Diane M. Janowicz

Experimental infection of human volunteers with Haemophilus ducreyi: Fifteen years of clinical data and experience

Haemophilus ducreyi causes chancroid, which facilitates transmission of human immunodeficiency virus type 1. To better understand the biology of H. ducreyi, we developed a human inoculation model. In the present article, we describe clinical outcomes for 267 volunteers who were infected with H. ducreyi. There was a relationship between papule formation and estimated delivered dose. The outcome (either pustule formation or resolution) of infected sites for a given subject was not independent; the most important determinants of pustule formation were sex and host effects. When 41 subjects were infected a second time, their outcomes segregated toward their initial outcome, confirming the host effect. Subjects with pustules developed local symptoms that required withdrawal from the study after a mean of 8.6 days. There were 191 volunteers who had tissue biopsy performed, 173 of whom were available for follow-up analysis; 28 (16.2%) of these developed hypertrophic scars, but the model was otherwise safe. Mutant-parent trials confirmed key features in H. ducreyi pathogenesis, and the model has provided an opportunity to study differential human susceptibility to a bacterial infection.

Activation of the CpxRA System by Deletion of cpxA Impairs the Ability of Haemophilus ducreyi To Infect Humans

ABSTRACT Haemophilus ducreyi must adapt to the environment of the human host to establish and maintain infection in the skin. Bacteria generally utilize stress response systems, such as the CpxRA two-component system, to adapt to hostile environments. CpxRA is the only obvious two-component system contained in the H. ducreyi genome and negatively regulates the lspB-lspA2 operon, which encodes proteins that enable the organism to resist phagocytosis. We constructed an unmarked, in-frame H. ducreyi cpxA deletion mutant, 35000HPΔcpxA. In human inoculation experiments, 35000HPΔcpxA formed papules at a rate and size that were significantly less than its parent and was unable to form pustules compared to the parent. CpxA usually has kinase and phosphatase activities for CpxR, and the deletion of CpxA leads to the accumulation of activated CpxR due to the loss of phosphatase activity and the ability of CpxR to accept phosphate groups from other donors. Using a reporter construct, the lspB-lspA2 promoter was downregulated in 35000HPΔcpxA, confirming that CpxR was activated. Deletion of cpxA downregulated DsrA, the major determinant of serum resistance in the organism, causing the mutant to become serum susceptible. Complementation in trans restored parental phenotypes. 35000HPΔcpxA is the first H. ducreyi mutant that is impaired in its ability to form both papules and pustules in humans. Since a major function of CpxRA is to control the flow of protein traffic across the periplasm, uncontrolled activation of this system likely causes dysregulated expression of multiple virulence determinants and cripples the ability of the organism to adapt to the host.

Expression of the LspA1 and LspA2 Proteins by Haemophilus ducreyi Is Required for Virulence in Human Volunteers

ABSTRACT Haemophilus ducreyi colocalizes with polymorphonuclear leukocytes and macrophages and evades phagocytosis during experimental infection of human volunteers. H. ducreyi contains two genes, lspA1 and lspA2, which encode predicted proteins of 456 and 543 kDa, respectively. Compared to its wild-type parent, an lspA1 lspA2 double mutant does not inhibit phagocytosis by macrophage and myelocytic cell lines in vitro and is attenuated in an experimental rabbit model of chancroid. To test whether expression of LspA1 and LspA2 was necessary for virulence in humans, six volunteers were experimentally infected. Each volunteer was inoculated with three doses (ranging from 85 to 112 CFU) of the parent (35000HP) in one arm and three doses (ranging from 60 to 822 CFU) of the mutant (35000HPΩ12) in the other arm. The papule formation rates were 88% (95% confidence interval [95% CI], 76.8 to 99.9%) at 18 parent sites and 72% (95% CI, 44.4 to 99.9%) at 18 mutant sites (P = 0.19). However, papules were significantly smaller at mutant sites (mean size, 24.8 mm2) than at parent sites (mean size, 39.1 mm2) 24 h after inoculation (P = 0.0002). The pustule formation rates were 44% (95% CI, 5.8 to 77.6%) at parent sites and 0% (95% CI, 0 to 39.4%) at mutant sites (P = 0.009). With the caveat that biosafety regulations preclude testing of a complemented mutant in human subjects, these results indicate that expression of LspA1 and LspA2 facilitates the ability of H. ducreyi to initiate disease and to progress to pustule formation in humans.

The Human Skin Microbiome Associates with the Outcome of and Is Influenced by Bacterial Infection

ABSTRACT The influence of the skin microbiota on host susceptibility to infectious agents is largely unexplored. The skin harbors diverse bacterial species that may promote or antagonize the growth of an invading pathogen. We developed a human infection model for Haemophilus ducreyi in which human volunteers are inoculated on the upper arm. After inoculation, papules form and either spontaneously resolve or progress to pustules. To examine the role of the skin microbiota in the outcome of H. ducreyi infection, we analyzed the microbiomes of four dose-matched pairs of “resolvers” and “pustule formers” whose inoculation sites were swabbed at multiple time points. Bacteria present on the skin were identified by amplification and pyrosequencing of 16S rRNA genes. Nonmetric multidimensional scaling (NMDS) using Bray-Curtis dissimilarity between the preinfection microbiomes of infected sites showed that sites from the same volunteer clustered together and that pustule formers segregated from resolvers (P = 0.001, permutational multivariate analysis of variance [PERMANOVA]), suggesting that the preinfection microbiomes were associated with outcome. NMDS using Bray-Curtis dissimilarity of the endpoint samples showed that the pustule sites clustered together and were significantly different than the resolved sites (P = 0.001, PERMANOVA), suggesting that the microbiomes at the endpoint differed between the two groups. In addition to H. ducreyi, pustule-forming sites had a greater abundance of Proteobacteria, Bacteroidetes, Micrococcus, Corynebacterium, Paracoccus, and Staphylococcus species, whereas resolved sites had higher levels of Actinobacteria and Propionibacterium species. These results suggest that at baseline, resolvers and pustule formers have distinct skin bacterial communities which change in response to infection and the resultant immune response. IMPORTANCE Human skin is home to a diverse community of microorganisms, collectively known as the skin microbiome. Some resident bacteria are thought to protect the skin from infection by outcompeting pathogens for resources or by priming the immune systems response to invaders. However, the influence of the skin microbiome on the susceptibility to or protection from infection has not been prospectively evaluated in humans. We characterized the skin microbiome before, during, and after experimental inoculation of the arm with Haemophilus ducreyi in matched volunteers who subsequently resolved the infection or formed abscesses. Our results suggest that the preinfection microbiomes of pustule formers and resolvers have distinct community structures which change in response to the progression of H. ducreyi infection to abscess formation. Human skin is home to a diverse community of microorganisms, collectively known as the skin microbiome. Some resident bacteria are thought to protect the skin from infection by outcompeting pathogens for resources or by priming the immune systems response to invaders. However, the influence of the skin microbiome on the susceptibility to or protection from infection has not been prospectively evaluated in humans. We characterized the skin microbiome before, during, and after experimental inoculation of the arm with Haemophilus ducreyi in matched volunteers who subsequently resolved the infection or formed abscesses. Our results suggest that the preinfection microbiomes of pustule formers and resolvers have distinct community structures which change in response to the progression of H. ducreyi infection to abscess formation.

Expression of Haemophilus ducreyi Collagen Binding Outer Membrane Protein NcaA Is Required for Virulence in Swine and Human Challenge Models of Chancroid

ABSTRACT Haemophilus ducreyi, the etiologic agent of the sexually transmitted genital ulcer disease chancroid, has been shown to associate with dermal collagen fibers within infected skin lesions. Here we describe NcaA, a previously uncharacterized outer membrane protein that is important for H. ducreyi collagen binding and host colonization. An H. ducreyi strain lacking the ncaA gene was impaired in adherence to type I collagen but not fibronectin (plasma or cellular form) or heparin. The mutation had no effect on serum resistance or binding to HaCaT keratinocytes or human foreskin fibroblasts in vitro. Escherichia coli expressing H. ducreyi NcaA bound to type I collagen, demonstrating that NcaA is sufficient to confer collagen attachment. The importance of NcaA in H. ducreyi pathogenesis was assessed using both swine and human experimental models of chancroid. In the swine model, 20% of lesions from sites inoculated with the ncaA mutant were culture positive for H. ducreyi 7 days after inoculation, compared to 73% of wild-type-inoculated sites. The average number of CFU recovered from mutant-inoculated lesions was also significantly reduced compared to that recovered from wild-type-inoculated sites at both 2 and 7 days after inoculation. In the human challenge model, 8 of 30 sites inoculated with wild-type H. ducreyi progressed to the pustular stage, compared to 0 of 30 sites inoculated with the ncaA mutant. Together these results demonstrate that the collagen binding protein NcaA is required for H. ducreyi infection.

Haemophilus ducreyi SapA Contributes to Cathelicidin Resistance and Virulence in Humans

ABSTRACT Haemophilus ducreyi is an extracellular pathogen of human epithelial surfaces that resists human antimicrobial peptides (APs). The organisms genome contains homologs of genes sensitive to antimicrobial peptides (sap operon) in nontypeable Haemophilus influenzae. In this study, we characterized the sap-containing loci of H. ducreyi 35000HP and demonstrated that sapA is expressed in broth cultures and H. ducreyi-infected tissue; sapA is also conserved among both class I and class II H. ducreyi strains. We constructed a nonpolar sapA mutant of H. ducreyi 35000HP, designated 35000HPsapA, and compared the percent survival of wild-type 35000HP and 35000HPsapA exposed to several human APs, including α-defensins, β-defensins, and the cathelicidin LL-37. Unlike an H. influenzae sapA mutant, strain 35000HPsapA was not more susceptible to defensins than strain 35000HP was. However, we observed a significant decrease in the survival of strain 35000HPsapA after exposure to LL-37, which was complemented by introducing sapA in trans. Thus, the Sap transporter plays a role in resistance of H. ducreyi to LL-37. We next compared mutant strain 35000HPsapA with strain 35000HP for their ability to cause disease in human volunteers. Although both strains caused papules to form at similar rates, the pustule formation rate at sites inoculated with 35000HPsapA was significantly lower than that of sites inoculated with 35000HP (33.3% versus 66.7%; P = 0.007). Together, these data establish that SapA acts as a virulence factor and as one mechanism for H. ducreyi to resist killing by antimicrobial peptides. To our knowledge, this is the first demonstration that an antimicrobial peptide resistance mechanism contributes to bacterial virulence in humans.

Dysregulated immune profiles for skin and dendritic cells are associated with increased host susceptibility to Haemophilus ducreyi infection in human volunteers.

ABSTRACT In experimentally infected human volunteers, the cutaneous immune response to Haemophilus ducreyi is orchestrated by serum, polymorphonuclear leukocytes, macrophages, T cells, and myeloid dendritic cells (DC). This response either leads to spontaneous resolution of infection or progresses to pustule formation, which is associated with the failure of phagocytes to ingest the organism and the presence of Th1 and regulatory T cells. In volunteers who are challenged twice, some subjects form at least one pustule twice (PP group), while others have all inoculated sites resolve twice (RR group). Here, we infected PP and RR subjects with H. ducreyi and used microarrays to profile gene expression in infected and wounded skin. The PP and RR groups shared a core response to H. ducreyi. Additional transcripts that signified effective immune function were differentially expressed in RR infected sites, while those that signified a hyperinflammatory, dysregulated response were differentially expressed in PP infected sites. To examine whether DC drove these responses, we profiled gene expression in H. ducreyi-infected and uninfected monocyte-derived DC. Both groups had a common response that was typical of a type 1 DC (DC1) response. RR DC exclusively expressed many additional transcripts indicative of DC1. PP DC exclusively expressed differentially regulated transcripts characteristic of DC1 and regulatory DC. The data suggest that DC from the PP and RR groups respond differentially to H. ducreyi. PP DC may promote a dysregulated T-cell response that contributes to phagocytic failure, while RR DC may promote a Th1 response that facilitates bacterial clearance.

Identification of Haemophilus ducreyi genes expressed during human infection.

To identify Haemophilus ducreyi transcripts that are expressed during human infection, we used selective capture of transcribed sequences (SCOTS) with RNA isolated from pustules obtained from three volunteers infected with H. ducreyi, and with RNA isolated from broth-grown bacteria used to infect volunteers. With SCOTS, competitive hybridization of tissue-derived and broth-derived sequences identifies genes that may be preferentially expressed in vivo. Among the three tissue specimens, we identified 531 genes expressed in vivo. Southern blot analysis of 60 genes from each tissue showed that 87 % of the identified genes hybridized better with cDNA derived from tissue specimens than with cDNA derived from broth-grown bacteria. RT-PCR on nine additional pustules confirmed in vivo expression of 10 of 11 selected genes in other volunteers. Of the 531 genes, 139 were identified in at least two volunteers. These 139 genes fell into several functional categories, including biosynthesis and metabolism, regulation, and cellular processes, such as transcription, translation, cell division, DNA replication and repair, and transport. Detection of genes involved in anaerobic and aerobic respiration indicated that H. ducreyi likely encounters both microenvironments within the pustule. Other genes detected suggest an increase in DNA damage and stress in vivo. Genes involved in virulence in other bacterial pathogens and 32 genes encoding hypothetical proteins were identified, and may represent novel virulence factors. We identified three genes, lspA1, lspA2 and tadA, known to be required for virulence in humans. This is the first study to broadly define transcripts expressed by H. ducreyi in humans.

A DltA mutant of Haemophilus ducreyi Is partially attenuated in its ability to cause pustules in human volunteers.

ABSTRACT Haemophilus ducreyi produces two outer membrane proteins, called DltA (H. ducreyi lectin A) and DsrA (H. ducreyi serum resistance A), that contribute to the ability of the organism to evade complement-mediated serum killing. In contrast to their isogenic parent strain, 35000HP, the DsrA mutant FX517 exhibits 0% survival in 50% normal human serum and the DltA mutant FX533 exhibits 23% survival. Compared to 35000HP, FX517 does not cause pustule formation in human volunteers. To test whether DltA was required for virulence in humans, seven volunteers were experimentally infected with 35000HP and FX533. Four subjects were inoculated with fixed doses of 35000HP (101 CFU or 130 CFU) at three sites on one arm and escalating doses of FX533 (range, 46 CFU to 915 CFU) at three sites on the other arm. Pustules only developed at mutant-injected sites at doses nearly twofold higher than that of the parent, suggesting that FX533 was partially attenuated. Three subjects were inoculated with similar doses of the parent (67 CFU) and mutant (104 CFU) at three sites. Pustules formed at five of nine parent sites and one of nine mutant sites. Overall, the papule and pustule formation rates for 35000HP and FX533 were similar for the trial. However, for the five subjects who received similar doses of the parent and mutant, pustules developed at 7 of 15 sites (46.7%; 95% confidence interval [CI], 16.9% to 76.5%) inoculated with the parent and at 1 of 15 (6.7%; 95% CI, 0.1% to 18.4%) sites inoculated with the mutant (P = 0.043). We concluded that the DltA mutant was attenuated in its ability to cause disease at doses similar to that of the parent.

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.