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Berlin Brandenburg

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  • 1
    Language: English
    In: mBio, 11 February 2014, Vol.5(1), pp.e01081-13
    Description: 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. 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.
    Keywords: Gene Expression Regulation, Bacterial ; Haemophilus Ducreyi -- Genetics ; Host Factor 1 Protein -- Metabolism ; Virulence Factors -- Biosynthesis
    E-ISSN: 2150-7511
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  • 2
    Language: English
    In: mBio, 15 September 2015, Vol.6(5), pp.e01315-15
    Description: 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. 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.
    Keywords: Microbiota ; Haemophilus Ducreyi -- Growth & Development ; Skin -- Microbiology ; Skin Diseases, Bacterial -- Microbiology
    E-ISSN: 2150-7511
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  • 3
    Language: English
    In: mBio, 01 June 2019, Vol.10(3), p.e01193-19
    Description: 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.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.
    Keywords: Dual RNA-Seq ; Haemophilus Ducreyi ; Human Infection Model ; Interactome ; Metabolome ; Biology
    ISSN: 21612129
    E-ISSN: 2150-7511
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