RESUMEN
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.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Chancroide/prevención & control , Vacunas contra Haemophilus/inmunología , Haemophilus ducreyi/inmunología , Adulto , Alelos , Proteínas Bacterianas/administración & dosificación , Proteínas Portadoras/administración & dosificación , Chancroide/inmunología , Chancroide/microbiología , Femenino , Vacunas contra Haemophilus/administración & dosificación , Vacunas contra Haemophilus/genética , Haemophilus ducreyi/genética , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
UNLABELLED: 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 system's 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.
Asunto(s)
Haemophilus ducreyi/crecimiento & desarrollo , Microbiota , Enfermedades Cutáneas Bacterianas/microbiología , Enfermedades Cutáneas Bacterianas/patología , Piel/microbiología , Análisis por Conglomerados , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Humanos , Datos de Secuencia Molecular , Filogenia , Estudios Prospectivos , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Etanolaminofosfotransferasa/genética , Haemophilus ducreyi/genética , Lípido A/metabolismo , Administración Oral , Adulto , Antibacterianos/uso terapéutico , Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas Bacterianas/metabolismo , Chancroide/tratamiento farmacológico , Chancroide/microbiología , Chancroide/patología , Ciprofloxacina/uso terapéutico , Etanolaminofosfotransferasa/metabolismo , Etanolaminas/metabolismo , Femenino , Eliminación de Gen , Expresión Génica , Prueba de Complementación Genética , Haemophilus ducreyi/efectos de los fármacos , Haemophilus ducreyi/metabolismo , Haemophilus ducreyi/patogenicidad , Voluntarios Sanos , Humanos , Lípido A/química , Masculino , Mutación , Unión Proteica , Electricidad Estática , alfa-Defensinas/farmacología , beta-Defensinas/farmacología , CatelicidinasRESUMEN
(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.
Asunto(s)
Guanosina Pentafosfato/deficiencia , Haemophilus ducreyi/patogenicidad , Ligasas/metabolismo , Pirofosfatasas/metabolismo , Adulto , Dermatitis/microbiología , Dermatitis/patología , Femenino , Eliminación de Gen , Prueba de Complementación Genética , Haemophilus ducreyi/genética , Voluntarios Sanos , Humanos , Ligasas/genética , Masculino , Persona de Mediana Edad , Pirofosfatasas/genéticaRESUMEN
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.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Chancroide/microbiología , Haemophilus ducreyi/patogenicidad , Factores de Virulencia/metabolismo , Adulto , Animales , Anticuerpos Antibacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Actividad Bactericida de la Sangre , Femenino , Eliminación de Gen , Humanos , Lipoproteínas/genética , Lipoproteínas/inmunología , Lipoproteínas/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Modelos Teóricos , Fagocitosis , Factores de Virulencia/genética , Factores de Virulencia/inmunología , Adulto JovenRESUMEN
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.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Haemophilus ducreyi/crecimiento & desarrollo , Haemophilus ducreyi/genética , Proteína de Factor 1 del Huésped/metabolismo , Factores de Virulencia/biosíntesis , Adulto , Chancroide/microbiología , Chancroide/patología , Femenino , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Haemophilus ducreyi/patogenicidad , Voluntarios Sanos , Proteína de Factor 1 del Huésped/genética , Humanos , Masculino , Persona de Mediana EdadRESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Farmacorresistencia Bacteriana , Haemophilus ducreyi/enzimología , Proteínas de Transporte de Membrana/metabolismo , Factores de Virulencia/metabolismo , Adulto , Chancroide/microbiología , Chancroide/patología , Femenino , Eliminación de Gen , Haemophilus ducreyi/efectos de los fármacos , Haemophilus ducreyi/genética , Haemophilus ducreyi/patogenicidad , Experimentación Humana , Humanos , Masculino , Proteínas de Transporte de Membrana/genética , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Virulencia , Adulto Joven , CatelicidinasRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Chancroide/microbiología , Haemophilus ducreyi/metabolismo , Haemophilus ducreyi/patogenicidad , Lipopolisacáridos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Adulto , Proteínas Bacterianas/genética , Células Dendríticas , Femenino , Regulación Bacteriana de la Expresión Génica/fisiología , Humanos , Macrófagos/fisiología , Masculino , Persona de Mediana Edad , Mutación , Fagocitosis , Virulencia , Adulto JovenRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Chancroide/microbiología , Haemophilus ducreyi/fisiología , Haemophilus ducreyi/patogenicidad , Adulto , Adhesión Bacteriana , Proteínas Bacterianas/genética , Femenino , Regulación Bacteriana de la Expresión Génica , Haemophilus ducreyi/genética , Experimentación Humana , Humanos , Masculino , Persona de Mediana Edad , Operón , Eliminación de Secuencia , VirulenciaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/genética , Chancroide/microbiología , Haemophilus ducreyi/genética , Haemophilus ducreyi/patogenicidad , Western Blotting , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fagocitosis , Eliminación de Secuencia , Factores de Virulencia/genéticaRESUMEN
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, 35000HPDeltacpxA. In human inoculation experiments, 35000HPDeltacpxA 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 35000HPDeltacpxA, 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. 35000HPDeltacpxA 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.
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Proteínas Bacterianas/fisiología , Haemophilus ducreyi/patogenicidad , Proteínas Quinasas/fisiología , Adulto , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Femenino , Eliminación de Gen , Humanos , Lectinas/genética , Masculino , Persona de Mediana EdadRESUMEN
The massive depletion of gastrointestinal-tract CD4 T cells is a hallmark of the acute phase of HIV infection. In contrast, the depletion of the lower-respiratory-tract mucosal CD4 T cells as measured in bronchoalveolar lavage (BAL) fluid is more moderate and similar to the depletion of CD4 T cells observed in peripheral blood (PB). To understand better the dynamics of disease pathogenesis and the potential for the reconstitution of CD4 T cells in the lung and PB following the administration of effective antiretroviral therapy, we studied cell-associated viral loads, CD4 T-cell frequencies, and phenotypic and functional profiles of antigen-specific CD4 T cells from BAL fluid and blood before and after the initiation of highly active antiretroviral therapy (HAART). The major findings to emerge were the following: (i) BAL CD4 T cells are not massively depleted or preferentially infected by HIV compared to levels for PB; (ii) BAL CD4 T cells reconstitute after the initiation of HAART, and their infection frequencies decrease; (iii) BAL CD4 T-cell reconstitution appears to occur via the local proliferation of resident BAL CD4 T cells rather than redistribution; and (iv) BAL CD4 T cells are more polyfunctional than CD4 T cells in blood, and their functional profile is relatively unchanged after the initiation of HAART. Taken together, these data suggest mechanisms for mucosal CD4 T-cell depletion and interventions that might aid in the reconstitution of mucosal CD4 T cells.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Terapia Antirretroviral Altamente Activa/métodos , Líquido del Lavado Bronquioalveolar/citología , Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Adulto , Sangre/inmunología , Recuento de Linfocito CD4 , Femenino , Humanos , Masculino , Persona de Mediana Edad , Carga ViralRESUMEN
Haemophilus ducreyi is an extracellular pathogen of human epithelial surfaces that resists human antimicrobial peptides (APs). The organism's 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 alpha-defensins, beta-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.
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Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas Bacterianas/fisiología , Farmacorresistencia Bacteriana , Haemophilus ducreyi/efectos de los fármacos , Haemophilus ducreyi/patogenicidad , Factores de Virulencia/fisiología , Adulto , Proteínas Bacterianas/genética , Chancroide/microbiología , Chancroide/patología , Secuencia Conservada , Femenino , Eliminación de Gen , Perfilación de la Expresión Génica , Prueba de Complementación Genética , Experimentación Humana , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Persona de Mediana Edad , Piel/patología , Virulencia , Factores de Virulencia/genética , CatelicidinasRESUMEN
Haemophilus ducreyi 35000HP contains a homologue of the luxS gene, which encodes an enzyme that synthesizes autoinducer 2 (AI-2) in other gram-negative bacteria. H. ducreyi 35000HP produced AI-2 that functioned in a Vibrio harveyi-based reporter system. A H. ducreyi luxS mutant was constructed by insertional inactivation of the luxS gene and lost the ability to produce AI-2. Provision of the H. ducreyi luxS gene in trans partially restored AI-2 production by the mutant. The luxS mutant was compared with its parent for virulence in the human challenge model of experimental chancroid. The pustule-formation rate in 5 volunteers was 93.3% (95% confidence interval, 81.7%-99.9%) at 15 parent sites and 60.0% (95% confidence interval, 48.3%-71.7%) at 15 mutant sites (1-tailed P < .001). Thus, the luxS mutant was partially attenuated for virulence. This is the first report of AI-2 production contributing to the pathogenesis of a genital ulcer disease.
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Proteínas Bacterianas/metabolismo , Liasas de Carbono-Azufre/metabolismo , Chancroide/microbiología , Haemophilus ducreyi/genética , Haemophilus ducreyi/patogenicidad , Adulto , Proteínas Bacterianas/genética , Bioensayo , Liasas de Carbono-Azufre/genética , Chancroide/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Piel/microbiología , Piel/patología , VirulenciaRESUMEN
A gene expression study of Haemophilus ducreyi identified the hypothetical lipoprotein HD0192, renamed here "fibrinogen binder A" (FgbA), as being preferentially expressed in vivo. To test the role played by fgbA in virulence, an isogenic fgbA mutant (35000HPfgbA) was constructed using H. ducreyi 35000HP, and 6 volunteers were experimentally infected with 35000HP or 35000HPfgbA. The overall pustule-formation rate was 61.1% at parent sites and 22.2% at mutant sites (P = .019). Papules were significantly smaller at mutant sites than at parent sites (13.3 vs. 37.9 mm(2); P = .002) 24 h after inoculation. Thus, fgbA contributed significantly to the virulence of H. ducreyi in humans. In vitro experiments demonstrated that fgbA encodes a fibrinogen-binding protein; no other fibrinogen-binding proteins were identified in 35000HP. fgbA was conserved among clinical isolates of both class I and II H. ducreyi strains, supporting the finding that fgbA is important for H. ducreyi infection.
Asunto(s)
Proteínas Bacterianas/metabolismo , Chancroide/microbiología , Fibrinógeno/metabolismo , Haemophilus ducreyi/genética , Haemophilus ducreyi/patogenicidad , Lipoproteínas/metabolismo , Adulto , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Femenino , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica/fisiología , Humanos , Lipoproteínas/química , Lipoproteínas/genética , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Unión Proteica , Enfermedades Cutáneas Bacterianas/microbiología , Virulencia , Adulto JovenRESUMEN
This study evaluated an intervention to facilitate adaptive coping by persons living with HIV (PLWH), with the participation of their cohabiting partners as a dimension of the intervention. An experimental design with randomization was used, and 84 PLWH and their partners were recruited. The intervention was based on a psychosocial educational model that incorporated four 2-hour sessions focused on communication, stress appraisal, adaptive coping strategies, and building social support. Both members of the dyad were included in each session. The comparison control included four supportive phone calls to the PLWH alone. Data were collected from both the PLWH and their partner in each of the two groups at baseline, immediately following the intervention, and 3 months and 6 months posttreatment. Data were analyzed using repeated measures analysis of variance, with change scores from the partner data being covariates. Results indicated that the design was a feasible model, which demonstrated potential for the management of stress and possibly problems such as adherence in PLWH. A manual was developed as a part of this intervention and is currently available.
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Adaptación Psicológica , Infecciones por VIH/psicología , Educación en Salud/organización & administración , Modelos Educacionales , Apoyo Social , Esposos/educación , Adulto , Análisis de Varianza , Actitud Frente a la Salud , Comunicación , Conflicto Psicológico , Curriculum , Estudios de Factibilidad , Femenino , Infecciones por VIH/complicaciones , Infecciones por VIH/prevención & control , Conocimientos, Actitudes y Práctica en Salud , Promoción de la Salud , Humanos , Masculino , Persona de Mediana Edad , Modelos Psicológicos , Investigación en Evaluación de Enfermería , Evaluación de Programas y Proyectos de Salud , Esposos/psicología , Estrés Psicológico/etiología , Estrés Psicológico/prevención & controlRESUMEN
BACKGROUND: Several commonly used antiretrovirals (ARVs) require dose adjustments to prevent toxicities in the presence of renal insufficiency. Because no prospective studies of the prevalence or risk factors for kidney disease in stable outpatient human immunodeficiency virus (HIV)-infected indigenous African populations have been published to date, it is not known if already scarce resources should be allocated to detect renal dysfunction, in those without risk factors for kidney disease, prior to initiation of increasingly available antiretrovirals in developing countries. METHODS: A cross-sectional study to determine the prevalence of and risk factors for renal disease in a cohort of medically stable, HIV-infected, antiretroviral-naïve adults, without diabetes or hypertension, presenting to an HIV clinic in western Kenya. RESULTS: Of 373 patients with complete data, renal insufficiency (CrCl <60 ml/min) was identified in 43 (11.5%) [18 (4.8%) had a CrCl <50 ml/min]. Despite high correlation coefficients between the three renal function estimating equations used, when compared to creatinine clearance as calculated by Cockcroft-Gault, lower rates of moderate to severe renal insufficiency were identified by the Modification of Diet in Renal Disease equations. Proteinuria, defined as a urine dipstick protein of equal to or greater than 1+, was detected in only 23 subjects (6.2%). CONCLUSIONS: Renal insufficiency is not uncommon, even in stable patients without diabetes or hypertension. Conversely, proteinuria was unexpectedly infrequent in this population. Utilizing resources to assess renal function prior to initiation of antiretrovirals in order to identify those likely to benefit from dosage adjustment is justified.
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Infecciones por VIH/complicaciones , Enfermedades Renales/complicaciones , Enfermedades Renales/virología , Adulto , Antirretrovirales/uso terapéutico , Estudios de Cohortes , Creatina/orina , Femenino , Infecciones por VIH/virología , Humanos , Kenia , Enfermedades Renales/diagnóstico , Enfermedades Renales/patología , Masculino , Persona de Mediana Edad , Pacientes Ambulatorios , Estudios Prospectivos , Proteinuria/diagnóstico , Factores de RiesgoRESUMEN
We infected 11 HIV-seropositive volunteers whose CD4(+) cell counts were >350 cells/ microL (7 of whom were receiving antiretrovirals) with Haemophilus ducreyi. The papule and pustule formation rates were similar to those observed in HIV-seronegative historical control subjects. No subject experienced a sustained change in CD4(+) cell count or HIV RNA level. The cellular infiltrate in biopsy samples obtained from the HIV-seropositive and HIV-seronegative subjects did not differ with respect to the percentage of leukocytes, neutrophils, macrophages, or T cells. The CD4(+):CD8(+) cell ratio in biopsy samples from the HIV-seropositive subjects was 1:3, the inverse of the ratio seen in the HIV-seronegative subjects (P<.0001). Although CD4(+) cells proliferated in lesions, in situ hybridization and reverse-transcription polymerase chain reaction for HIV RNA was negative. We conclude that experimental infection in HIV-seropositive persons is clinically similar to infection in HIV-seronegative persons and does not cause local or augment systemic viral replication. Thus, prompt treatment of chancroid may abrogate increases in viral replication associated with natural disease.
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Chancroide/complicaciones , Infecciones por VIH/complicaciones , Haemophilus ducreyi/fisiología , Adulto , Terapia Antirretroviral Altamente Activa , Recuento de Linfocito CD4 , Chancroide/sangre , Chancroide/patología , Femenino , Infecciones por VIH/sangre , Infecciones por VIH/tratamiento farmacológico , Humanos , Recuento de Leucocitos , Recuento de Linfocitos , Masculino , ARN Viral/sangre , Carga Viral , Replicación ViralRESUMEN
With human volunteers inoculated at two sites with Haemophilus ducreyi, outcomes for a subject were not independent. In a reinfection trial, 2 of 11 previous pustule formers and 6 of 10 previous resolvers resolved all sites of infection. There was no correlation between serum bactericidal or phagocytic activity and outcome in the trial. These data indicate that different hosts are differentially susceptible to disease progression versus resolution in the model.