RESUMEN
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.
Asunto(s)
Chancroide , Haemophilus ducreyi , Humanos , Haemophilus ducreyi/genética , Virulencia , Chancroide/genética , Ácido Ascórbico , OperónRESUMEN
BACKGROUND: In humans inoculated with Haemophilus ducreyi, there are host effects on the possible clinical outcomes-pustule formation versus spontaneous resolution of infection. However, the immunogenetic factors that influence these outcomes are unknown. Here we examined the role of 14 single-nucleotide polymorphisms (SNPs) in 7 selected pathogen-recognition pathways and cytokine genes on the gradated outcomes of experimental infection. METHODS: DNAs from 105 volunteers infected with H. ducreyi at 3 sites were genotyped for SNPs, using real-time polymerase chain reaction. The participants were classified into 2 cohorts, by race, and into 4 groups, based on whether they formed 0, 1, 2, or 3 pustules. χ(2) tests for trend and logistic regression analyses were performed on the data. RESULTS: In European Americans, the most significant findings were a protective association of the TLR9 +2848 GG genotype and a risk-enhancing association of the TLR9 TA haplotype with pustule formation; logistic regression showed a trend toward protection for the TLR9 +2848 GG genotype. In African Americans, logistic regression showed a protective effect for the IL10 -2849 AA genotype and a risk-enhancing effect for the IL10 AAC haplotype. CONCLUSIONS: Variations in TLR9 and IL10 are associated with the outcome of H. ducreyi infection.
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Chancroide/genética , Haemophilus ducreyi/inmunología , Interleucina-10/genética , Polimorfismo de Nucleótido Simple , Receptor Toll-Like 9/genética , Adulto , Negro o Afroamericano , Chancroide/inmunología , Estudios de Cohortes , Femenino , Estudios de Asociación Genética , Genotipo , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Estados Unidos , Población Blanca , Adulto JovenRESUMEN
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.
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Proteínas Bacterianas/metabolismo , Carbono/metabolismo , Chancroide/metabolismo , Chancroide/microbiología , Haemophilus ducreyi/metabolismo , Haemophilus ducreyi/patogenicidad , Adulto , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Chancroide/genética , Fibroblastos/metabolismo , Fibroblastos/microbiología , Haemophilus ducreyi/genética , Humanos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/microbiología , Macrófagos/metabolismo , Macrófagos/microbiología , Datos de Secuencia Molecular , Mutación , Estrés Oxidativo/genética , Proteínas Tirosina Fosfatasas no Receptoras/genética , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Eliminación de Secuencia/genética , Virulencia , Adulto JovenRESUMEN
Few studies have investigated host-bacterial interactions at sites of infection in humans using transcriptomics and metabolomics. Haemophilus ducreyi causes cutaneous ulcers in children and the genital ulcer disease chancroid in adults. We developed a human challenge model in which healthy adult volunteers are infected with H. ducreyi on the upper arm until they develop pustules. Here, we characterized host-pathogen interactions in pustules using transcriptomics and metabolomics and examined interactions between the host transcriptome and metabolome using integrated omics. In a previous pilot study, we determined the human and H. ducreyi transcriptomes and the metabolome of pustule and wounded sites of 4 volunteers (B. Griesenauer, T. M. Tran, K. R. Fortney, D. M. Janowicz, et al., mBio 10:e01193-19, 2019, https://doi.org/10.1128/mBio.01193-19). While we could form provisional transcriptional networks between the host and H. ducreyi, the study was underpowered to integrate the metabolome with the host transcriptome. To better define and integrate the transcriptomes and metabolome, we used samples from both the pilot study (n = 4) and new volunteers (n = 8) to identify 5,495 human differentially expressed genes (DEGs), 123 H. ducreyi DEGs, 205 differentially abundant positive ions, and 198 differentially abundant negative ions. We identified 42 positively correlated and 29 negatively correlated human-H. ducreyi transcriptome clusters. In addition, we defined human transcriptome-metabolome networks consisting of 9 total clusters, which highlighted changes in fatty acid metabolism and mitigation of oxidative damage. Taken together, the data suggest a mixed pro- and anti-inflammatory environment and rewired central metabolism in the host that provides a hostile, nutrient-limited environment for H. ducreyi. IMPORTANCE Interactions between the host and bacteria at sites of infection in humans are poorly understood. We inoculated human volunteers on the upper arm with the skin pathogen H. ducreyi or a buffer control and biopsied the resulting infected and sham-inoculated sites. We performed dual transcriptome sequencing (RNA-seq) and metabolic analysis on the biopsy samples. Network analyses between the host and bacterial transcriptomes and the host transcriptome-metabolome network were used to identify molecules that may be important for the virulence of H. ducreyi in the human host. Our results suggest that the pustule is highly oxidative, contains both pro- and anti-inflammatory components, and causes metabolic shifts in the host, to which H. ducreyi adapts to survive. To our knowledge, this is the first study to integrate transcriptomic and metabolomic responses to a single bacterial pathogen in the human host.
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Chancroide , Haemophilus ducreyi , Adulto , Niño , Humanos , Haemophilus ducreyi/genética , Proyectos Piloto , Chancroide/genética , Piel/microbiología , Estrés OxidativoRESUMEN
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.
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Chancroide/genética , Redes Reguladoras de Genes , Haemophilus ducreyi/genética , Haemophilus ducreyi/patogenicidad , Interacciones Huésped-Patógeno/genética , Metaboloma , Adulto , Anaerobiosis , Ácido Ascórbico/metabolismo , Proteínas Bacterianas/genética , Chancroide/inmunología , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Metabolómica , Persona de Mediana Edad , RNA-SeqRESUMEN
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.
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Proteínas Bacterianas , Chancroide , Genoma Bacteriano , Islas Genómicas , Vacunas contra Haemophilus , Haemophilus ducreyi , Factores de Virulencia , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Chancroide/genética , Chancroide/inmunología , Chancroide/prevención & control , Vacunas contra Haemophilus/genética , Vacunas contra Haemophilus/inmunología , Vacunas contra Haemophilus/metabolismo , Haemophilus ducreyi/genética , Haemophilus ducreyi/inmunología , Haemophilus ducreyi/metabolismo , Haemophilus ducreyi/patogenicidad , Humanos , Vacunología , Factores de Virulencia/genética , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismoRESUMEN
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.
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Chancroide/inmunología , Haemophilus ducreyi/inmunología , Células de Langerhans/inmunología , Adulto , Proliferación Celular , Chancroide/genética , Chancroide/microbiología , Chancroide/patología , Citocinas/biosíntesis , Citocinas/genética , Citocinas/inmunología , Susceptibilidad a Enfermedades , Femenino , Humanos , Células de Langerhans/patología , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Linfocitos T/inmunologíaRESUMEN
Chancroid, a sexually transmitted genital ulcer disease caused by the Gram-negative bacterium Haemophilus ducreyi, facilitates the acquisition and transmission of HIV. An effective vaccine against chancroid has not been developed. In this preliminary study, the gene encoding the H. ducreyi outer membrane hemoglobin receptor HgbA was cloned into the plasmid pTETnir15. The recombinant construct was introduced into the attenuated Salmonella typhimurium SL3261 strain and stable expression was induced in vitro under anaerobic conditions. The vaccine strain was delivered into the temperature-dependent rabbit model of chancroid by intragastric immunization as a single dose, or as three doses administered at two-weekly intervals. No specific antibody to HgbA was elicited after either dose schedule. Although the plasmid vector survived in vivo passage for up to 15 days following single oral challenge, HgbA expression was restricted to plasmid isolates recovered one day after immunization. Rabbits inoculated with the 3-dose booster regimen achieved no protective immunity from homologous challenge. These results emphasize that refinements in plasmid design to enhance a durable heterologous protein expression are necessary for the development of a live oral vaccine against chancroid.
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Proteínas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Proteínas Portadoras/inmunología , Chancroide/inmunología , Salmonella typhimurium/inmunología , Administración Oral , Animales , Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas/genética , Vacunas Bacterianas/genética , Proteínas Portadoras/genética , Chancroide/genética , Chancroide/prevención & control , Haemophilus ducreyi/genética , Haemophilus ducreyi/inmunología , Inmunización/métodos , Masculino , Conejos , Salmonella typhimurium/genética , Vacunación/métodos , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Haemophilus ducreyi and Klebsiella (Calymmatobacterium) granulomatis are sexually transmitted bacteria that cause characteristic, persisting ulceration on external genitals called chancroid and granuloma inguinale, respectively. Those ulcers are endemic in developing countries or exist, as does granuloma inguinale, only in some geographic "hot spots."H. ducreyi is placed in the genus Haemophilus (family Pasteurellacae); however, this phylogenetic position is not obvious. The multiple ways in which the bacterium may be adapted to its econiche through specialized nutrient acquisitions; defenses against the immune system; and virulence factors that increase attachment, fitness, and persistence within genital tissue are discussed below. The analysis of K. granulomatis phylogeny demonstrated a high degree of identity with other Klebsiella species, and the name K. granulomatis comb. nov. was proposed. Because of the difficulty in growing this bacterium on artificial media, its characteristics have not been sufficiently defined. More studies are needed to understand bacterial genetics related to the pathogenesis and evolution of K. granulomatis.
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Evolución Molecular , Haemophilus ducreyi/genética , Klebsiella/genética , Enfermedades Bacterianas de Transmisión Sexual/microbiología , Animales , Chancroide/genética , Chancroide/microbiología , Chancroide/transmisión , Variación Genética , Haemophilus ducreyi/patogenicidad , Haemophilus ducreyi/fisiología , Humanos , Klebsiella/patogenicidad , Klebsiella/fisiología , Infecciones por Klebsiella/genética , Infecciones por Klebsiella/microbiología , Infecciones por Klebsiella/transmisión , Filogenia , Factores de Virulencia/genética , Factores de Virulencia/fisiologíaRESUMEN
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.