Multiple proteins arising from a single gene: The role of the Spa33 variants in Shigella T3SS regulation

Abstract

Shigella invasion and dissemination in intestinal epithelial cells relies on a type 3 secretion system (T3SS), which mediates translocation of virulence proteins into host cells. T3SSs are composed of three major parts: an extracellular needle, a basal body, and a cytoplasmic complex. Three categories of proteins are hierarchically secreted: (a) the needle components, (b) the translocator proteins which form a pore (translocon) inside the host cell membrane and (c) the effectors interfering with the host cell signaling pathways. In the absence of host cell contact, the T3SS is maintained in an “off” state by the presence of a tip complex. Secretion is activated by host cell contact which allows the release of a gatekeeper protein called MxiC. In this work, we have investigated the role of Spa33, a component of the cytoplasmic complex, in the regulation of secretion. The spa33 gene encodes a 33‐kDa protein and a smaller fragment of 12 kDa (Spa33C) which are both essential components of the cytoplasmic complex. We have shown that the spa33 gene gives rise to 5 fragments of various sizes. Among them, three are necessary for T3SS. Interestingly, we have shown that Spa33 is implicated in the regulation of secretion. Indeed, the mutation of a single residue in Spa33 induces an effector mutant phenotype, in which MxiC is sequestered. Moreover, we have shown a direct interaction between Spa33 and MxiC.

Composition and Biophysical Properties of the Sorting Platform Pods in the Shigella Type III Secretion System

“…The contrasting findings indicate a need to better understand the actual makeup and organization of the SP pods in Shigella . Even more recently, additional Spa33 protein variants were identified, including a 6.5 kDa form called Spa33 CC (found to be important for injectisome activity) and two other Spa33 variants (called Spa33 N and Spa33 X ) that have no obvious functions and may be translational artifacts ( Kadari et al., 2019 ). Although we have also observed production of Spa33 CC (unpublished observation), it will not be considered further here since it does not associate with the Spa33 heterotrimer.…”

The Shigella Type III Secretion System: An Overview from Top to Bottom

“…When purified as a recombinant protein, Spa33 gives rise to a heterotrimeric structure containing one full-length copy of the protein and two smaller C-terminal fragments, Spa33 C-term , generated from an internal ribosome-binding site present in the mRNA [127]. This also appears to be the case for other members of the SctQ family [129][130][131][132]. While the structure of full-length Spa33 has not yet been determined, the crystal structure of Spa33 C-term was solved and, based on the known structure of the homologous flagellar C-ring protein FliN from Thermatoga maritima, initially led to a model in which the main component of the sorting platform is a toroid structure [127].…”

“…Nevertheless, it does appear that the C-terminal variant is required to maintain the solubility of the full-length protein and for interactions involving other sorting platform components outside of the context of the sorting platform itself [ 130 ]. Interestingly, a report by Kadari et al suggests that there are additional Spa33 variants and some may interact at other sites within the injectisome [ 132 ]. Based on the size of the Spa33 pods in the Shigella sorting platform, it is not clear that 24 copies of full-length Spa33 could be accommodated [ 28 ], which indirectly suggests that Spa33 C-term is a likely component of the pods.…”

Adaptivity and dynamics in type III secretion systems

“…Another possibility is that-just like in the flagellum-the cytosolic components have a regulatory role. In support of such a function, SctQ binds the main gatekeeper protein SctW that prevents premature secretion of effectors (Kadari et al, 2019).…”

“…SctQ harbors two SpoA domains which can engage in a variety of protein interactions, both within the T3SS (e.g., to SctQ C , which has one SpoA domain) and to external components. In line with its high degree of structural and functional variety, SctQ may therefore be a hotspot for structural and functional adaptations of the T3SS (Diepold, 2019;Kadari et al, 2019;Lam et al, 2018;Lara-Tejero, 2019). and Berg, 1984;Reid et al, 2006).…”