Role of the Type III Secretion System in a Hypervirulent Lineage of Bordetella bronchiseptica

Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Infection and immunity (Impact Factor: 3.73). 08/2009; 77(9):3969-77. DOI: 10.1128/IAI.01362-08
Source: PubMed


Despite the fact that closely related bacteria can cause different levels of disease, the genetic changes that cause some
isolates to be more pathogenic than others are generally not well understood. We use a combination of approaches to determine
which factors contribute to the increased virulence of a Bordetella bronchiseptica lineage. A strain isolated from a host with B. bronchiseptica-induced disease, strain 1289, was 60-fold more virulent in mice than one isolated from an asymptomatically infected host,
strain RB50. Transcriptome analysis and quantitative reverse transcription-PCR showed that the type III secretion system (TTSS)
genes were more highly expressed by strain 1289 than strain RB50. Compared to strain RB50, strain 1289 exhibited greater TTSS-mediated
cytotoxicity of a mammalian cell line. Additionally, we show that the increase in virulence of strain 1289 compared to that
of RB50 was partially attributable to the TTSS. Using multilocus sequence typing, we identified another strain from the same
lineage as strain 1289. Similar to strain 1289, we implicate the TTSS in the increased virulence of this strain. Together,
our data suggest that the TTSS is involved in the increased virulence of a B. bronchiseptica lineage which appears to be disproportionately associated with disease. These data are consistent with the view that B. bronchiseptica lineages can have different levels of virulence, which may contribute to this species' ability to cause different severities
of respiratory disease.

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Available from: Eric T Harvill, Mar 21, 2014
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    • "Additionally, dye-swap experiments were performed analogously, in which the fluorescent labels were exchanged to ensure that uneven incorporation did not confound our results. Fluorescently-labeled cDNA copies of the total RNA pool were prepared and the two differentially labeled reactions to be compared were then mixed, followed by buffer exchange, purification, and concentration as described [45], [46], [47], [48]. The two differentially labeled reactions to be compared were combined and hybridized to a B. bronchiseptica strain RB50 specific long-oligonucleotide microarray [48]. "
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    ABSTRACT: We have used microarray analysis to study the transcriptome of the bacterial pathogen Bordetella bronchiseptica over the course of five time points representing distinct stages of biofilm development. The results suggest that B. bronchiseptica undergoes a coordinately regulated gene expression program similar to a bacterial developmental process. Expression and subsequent production of the genes encoding flagella, a classical Bvg(-) phase phenotype, occurs and is under tight regulatory control during B. bronchiseptica biofilm development. Using mutational analysis, we demonstrate that flagella production at the appropriate stage of biofilm development, i.e. production early subsequently followed by repression, is required for robust biofilm formation and maturation. We also demonstrate that flagella are necessary and enhance the initial cell-surface interactions, thereby providing mechanistic information on the initial stages of biofilm development for B. bronchiseptica. Biofilm formation by B. bronchiseptica involves the production of both Bvg-activated and Bvg-repressed factors followed by the repression of factors that inhibit formation of mature biofilms.
    PLoS ONE 11/2012; 7(11):e49166. DOI:10.1371/journal.pone.0049166 · 3.23 Impact Factor
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    • "Additionally, dye-swap experiments were performed analogously, in which the fluorescent labels were exchanged to ensure that uneven incorporation did not confound our results. Fluorescently-labeled cDNA copies of the total RNA pool were prepared by direct incorporation of fluorescent nucleotide analogs during a first-strand reverse transcription (RT) reaction [39], [45]–[47]. The two differentially labeled reactions were then combined and directly hybridized to a B. bronchiseptica strain RB50-specific long-oligonucleotide microarray [46]. "
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    ABSTRACT: Sensing the environment allows pathogenic bacteria to coordinately regulate gene expression to maximize survival within or outside of a host. Here we show that Bordetella species regulate virulence factor expression in response to carbon dioxide levels that mimic in vivo conditions within the respiratory tract. We found strains of Bordetella bronchiseptica that did not produce adenylate cyclase toxin (ACT) when grown in liquid or solid media with ambient air aeration, but produced ACT and additional antigens when grown in air supplemented to 5% CO(2). Transcriptome analysis and quantitative real time-PCR analysis revealed that strain 761, as well as strain RB50, increased transcription of genes encoding ACT, filamentous hemagglutinin (FHA), pertactin, fimbriae and the type III secretion system in 5% CO(2) conditions, relative to ambient air. Furthermore, transcription of cyaA and fhaB in response to 5% CO(2) was increased even in the absence of BvgS. In vitro analysis also revealed increases in cytotoxicity and adherence when strains were grown in 5% CO(2). The human pathogens B. pertussis and B. parapertussis also increased transcription of several virulence factors when grown in 5% CO(2), indicating that this response is conserved among the classical bordetellae. Together, our data indicate that Bordetella species can sense and respond to physiologically relevant changes in CO(2) concentrations by regulating virulence factors important for colonization, persistence and evasion of the host immune response.
    PLoS ONE 10/2012; 7(10):e47635. DOI:10.1371/journal.pone.0047635 · 3.23 Impact Factor
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    • "When macrophages were infected with either RB50 at a MOI of 1, we observed increasing levels of cytotoxicity over the first 6 hours of incubation (Figure 3A). By six hours, 71% of macrophages were lysed by RB50, as previously described [35], [61], [62]. Minimal cytotoxicity was observed in macrophages exposed to RB50ΔclpV at any point throughout the 6 hour incubation (Figure 3A). "
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    ABSTRACT: Type VI Secretion Systems (T6SSs) have been identified in numerous Gram-negative pathogens, but the lack of a natural host infection model has limited analysis of T6SS contributions to infection and pathogenesis. Here, we describe disruption of a gene within locus encoding a putative T6SS in Bordetella bronchiseptica strain RB50, a respiratory pathogen that circulates in a broad range of mammals, including humans, domestic animals, and mice. The 26 gene locus encoding the B. bronchiseptica T6SS contains apparent orthologs to all known core genes and possesses thirteen novel genes. By generating an in frame deletion of clpV, which encodes a putative ATPase required for some T6SS-dependent protein secretion, we observe that ClpV contributes to in vitro macrophage cytotoxicity while inducing several eukaryotic proteins associated with apoptosis. Additionally, ClpV is required for induction of IL-1β, IL-6, IL-17, and IL-10 production in J774 macrophages infected with RB50. During infections in wild type mice, we determined that ClpV contributes to altered cytokine production, increased pathology, delayed lower respiratory tract clearance, and long term nasal cavity persistence. Together, these results reveal a natural host infection system in which to interrogate T6SS contributions to immunomodulation and pathogenesis.
    PLoS ONE 10/2012; 7(10):e45892. DOI:10.1371/journal.pone.0045892 · 3.23 Impact Factor
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