human volunteers.

Haemophilus ducreyi causes the genital ulcer disease chancroid and painful cutaneous ulcers in children who live in the tropics. To acquire heme from the host, H. ducreyi expresses a TonB-dependent hemoglobin receptor, HgbA, which is necessary and sufficient for H. ducreyi to progress to the pustular stage of disease in a controlled human infection model. HgbA transports hemoglobin across the outer membrane; how heme is transported across the cytoplasmic membrane is unclear. In previous studies, transcripts encoding the YfeABCD heme transporter were upregulated in experimental lesions caused by H. ducreyi in human volunteers, suggesting the latter may have a role in virulence. Here we constructed a double deletion mutant, 35000HPΔyfeABΔyfeCD, which exhibited growth defects relative to its parent 35000HP in media containing human hemoglobin as an iron source. Five human volunteers were inoculated at three sites on the skin overlying the deltoid with each strain. The results of the trial showed that papules formed at 100% (95% CI, 71.5, 100) at both 35000HP and 35000HPΔyfeABΔyfeCD-inoculated sites (P = 1.0). Pustules formed at 60% (95% CI, 25.9, 94.1) at parent-inoculated sites and 53% (95% CI, 18.3, 88.4) at mutant-inoculated sites (P = 0.79). Thus, the ABC transporter encoded by yfeAB and yfeCD was dispensable for H. ducreyi virulence in humans. In the absence of YfeABCD, H. ducreyi likely utilizes other periplasmic binding proteins and ABC-transporters such as HbpA, SapABCDF, and DppBCDF to shuttle heme from the periplasm into the cytoplasm, underscoring the importance of redundancy of such systems in gram-negative pathogens.

Interactions of the Skin Pathogen Haemophilus ducreyi With the Human Host.

The obligate human pathogen Haemophilus ducreyi causes both cutaneous ulcers in children and sexually transmitted genital ulcers (chancroid) in adults. Pathogenesis is dependent on avoiding phagocytosis and exploiting the suppurative granuloma-like niche, which contains a myriad of innate immune cells and memory T cells. Despite this immune infiltrate, long-lived immune protection does not develop against repeated H. ducreyi infections-even with the same strain. Most of what we know about infectious skin diseases comes from naturally occurring infections and/or animal models; however, for H. ducreyi, this information comes from an experimental model of infection in human volunteers that was developed nearly three decades ago. The model mirrors the progression of natural disease and serves as a valuable tool to determine the composition of the immune cell infiltrate early in disease and to identify host and bacterial factors that are required for the establishment of infection and disease progression. Most recently, holistic investigation of the experimentally infected skin microenvironment using multiple "omics" techniques has revealed that non-canonical bacterial virulence factors, such as genes involved in central metabolism, may be relevant to disease progression. Thus, the immune system not only defends the host against H. ducreyi, but also dictates the nutrient availability for the invading bacteria, which must adapt their gene expression to exploit the inflammatory metabolic niche. These findings have broadened our view of the host-pathogen interaction network from considering only classical, effector-based virulence paradigms to include adaptations to the metabolic environment. How both host and bacterial factors interact to determine infection outcome is a current focus in the field. Here, we review what we have learned from experimental H. ducreyi infection about host-pathogen interactions, make comparisons to what is known for other skin pathogens, and discuss how novel technologies will deepen our understanding of this infection.

Determination of an Interaction Network between an Extracellular Bacterial Pathogen and the Human Host.

A major gap in understanding infectious diseases is the lack of information about molecular interaction networks between pathogens and the human host. Haemophilus ducreyi causes the genital ulcer disease chancroid in adults and is a leading cause of cutaneous ulcers in children in the tropics. We developed a model in which human volunteers are infected on the upper arm with H. ducreyi until they develop pustules. To define the H. ducreyi and human interactome, we determined bacterial and host transcriptomic and host metabolomic changes in pustules. We found that in vivoH. ducreyi transcripts were distinct from those in the inocula, as were host transcripts in pustule and wounded control sites. Many of the upregulated H. ducreyi genes were found to be involved in ascorbic acid and anaerobic metabolism and inorganic ion/nutrient transport. The top 20 significantly expressed human pathways showed that all were involved in immune responses. We generated a bipartite network for interactions between host and bacterial gene transcription; multiple positively correlated networks contained H. ducreyi genes involved in anaerobic metabolism and host genes involved with the immune response. Metabolomic studies showed that pustule and wounded samples had different metabolite compositions; the top ion pathway involved ascorbate and aldarate metabolism, which correlated with the H. ducreyi transcriptional response and upregulation of host genes involved in ascorbic acid recycling. These data show that an interactome exists between H. ducreyi and the human host and suggest that H. ducreyi exploits the metabolic niche created by the host immune response.IMPORTANCE Dual RNA sequencing (RNA-seq) offers the promise of determining an interactome at a transcriptional level between a bacterium and the host but has yet to be done on any bacterial infection in human tissue. We performed dual RNA-seq and metabolomics analyses on wounded and infected sites following experimental infection of the arm with H. ducreyi Our results suggest that H. ducreyi survives in an abscess by utilizing l-ascorbate as an alternative carbon source, possibly taking advantage of host ascorbic acid recycling, and that H. ducreyi also adapts by upregulating genes involved in anaerobic metabolism and inorganic ion and nutrient transport. To our knowledge, this is the first description of an interaction network between a bacterium and the human host at a site of infection.

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.

Epidemiology of Haemophilus ducreyi Infections.

The global epidemiology of Haemophilus ducreyi infections is poorly documented because of difficulties in confirming microbiological diagnoses. We evaluated published data on the proportion of genital and nongenital skin ulcers caused by H. ducreyi before and after introduction of syndromic management for genital ulcer disease (GUD). Before 2000, the proportion of GUD caused by H. ducreyi ranged from 0.0% to 69.0% (35 studies in 25 countries). After 2000, the proportion ranged from 0.0% to 15.0% (14 studies in 13 countries). In contrast, H. ducreyi has been recently identified as a causative agent of skin ulcers in children in the tropical regions; proportions ranged from 9.0% to 60.0% (6 studies in 4 countries). We conclude that, although there has been a sustained reduction in the proportion of GUD caused by H. ducreyi, this bacterium is increasingly recognized as a major cause of nongenital cutaneous ulcers.

Haemophilus ducreyi: from sexually transmitted infection to skin ulcer pathogen.

PURPOSE OF REVIEW: This article provides an overview of the biology, epidemiology, clinical features, diagnostic tests, and treatment of Haemophilus ducreyi infection, with special reference to the decline of chancroid and the recent emergence of H. ducreyi as a pathogen responsible for chronic limb ulceration clinically similar to yaws. RECENT FINDINGS: Chancroid has declined in importance as a sexually transmitted infection in most countries where it was previously endemic. Chancroid may be caused by either class I or class II H. ducreyi isolates; these two classes diverged from each other approximately 1.95 million years ago. H. ducreyi has recently emerged as a cause of chronic skin ulceration in the Pacific region and Africa. Based on sequencing of whole genomes and defined genetic loci, it appears that the cutaneous H. ducreyi strains diverged from the class I genital strains relatively recently. SUMMARY: H. ducreyi should be considered as a major cause of chronic limb ulceration in both adults and children and appropriate molecular diagnostic assays are required to determine ulcer aetiology. The high prevalence of H. ducreyi-related cutaneous ulceration in yaws-endemic countries has challenged the validity of observational surveys to monitor the effectiveness of the WHO's yaws eradication campaign.

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.

Cytolethal distending toxins require components of the ER-associated degradation pathway for host cell entry.

Intracellular acting protein exotoxins produced by bacteria and plants are important molecular determinants that drive numerous human diseases. A subset of these toxins, the cytolethal distending toxins (CDTs), are encoded by several Gram-negative pathogens and have been proposed to enhance virulence by allowing evasion of the immune system. CDTs are trafficked in a retrograde manner from the cell surface through the Golgi apparatus and into the endoplasmic reticulum (ER) before ultimately reaching the host cell nucleus. However, the mechanism by which CDTs exit the ER is not known. Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs. Complementation of Derl2-deficient cells with Derl2:Derl1 chimeras identified two previously uncharacterized functional domains in Derl2, the N-terminal 88 amino acids and the second ER-luminal loop, as required for intoxication by the CDT encoded by Haemophilus ducreyi (Hd-CDT). In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication. Interestingly, this previously undescribed mechanism is shared with the plant toxin ricin. These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins.

A (p)ppGpp-null mutant of Haemophilus ducreyi is partially attenuated in humans due to multiple conflicting phenotypes.

(p)ppGpp responds to nutrient limitation through a global change in gene regulation patterns to increase survival. The stringent response has been implicated in the virulence of several pathogenic bacterial species. Haemophilus ducreyi, the causative agent of chancroid, has homologs of both relA and spoT, which primarily synthesize and hydrolyze (p)ppGpp in Escherichia coli. We constructed relA and relA spoT deletion mutants to assess the contribution of (p)ppGpp to H. ducreyi pathogenesis. Both the relA single mutant and the relA spoT double mutant failed to synthesize (p)ppGpp, suggesting that relA is the primary synthetase of (p)ppGpp in H. ducreyi. Compared to the parent strain, the double mutant was partially attenuated for pustule formation in human volunteers. The double mutant had several phenotypes that favored attenuation, including increased sensitivity to oxidative stress. The increased sensitivity to oxidative stress could be complemented in trans. However, the double mutant also exhibited phenotypes that favored virulence. When grown to the mid-log phase, the double mutant was significantly more resistant than its parent to being taken up by human macrophages and exhibited increased transcription of lspB, which is involved in resistance to phagocytosis. Additionally, compared to the parent, the double mutant also exhibited prolonged survival in the stationary phase. In E. coli, overexpression of DksA compensates for the loss of (p)ppGpp; the H. ducreyi double mutant expressed higher transcript levels of dksA than the parent strain. These data suggest that the partial attenuation of the double mutant is likely the net result of multiple conflicting phenotypes.

Outer membrane protein P4 is not required for virulence in the human challenge model of Haemophilus ducreyi infection.

BACKGROUND: Bacterial lipoproteins often play important roles in pathogenesis and can stimulate protective immune responses. Such lipoproteins are viable vaccine candidates. Haemophilus ducreyi, which causes the sexually transmitted disease chancroid, expresses a number of lipoproteins during human infection. One such lipoprotein, OmpP4, is homologous to the outer membrane lipoprotein e (P4) of H. influenzae. In H. influenzae, e (P4) stimulates production of bactericidal and protective antibodies and contributes to pathogenesis by facilitating acquisition of the essential nutrients heme and nicotinamide adenine dinucleotide (NAD). Here, we tested the hypothesis that, like its homolog, H. ducreyi OmpP4 contributes to virulence and stimulates production of bactericidal antibodies. RESULTS: We determined that OmpP4 is broadly conserved among clinical isolates of H. ducreyi. We next constructed and characterized an isogenic ompP4 mutant, designated 35000HPompP4, in H. ducreyi strain 35000HP. To test whether OmpP4 was necessary for virulence in humans, eight healthy adults were experimentally infected. Each subject was inoculated with a fixed dose of 35000HP on one arm and three doses of 35000HPompP4 on the other arm. The overall parent and mutant pustule formation rates were 52.4% and 47.6%, respectively (P = 0.74). These results indicate that expression of OmpP4 in not necessary for H. ducreyi to initiate disease or progress to pustule formation in humans. Hyperimmune mouse serum raised against purified, recombinant OmpP4 did not promote bactericidal killing of 35000HP or phagocytosis by J774A.1 mouse macrophages in serum bactericidal and phagocytosis assays, respectively. CONCLUSIONS: Our data suggest that, unlike e (P4), H. ducreyi OmpP4 is not a suitable vaccine candidate. OmpP4 may be dispensable for virulence because of redundant mechanisms in H. ducreyi for heme acquisition and NAD utilization.

Haemophilus ducreyi Hfq contributes to virulence gene regulation as cells enter stationary phase.

UNLABELLED: To adapt to stresses encountered in stationary phase, Gram-negative bacteria utilize the alternative sigma factor RpoS. However, some species lack RpoS; thus, it is unclear how stationary-phase adaptation is regulated in these organisms. Here we defined the growth-phase-dependent transcriptomes of Haemophilus ducreyi, which lacks an RpoS homolog. Compared to mid-log-phase organisms, cells harvested from the stationary phase upregulated genes encoding several virulence determinants and a homolog of hfq. Insertional inactivation of hfq altered the expression of ~16% of the H. ducreyi genes. Importantly, there were a significant overlap and an inverse correlation in the transcript levels of genes differentially expressed in the hfq inactivation mutant relative to its parent and the genes differentially expressed in stationary phase relative to mid-log phase in the parent. Inactivation of hfq downregulated genes in the flp-tad and lspB-lspA2 operons, which encode several virulence determinants. To comply with FDA guidelines for human inoculation experiments, an unmarked hfq deletion mutant was constructed and was fully attenuated for virulence in humans. Inactivation or deletion of hfq downregulated Flp1 and impaired the ability of H. ducreyi to form microcolonies, downregulated DsrA and rendered H. ducreyi serum susceptible, and downregulated LspB and LspA2, which allow H. ducreyi to resist phagocytosis. We propose that, in the absence of an RpoS homolog, Hfq serves as a major contributor of H. ducreyi stationary-phase and virulence gene regulation. The contribution of Hfq to stationary-phase gene regulation may have broad implications for other organisms that lack an RpoS homolog. IMPORTANCE: Pathogenic bacteria encounter a wide range of stresses in their hosts, including nutrient limitation; the ability to sense and respond to such stresses is crucial for bacterial pathogens to successfully establish an infection. Gram-negative bacteria frequently utilize the alternative sigma factor RpoS to adapt to stresses and stationary phase. However, homologs of RpoS are absent in some bacterial pathogens, including Haemophilus ducreyi, which causes chancroid and facilitates the acquisition and transmission of HIV-1. Here, we provide evidence that, in the absence of an RpoS homolog, Hfq serves as a major contributor of stationary-phase gene regulation and that Hfq is required for H. ducreyi to infect humans. To our knowledge, this is the first study describing Hfq as a major contributor of stationary-phase gene regulation in bacteria and the requirement of Hfq for the virulence of a bacterial pathogen in humans.

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.

Etiologic pattern of genital ulcers in Lusaka, Zambia: has chancroid been eliminated?

BACKGROUND: Genital ulcers are a public health problem in developing countries. The World Health Organization recommends the use of syndromic guidelines for sexually transmitted infection treatment in resource-constrained countries. Monitoring local etiologies provides information that may aid policy for sexually transmitted infection treatment. We investigated the etiology of genital ulcer disease among outpatients in Lusaka, Zambia. METHODOLOGY: Swabs from genital ulcers of 200 patients were tested using polymerase chain reaction for Treponema pallidum, herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), Haemophilus ducreyi, and Chlamydia trachomatis. RESULTS: The prevalence of the detected pathogens was as follows; HSV-2, 28%; T. pallidum, 11.5%; C. trachomatis, 3%; HSV-1, 0.5%; and H. ducreyi, 0%. Coinfection with HSV-2 and T. pallidum was 1.5%, and coinfection of HSV-2 and C. trachomatis was 1%. In 55% of the patients, no etiologic diagnosis could be established. CONCLUSIONS: H. ducreyi was not detected, whereas HSV-2 and T. pallidum were the commonest pathogens. Nondetection of H. ducreyi requires further studies. If the present findings are validated, treatment guidelines would require to be revised in Zambia.

Permeases of the sap transporter are required for cathelicidin resistance and virulence of Haemophilus ducreyi in humans.

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.

The molecular diagnosis of sexually transmitted genital ulcer disease.

Highly sensitive and specific nucleic acid amplification tests (NAATs) have emerged as the gold standard diagnostic tests for many infectious diseases. Real-time PCR has further refined the technology of nucleic acid amplification with detection in a closed system and enabled multiplexing to simultaneously detect multiple pathogens. It is a versatile, fast, and high-throughput system for pathogen detection that has reduced the risk of PCR contamination, eliminated post-PCR manipulations, and improved the cost-effectiveness of testing. In addition, real-time PCR can be applied to self-collected noninvasive specimens. Here, we describe an in-house developed TaqMan-based real-time multiplex PCR (M-PCR) assay for the diagnosis of sexually transmitted genital ulcer disease (GUD) and discuss briefly on issues associated with validation of assay performance.

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.

Expression of the Flp proteins by Haemophilus ducreyi is necessary for virulence in human volunteers.

BACKGROUND: Haemophilus ducreyi, the causative agent of the sexually transmitted disease chancroid, contains a flp (fimbria like protein) operon that encodes proteins predicted to contribute to adherence and pathogenesis. H. ducreyi mutants that lack expression of Flp1 and Flp2 or TadA, which has homology to NTPases of type IV secretion systems, have decreased abilities to attach to and form microcolonies on human foreskin fibroblasts (HFF). A tadA mutant is attenuated in its ability to cause disease in human volunteers and in the temperature dependent rabbit model, but a flp1flp2 mutant is virulent in rabbits. Whether a flp deletion mutant would cause disease in humans is not clear. RESULTS: We constructed 35000HPΔflp1-3, a deletion mutant that lacks expression of all three Flp proteins but has an intact tad secretion system. 35000HPΔflp1-3 was impaired in its ability to form microcolonies and to attach to HFF in vitro when compared to its parent (35000HP). Complementation of the mutant with flp1-3 in trans restored the parental phenotype. To test whether expression of Flp1-3 was necessary for virulence in humans, ten healthy adult volunteers were experimentally infected with a fixed dose of 35000HP (ranging from 54 to 67 CFU) on one arm and three doses of 35000HPΔflp1-3 (ranging from 63 to 961 CFU) on the other arm. The overall papule formation rate for the parent was 80% (95% confidence interval, CI, 55.2%-99.9%) and for the mutant was 70.0% (95% CI, 50.5%-89.5%) (P = 0.52). Mutant papules were significantly smaller (mean, 11.2 mm2) than were parent papules (21.8 mm2) 24 h after inoculation (P = 0.018). The overall pustule formation rates were 46.7% (95% CI 23.7-69.7%) at 30 parent sites and 6.7% (95% CI, 0.1-19.1%) at 30 mutant sites (P = 0.001). CONCLUSION: These data suggest that production and secretion of the Flp proteins contributes to microcolony formation and attachment to HFF cells in vitro. Expression of flp1-3 is also necessary for H. ducreyi to initiate disease and progress to pustule formation in humans. Future studies will focus on how Flp proteins contribute to microcolony formation and attachment in vivo.

Passive immunization with a polyclonal antiserum to the hemoglobin receptor of Haemophilus ducreyi confers protection against a homologous challenge in the experimental swine model of chancroid.

Haemophilus ducreyi, the etiologic agent of chancroid, has an obligate requirement for heme. Heme is acquired by H. ducreyi from its human host via TonB-dependent transporters expressed at its bacterial surface. Of 3 TonB-dependent transporters encoded in the genome of H. ducreyi, only the hemoglobin receptor, HgbA, is required to establish infection during the early stages of the experimental human model of chancroid. Active immunization with a native preparation of HgbA (nHgbA) confers complete protection in the experimental swine model of chancroid, using either Freund's or monophosphoryl lipid A as adjuvants. To determine if transfer of anti-nHgbA serum is sufficient to confer protection, a passive immunization experiment using pooled nHgbA antiserum was conducted in the experimental swine model of chancroid. Pigs receiving this pooled nHgbA antiserum were protected from a homologous, but not a heterologous, challenge. Passively transferred polyclonal antibodies elicited to nHgbA bound the surface of H. ducreyi and partially blocked hemoglobin binding by nHgbA, but were not bactericidal. Taken together, these data suggest that the humoral immune response to the HgbA vaccine is protective against an H. ducreyi infection, possibly by preventing acquisition of the essential nutrient heme.

A Haemophilus ducreyi CpxR deletion mutant is virulent in human volunteers.

Haemophilus ducreyi 35000HP contains a homolog of the CpxRA 2-component signal transduction system, which controls the cell envelope stress response system in other gram-negative bacteria and regulates some important H. ducreyi virulence factors. A H. ducreyi cpxR mutant was compared with its parent for virulence in the human challenge model of experimental chancroid. The pustule formation rate in 5 volunteers was 33% (95% confidence interval [CI], 1.3%-65.3%) at 15 parent sites and 40% (95% CI, 18.1%-61.9%) at 15 mutant sites (P = .35). Thus, the cpxR mutant was not attenuated for virulence. Inactivation of the H. ducreyi cpxR gene did not reduce the ability of this mutant to express certain proven virulence factors, including the DsrA serum resistance protein and the LspA2 protein, which inhibits phagocytosis. These results expand our understanding of the involvement of the CpxRA system in regulating virulence expression in H. ducreyi.