New insights on SepL, the gatekeeper component in Type 3 secretion system of enteropathogenic E. coli, though functional interchangeability studies

Abstract

PhD ThesisThe pathogenesis of many gram-negative bacteria depends on Type Three secretion systems (T3SSs) that deliver subversive effector proteins into the infected host cell. These injectosomes evolved from flagellar export apparatus with significant homology remaining between components that form the T3SS/flagella export channel (which spans the bacterial envelope) and the sorting platform that controls the timing and hierarchy of substrate export. There are distinct T3SS families where high protein homology is a feature within, but not between families. One family is represented by the enteropathogenic E. coli (EPEC) T3SS encoded-alongside genes for T3SS substrates (several T3SS components; translocators (link T3SS to host cell); effectors, chaperones (aid stability/export of T3SS substrates), regulators, and intimin surface protein-on a 41-gene region called LEE (Locus of Enterocyte Effacement). Unexpectedly, LEE was found in an Edwardsiella tarda (E. tarda) strain with genetic rearrangement linked to gene loss and disruption. However, recent studies support functionality with the discovery of unprecedented divergence indicative of a district T3SS family. Preliminary functional interchangeability studies identified E. tarda T3SS proteins that could and, more interestingly, could not functionally substitute their EPEC counterparts. The divergence level did not predict functionality. Studies with support complementation defects for five E. tarda T3SS proteins are described here, which revealed unexpectedly novel functions for SepL (the gatekeeper controlling switching from translocator to effector substrates). Further investigation revealed that i) SepL, 3 (CesT, CesAB, CesD2), an effector (EspF), and two T3SS components (EscC, EscD) each control the cellular O127-antigen level; ii) SepL protects Tir from cleavage; and iii) SepL, CesT, CesAB, and CesD2 protect EspF from cleavage. Cleavage event requires EscU; the latter has auto-proteolytic activity linked to regulating substrate export hierarchy. Importantly, these activities were not shared by the E. tarda homologs with domain swap experiments linking different SepL functionalities to one or more of its three X-bundling domains.Taibah University and the Saudi Arabian Cultural Bureau (SACB