IU Indianapolis ScholarWorks :: Browsing by Subject "Haemophilus ducreyi"

Browsing by Subject "Haemophilus ducreyi"

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A Haemophilus ducreyi strain lacking the yfeABCD iron transport system is virulent in human volunteers
(American Society for Microbiology, 2024) Fortney, Kate R.; Brothwell, Julie A.; Batteiger, Teresa A.; Duplantier, Rory; Katz, Barry P.; Spinola, Stanley M.; Medicine, School of Medicine
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.
A high-resolution view of the immune and stromal cell response to Haemophilus ducreyi infection in human volunteers
(American Society for Microbiology, 2025) Brothwell, Julie A.; Wei, Yuhui; Wang, Jia; Guo, Tingbo; Zhang, Chi; Fortney, Kate R.; Duplantier, Rory; Chen, Li; Batteiger, Teresa A.; Kaplan, Mark H.; Spinola, Stanley M.; Cao, Sha; Microbiology and Immunology, School of Medicine
Haemophilus ducreyi causes the genital ulcer disease chancroid and cutaneous ulcers in children. To study its pathogenesis, we developed a human challenge model in which we infect the skin on the upper arm of human volunteers with H. ducreyi to the pustular stage of disease. The model has been used to define lesional architecture, describe the immune infiltrate into the infected sites using flow cytometry, and explore the molecular basis of the immune response using bulk RNA-seq. Here, we used single cell RNA-seq (scRNA-seq) and spatial transcriptomics to simultaneously characterize multiple cell types within infected human skin and determine the cellular origin of differentially expressed transcripts that we had previously identified by bulk RNA-seq. We obtained paired biopsies of pustules and wounded (mock infected) sites from five volunteers for scRNA-seq. We identified 13 major cell types, including T- and NK-like cells, macrophages, dendritic cells, as well as other cell types typically found in the skin. Immune cell types were enriched in pustules, and some subtypes within the major cell types were exclusive to pustules. Sufficient tissue specimens for spatial transcriptomics were available from four of the volunteers. T- and NK-like cells were highly associated with multiple antigen presentation cell types. In pustules, type I interferon stimulation was high in areas that were high in antigen presentation-especially in macrophages near the abscess-compared to wounds. Together, our data provide a high-resolution view of the cellular immune response to the infection of the skin with a human pathogen. IMPORTANCE: A high-resolution view of the immune infiltrate due to infection with an extracellular bacterial pathogen in human skin has not yet been defined. Here, we used the human skin pathogen Haemophilus ducreyi in a human challenge model to identify on a single cell level the types of cells that are present in volunteers who fail to spontaneously clear infection and form pustules. We identified 13 major cell types. Immune cells and immune-activated stromal cells were enriched in pustules compared to wounded (mock infected) sites. Pustules formed despite the expression of multiple pro-inflammatory cytokines, such as IL-1β and type I interferon. Interferon stimulation was most evident in macrophages, which were proximal to the abscess. The pro-inflammatory response within the pustule may be tempered by regulatory T cells and cells that express indoleamine 2,3-dioxygenase, leading to failure of the immune system to clear H. ducreyi.
A Class I Haemophilus ducreyi Strain Containing a Class II hgbA Allele Is Partially Attenuated in Humans: Implications for HgbA Vaccine Efficacy Trials
(American Society for Microbiology, 2019-07) Leduc, Isabelle; Fortney, Kate R.; Janowicz, Diane M.; Zwickl, Beth; Ellinger, Sheila; Katz, Barry P.; Lin, Huaiying; Dong, Qunfeng; Spinola, Stanley M.; Microbiology and Immunology, School of Medicine
Haemophilus ducreyi causes chancroid and is a major cause of cutaneous ulcers in children. Due to environmental reservoirs, both class I and class II H. ducreyi strains persist in cutaneous ulcer regions of endemicity following mass drug administration of azithromycin, suggesting the need for a vaccine. The hemoglobin receptor (HgbA) is a leading vaccine candidate, but its efficacy in animal models is class specific. Controlled human infection models can be used to evaluate vaccines, but only a class I strain (35000HP) has been characterized in this model. As a prelude to evaluating HgbA vaccines in the human model, we tested here whether a derivative of 35000HP containing a class II hgbA allele (FX548) is as virulent as 35000HP in humans. In eight volunteers infected at three sites with each strain, the papule formation rate was 95.8% for 35000HP versus 62.5% for FX548 (P = 0.021). Excluding doses of FX548 that were ≥2-fold higher than those of 35000HP, the pustule formation rate was 25% for 35000HP versus 11.7% for FX548 (P = 0.0053). By Western blot analysis, FX548 and 35000HP expressed equivalent amounts of HgbA in whole-cell lysates and outer membranes. The growth of FX548 and 35000HP was similar in media containing hemoglobin or hemin. By whole-genome sequencing and single-nucleotide polymorphism analysis, FX548 contained no mutations in open reading frames other than hgbA. We conclude that by an unknown mechanism, FX548 is partially attenuated in humans and is not a suitable strain for HgbA vaccine efficacy trials in the model.
Determination of an Interaction Network between an Extracellular Bacterial Pathogen and the Human Host
(American Society for Microbiology, 2019-06-18) Griesenauer, Brad; Tran, Tuan M.; Fortney, Kate R.; Janowicz, Diane M.; Johnson, Paula; Gao, Hongyu; Barnes, Stephen; Wilson, Landon S.; Liu, Yunlong; Spinola, Stanley M.; Microbiology and Immunology, School of Medicine
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 vivo H. 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.
Dispensability of Ascorbic Acid Uptake and Utilization Encoded by ulaABCD for the Virulence of Haemophilus ducreyi in Humans
(Oxford University Press, 2023) Brothwell, Julie A.; Fortney, Kate R.; Batteiger, Teresa; Katz, Barry P.; Spinola, Stanley M.; Microbiology and Immunology, School of Medicine
Compared with wounded skin, ascorbic acid is enriched in pustules of humans experimentally infected with Haemophilus ducreyi. Compared with the broth-grown inocula, transcription of the H. ducreyi ulaABCD operon, which encodes genes for ascorbic acid uptake, is increased in pustules. We hypothesized that ascorbic acid uptake plays a role in H. ducreyi virulence. Five volunteers were infected with both H. ducreyi strain 35000HP and its isogenic ulaABCD deletion mutant at multiple sites; the papule and pustule formation rates of the mutant and parent strains were similar. Thus, ascorbic acid uptake is not essential for H. ducreyi virulence in humans.
Draft Whole-Genome Sequence of Haemophilus ducreyi Strain AUSPNG1, Isolated from a Cutaneous Ulcer of a Child from Papua New Guinea.
(ASM, 2016-02) Gangaiah, Dharanesh; Marinov, Georgi K.; Roberts, Sally A.; Robson, Jenny; Spinola, Stanley M.; Department of Microbiology & Immunology, IU School of Medicine
Haemophilus ducreyi has recently emerged as a leading cause of cutaneous ulcers in the yaws-endemic areas of Papua New Guinea and other South Pacific islands. Here, we report the draft genome sequence of the H. ducreyi strain AUSPNG1, isolated from a cutaneous ulcer of a child from Papua New Guinea.
Formate production is dispensable for Haemophilus ducreyi virulence in human volunteers
(American Society for Microbiology, 2023) Brothwell, Julie A.; Fortney, Kate R.; Williams, Jalan S.; Batteiger, Teresa A.; Duplantier, Rory; Grounds, Danielle; Jannasch, Amber S.; Katz, Barry P.; Spinola, Stanley M.; Microbiology and Immunology, School of Medicine
Haemophilus ducreyi is a causative agent of cutaneous ulcers in children who live in the tropics and of the genital ulcer disease chancroid in sexually active persons. In the anaerobic environment of abscesses and ulcers, anaerobic respiration and mixed acid fermentation (MAF) can be used to provide cellular energy. In Escherichia coli, MAF produces formate, acetate, lactate, succinate, and ethanol; however, MAF has not been studied in H. ducreyi. In human challenge experiments with H. ducreyi 35000HP, transcripts of the formate transporter FocA and pyruvate formate lyase (PflB) were upregulated in pustules compared to the inocula. We made single and double mutants of focA and pflB in 35000HP. Growth of 35000HPΔfocA was similar to 35000HP, but 35000HPΔpflB and 35000HPΔfocA-pflB had growth defects during both aerobic and anaerobic growth. Mutants lacking pflB did not secrete formate into the media. However, formate was secreted into the media by 35000HPΔfocA, indicating that H. ducreyi has alternative formate transporters. The pH of the media during anaerobic growth decreased for 35000HP and 35000HPΔfocA, but not for 35000HPΔpflB or 35000HPΔfocA-pflB, indicating that pflB is the main contributor to media acidification during anaerobic growth. We tested whether formate production and transport were required for virulence in seven human volunteers in a mutant versus parent trial between 35000HPΔfocA-pflB and 35000HP. The pustule formation rate was similar for 35000HP (42.9%)- and 35000HPΔfocA-pflB (62%)-inoculated sites. Although formate production occurs during in vitro growth and focA-pflB transcripts are upregulated during human infection, focA and pflB are not required for virulence in humans.
Genes Differentially Expressed by Haemophilus ducreyi during Anaerobic Growth Significantly Overlap Those Differentially Expressed during Experimental Infection of Human Volunteers
(American Society for Microbiology, 2022) Brothwell, Julie A.; Spinola, Stanley M.; Microbiology and Immunology, School of Medicine
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.
Haemophilus ducreyi Cutaneous Ulcer Strains Diverged from Both Class I and Class II Genital Ulcer Strains: Implications for Epidemiological Studies
(Plos, 2016-12-27) Gangaiah, Dharanesh; Spinola, Stanley M.; Department of Microbiology and Immunology, IU School of Medicine
BACKGROUND: Haemophilus ducreyi has emerged as a major cause of cutaneous ulcers (CU) in yaws-endemic regions of the tropics in the South Pacific, South East Asia and Africa. H. ducreyi was once thought only to cause the genital ulcer (GU) disease chancroid; GU strains belong to 2 distinct classes, class I and class II. Using whole-genome sequencing of 4 CU strains from Samoa, 1 from Vanuatu and 1 from Papua New Guinea, we showed that CU strains diverged from the class I strain 35000HP and that one CU strain expressed β-lactamase. Recently, the Center for Disease Control and Prevention released the genomes of 11 additional CU strains from Vanuatu and Ghana; however, the evolutionary relationship of these CU strains to previously-characterized CU and GU strains is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We performed phylogenetic analysis of 17 CU and 10 GU strains. Class I and class II GU strains formed two distinct clades. The class I strains formed two subclades, one containing 35000HP and HD183 and the other containing the remainder of the class I strains. Twelve of the CU strains formed a subclone under the class I 35000HP subclade, while 2 CU strains formed a subclone under the other class I subclade. Unexpectedly, 3 of the CU strains formed a subclone under the class II clade. Phylogenetic analysis of dsrA-hgbA-ncaA sequences yielded a tree similar to that of whole-genome phylogenetic tree. CONCLUSIONS/SIGNIFICANCE: CU strains diverged from multiple lineages within both class I and class II GU strains. Multilocus sequence typing of dsrA-hgbA-ncaA could be reliably used for epidemiological investigation of CU and GU strains. As class II strains grow relatively poorly and are relatively more susceptible to vancomycin than class I strains, these findings have implications for methods to recover CU strains. Comparison of contemporary CU and GU isolates would help clarify the relationship between these entities.
Haemophilus ducreyi Hfq Contributes to Virulence Gene Regulation as Cells Enter Stationary Phase
(American Society for Microbiology, 2014-02-11) Gangaiah, Dharanesh; Labandeira-Rey, Maria; Zhang, Xinjun; Fortney, Kate R.; Ellinger, Sheila; Zwickl, Beth; Baker, Beth; Liu, Yunlong; Janowicz, Diane M.; Katz, Barry P.; Brautigam, Chad A.; Munson, Robert S., Jr.; Hansen, Eric J.; Spinola, Stanley M.; Microbiology and Immunology, School of Medicine
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.

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