Abstract
BackgroundThe pathogenic Yersinia species exhibit a primarily extracellular lifestyle through manipulation of host signaling pathways that regulate pro-inflammatory gene expression and cytokine release. To identify host genes that are targeted by Yersinia during the infection process, we performed an RNA interference (RNAi) screen based on recovery of host NF-κB-mediated gene activation in response to TNF-α stimulation upon Y. enterocolitica infection.ResultsWe screened shRNAs against 782 genes in the human kinome and 26 heat shock genes, and identified 19 genes that exhibited ≥40% relative increase in NF-κB reporter gene activity. The identified genes function in multiple cellular processes including MAP and ERK signaling pathways, ion channel activity, and regulation of cell growth. Pre-treatment with small molecule inhibitors specific for the screen hits c-KIT and CKII recovered NF-κB gene activation and/or pro-inflammatory TNF-α cytokine release in multiple cell types, in response to either Y. enterocolitica or Y. pestis infection.ConclusionsWe demonstrate that pathogenic Yersinia exploits c-KIT signaling in a T3SS-dependent manner to downregulate expression of transcription factors EGR1 and RelA/p65, and pro-inflammatory cytokines. This study is the first major functional genomics RNAi screen to elucidate virulence mechanisms of a pathogen that is primarily dependent on extracellular-directed immunomodulation of host signaling pathways for suppression of host immunity.
Highlights
The pathogenic Yersinia species exhibit a primarily extracellular lifestyle through manipulation of host signaling pathways that regulate pro-inflammatory gene expression and cytokine release
We identified 19 host genes that are targeted by Y. enterocolitica to inhibit NF-κB-regulated gene expression and validated their role in host cells infected with Y. pestis, in addition to Y. enterocolitica
We designed our screen (Figure 1B) to select for short hairpin RNA (shRNA) that increased NF-κB-driven luciferase activity ≥40% compared to the mean of all assay reads in Y. enterocolitica-infected, TNF-α stimulated cells for each plate. (Figure 1C, black squares compared to grey squares) we applied a standard z-score method to identify shRNAs that produced a statisticallysignificant recovery (z score ≥3) of luciferase activity (Figure 1D, black diamonds)
Summary
The pathogenic Yersinia species exhibit a primarily extracellular lifestyle through manipulation of host signaling pathways that regulate pro-inflammatory gene expression and cytokine release. In spite of the differences in route of infection and severity of disease, the three species share similar pathogenic mechanisms, primarily the ~70 kb virulence plasmid (pCD1 in Y. pestis and pYV in Y. pseudotuberculosis and Y. enterocolitica) that encodes for the Type III secretion system (T3SS) [1]. Yersinia are unusual amongst other Gram-negative bacteria that express the T3SS, in that they do not actively induce phagocytosis for entry and intracellular growth in the host [5]. Pathogenic Yersinia have been reported to multiply within macrophages early in the infection process [6,7], Y. pestis exponential growth occurs primarily in the extracellular phase, causing acute septicemia with blood counts as high as 108 CFU/ml [8]. Identification of host cell factors that are targeted by Yersinia during infection would provide valuable molecular insights in understanding Yersinia pathogenesis, and in designing effective host-targeted therapies and antimicrobial agents
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