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.
Highlights
Shigellosis was the second leading cause of diarrheal mortality in 2016, accounting for more than 200,000 deaths worldwide (Khalil et al, 2018)
We presented evidence that multiple proteins result from the spa33 gene, some being required for type 3 secretion system (T3SS) function
Our results suggest that these two mutations, one on MxiC and one on Spa33, rescue each other's phenotypes and support that Spa33 is involved in T3SS regulation
Summary
Shigellosis was the second leading cause of diarrheal mortality in 2016, accounting for more than 200,000 deaths worldwide (Khalil et al, 2018). Upon activation of T3SS by host cell contact, the transloca‐ tors IpaB and IpaC, are inserted into the host cell membrane form‐ ing a translocation pore (Blocker et al, 1999; Veenendaal et al, 2007), and releasing IpgC in the cytoplasm. As shown for SpaO and YscQ, an internal translation start codon is present in spa, and leads to the expression of a short carboxy‐terminal variant, called Spa33C (12‐kDa), which interacts with the full‐length protein (Spa33FL, 33‐ kDa) (Bzymek, Hamaoka, & Ghosh, 2012; McDowell et al, 2016; Song et al, 2017).
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