Shigella virulence factor virk and its homologue ybjx contribute to membrane permeability and the host immune response

Background and Aims In Shigella flexneri, the etiological agent of shigellosis, pathogenesis is the sum of the actions of virulence factors mainly encoded by the virulence plasmid. We analysed the role played by the pINV-encoded VirK protein and by its structural homologue YbjX, encoded by a chromosomal gene and not characterised in Shigella. Methods We performed molecular analysis of ybjX and virK loci. Additionally, we evaluated the effect of gene deletion in vitro and in vivo by performing susceptibility assays to antimicrobial compounds and infection experiments. Results We observe that both ybjX and virK promoters are controlled by the PhoPQ two-component system. We also show that the loss of VirK and/or YbjX affects membrane permeability increasing the susceptibility to polymyxin-B. Both virK and ybjX genes are expressed during the infection of THP-1-derived macrophages and CaCo-2 epithelial cells; ybjX induction is significant during the infection of both cell types, while the induction of virK is significant only within epithelial cells. Infection with strains lacking virK and/or ybjX increases the release of IL-1b by THP-1 and IL-8 by CaCo-2, suggesting a modulation of the host inflammatory response. We noticed no difference when we infected HEK-293 cells, whose IL-8 secretion mostly depends on NOD receptors, suggesting that the increase in IL-8 by CaCo-2 cells may rely on the TLR stimulation probably due to an alteration of LPS structure and/or release. Conclusions Altogether, this study sheds light on poorly characterised Shigella virK and ybjX genes, suggesting a role in membrane integrity and, possibly, the evasion of the host immune response.