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.
Highlights
Conserved Type VI Secretion System (T6SS) gene clusters have been recently identified in 92 different strains of bacteria [1]
We characterize a contiguous 26 gene locus in B. bronchiseptica strain RB50 predicted to encode proteins sharing high amino acid sequence similarity with highly conserved T6SS proteins found in V. cholerae, S. enterica and P. aeruginosa (Figure 1)
As with multiple other pathogens, we find that mutation of clpV affects macrophages cytotoxicity in vitro and that ClpV-dependent interactions with macrophages result in proteomic changes consistent with apoptotic responses
Summary
Conserved Type VI Secretion System (T6SS) gene clusters have been recently identified in 92 different strains of bacteria [1]. T6SS loci are disproportionately associated with virulent strains, and multiple virulence-related phenotypes have been attributed to the T6SS in pathogenic bacteria, including mucosal adherence, intracellular growth within macrophages, survival within host cells, and the delivery of bacteriolytic proteins into competitor bacteria [1,2,3,4,5]. In Vibrio cholerae [6], Aeromonas hydrophila [7], and Legionella pneumophila [8], T6SS activity enables macrophage cytotoxicity, while T6SSs of Salmonella typhimurium and Yersinia pseudotuberculosis facilitate HEp-2 cell invasion [9]. Abrogating T6SS functions is associated with reduced virulence in vivo of Aeromonas hydrophila in a mouse model of septicemia [10], Pseudomonas aeruginosa in neutropenic mice [11], V. cholera in infant mice and rabbits [12,13], and Burkholderia mallei in hamsters [14]. Despite evidence that the T6SS enables virulence in multiple species, many of the discrete, in vivo interactions between the T6SS and host immunity have not yet been determined
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