Identification of host cell proteins involved in Shigella flexneri pathogenesis.

Abstract

Shigella flexneri is the etiological agent of bacillary dysentery (shigellosis). It is transmitted via the faecal-oral route and is a significant human pathogen due to the high morbidity among children <5 years in developing countries. The key pathogenic features of Shigella include cell death induction in myeloid immune cells and circumventing cell death in colonic epithelial cells, the site of bacterial infection. Shigella also interact with host proteins to initiate de novo actin synthesis to facilitate its intra- and intercellular spread to disseminate in the host. In this thesis, the role of three host proteins: myosin IIA, dynamin II, and dynamin-related protein 1 (Drp1) during Shigella cell-to-cell spreading was examined. The myosin IIA specific kinase, myosin like chain kinase (MLCK), was previously shown to be important for Shigella plaque formation. Myosin IIA and MLCK have also been implicated in septin caging of non-motile Shigella which are targeted for degradation. Chemical inhibition and siRNA knockdown of myosin IIA reduced Shigella plaque formation. Curiously HeLa cells infected with Shigella mutants defective in cell-to-cell spreading have significantly reduced myosin IIA levels when quantified by immunofluorescence microscopy. Dynamin II and Drp1 are members of the dynamin superfamily. Both proteins have self-assembly driven GTPase activation. Dynamin II is important for clathrin-mediated endocytosis and pinches the budding clathrin-coated vesicle, and Drp1 is essential for mitochondrial fission. It was hypothesized that Shigella protrusion formation into adjacent host cells resembles endocytic and exocytic processes, and components of these processes may facilitate Shigella dissemination. When dynamin II GTPase was inhibited with dynasore and dynamin II was knocked down with siRNA, Shigella cell-to-cell spreading was significantly reduced. The in vivo efficacy of dynasore was tested in a murine Sereny model. No significant reduction in inflammation was observed but mice were protected against weight loss during infection. Further experimentation suggested dynasore protected mice against cytotoxic effects from the three secretion system (TTSS) effectors expressed by Shigella during infection. Drp1 was investigated in this thesis as dynasore also inhibits the GTPase of this mitochondrial fission protein. Mitochondrial fission is important in maintaining mitochondrial dynamics and also in events downstream of intrinsic apoptosis and programmed necrosis pathways activation. Loss of mitochondrial function in Shigella-induced epithelial cell death has been reported previously. Hence the role of Drp1 in Shigella plaque formation and HeLa death was examined with the Drp1-specific inhibitor, Mdivi-1, and siRNA knockdown. HeLa cell death was significantly reduced; suggesting loss of mitochondrial function observed previously may now be attributed to Drp1 and subsequent Drp1-mediated mitochondrial fission. The impairment in Shigella cell-to-cell spreading in the absence of Drp1 suggests maintaining an intact mitochondrial network is essential for Shigella lateral spread since loss of Drp1 function would result in excessive mitochondrial fusion, leading to formation of netlike or perinuclear structures. The outcomes of this thesis highlight the importance of host proteins during different stages of Shigella infection. By improving our understanding on the host and bacteria interaction, future work on novel approaches to prevent Shigella dissemination can be developed.