Elucidating the role of Salmonella invasive protein (SIPC) in Salmonella pathogenesis

Sebenzile K Myeni, Purdue University

Abstract

Salmonella species are Gram-negative enteropathogenic bacteria that cause diseases in humans and other animals including gastroenteritis and typhoid fever. Key to the pathogenesis of Salmonella is the type III secretion system (TTSS) used by the bacteria to translocate effector proteins into target host cells to subvert various host cell functions. The targeting of effector proteins across the plasma membrane requires a translocon. This translocon is formed by at least two secreted effector proteins whose functions depend on customized class II TTSS chaperones. In addition, Salmonella can induce its own entry into non-phagocytic cells by engaging and manipulating the actin cytoskeleton machinery. Salmonella SipC is known to have actin-nucleation, F-actin-bundling, and type III effector translocation activities. This study aimed at further characterizing the contributions of the yet undefined domains of the SipC protein in its biochemical (actin bundling) and secretion/translocation activities. In this work, we have demonstrated for the first time that SipC can bind and bundle actin filaments using its C-terminal region. We have separated the actin-bundling activity from its nucleation activity and provide evidence that the SipC mediated F-actin bundling activity contributes to Salmonella invasion in tissue cultured cells and Salmonella -induced inflammation in a mouse infection model. We have also identified the N-terminus amino acid residues of Salmonella SipC that are necessary for its secretion, SicA chaperone binding and the stability of the protein in the bacterial cytosol. In addition, we observed that the loss of SipC's binding ability to its chaperone, SicA, leads to reduced production of SipC which subsequently results in compromised biological activities of the protein during Salmonella invasion. SipC together with SipB and SipD are TTSS translocators which form the translocon in the host cell membranes to allow for the subsequent entry of effectors into the host cell cytoplasm. The direct evidence of entry of effectors through the translocon and the molecular mechanism of the contributions of these proteins in this process is not available. We have found that the formation of the SipB-SipC complex is a prerequisite for plasma membrane insertion/association of SipC and SipB into the host plasma membrane during Salmonella infection. However, through indirect evidence, we have shown that this event is not sufficient for the formation of functional pores in eukaryotic membranes. Our data also illustrated that the oligomerization of SipC might be dispensable in the translocation activities of SipC or intricately regulated during bacterial infection. Finally, we have successfully mapped the secretion, the chaperone-binding, the SipB interaction or plasma membrane localization domains of SipC, and characterized the F-actin bundling modulating activities of SipC.

Degree

Ph.D.

Advisors

Zhou, Purdue University.

Subject Area

Cellular biology|Microbiology|Immunology

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