Steps for Shigella Gatekeeper Protein MxiC Function in Hierarchical Type III Secretion Regulation

Abstract

Type III secretion systems are complex nanomachines used for injection of proteins from Gram-negative bacteria into eukaryotic cells. Although they are assembled when the environmental conditions are appropriate, they only start secreting upon contact with a host cell. Secretion is hierarchical. First, the pore-forming translocators are released. Second, effector proteins are injected. Hierarchy between these protein classes is mediated by a conserved gatekeeper protein, MxiC, in Shigella. As its molecular mechanism of action is still poorly understood, we used its structure to guide site-directed mutagenesis and to dissect its function. We identified mutants predominantly affecting all known features of MxiC regulation as follows: secretion of translocators, MxiC and/or effectors. Using molecular genetics, we then mapped at which point in the regulatory cascade the mutants were affected. Analysis of some of these mutants led us to a set of electron paramagnetic resonance experiments that provide evidence that MxiC interacts directly with IpaD. We suggest how this interaction regulates a switch in its conformation that is key to its functions.

The inner‐rod component of Shigella flexneri type 3 secretion system, MxiI, is involved in the transmission of the secretion activation signal by its interaction with MxiC

“…Indeed, mutation in MxiC that abolishes MxiI interaction (MxiC F206S ) leads to a mxiC ‐mutant phenotype in terms of early effectors secretion (Cherradi et al., ). Nevertheless, this mutation still allows CR response as a wild‐type strain suggesting that the two functions of MxiC can be uncoupled, as confirmed recently (Roehrich et al., ). To strengthen our model, we have looked for a point mutation in MxiI that, by losing MxiC binding capacities, will also lead to the loss of effectors secretion control ( mxiC ‐like phenotype).…”

“…Eluted fractions (EF) were resolved by SDS-PAGE and analyzed by Coomassie blue staining and by Western blot using monoclonal antibodies against His-tag. All experiments were performed at least three times strain suggesting that the two functions of MxiC can be uncoupled, as confirmed recently (Roehrich et al, 2016). To strengthen our model, we have looked for a point mutation in MxiI that, by losing MxiC binding capacities, will also lead to the loss of effectors secretion control (mxiC-like phenotype).…”

Hierarchical protein targeting and secretion is controlled by an affinity switch in the type III secretion system of enteropathogenic Escherichia coli

“…SepL and homologous gatekeepers (e.g., MxiC, CopN, YopN/TyeA) are considered as exported, effector-like proteins with N-terminal signal peptides (Fields & Hackstadt, 2000;Botteaux et al, 2009;Younis et al, 2010;Amer et al, 2016). However, if and how their own export relates to that of translocators or effectors is unclear (Roehrich et al, 2017). Gatekeepers might recognize specific sequences that lie downstream of the N-terminal signal sequences of translocators, for example, in EspB (Deng et al, 2015;Roehrich et al, 2017).…”

“…However, if and how their own export relates to that of translocators or effectors is unclear (Roehrich et al, 2017). Gatekeepers might recognize specific sequences that lie downstream of the N-terminal signal sequences of translocators, for example, in EspB (Deng et al, 2015;Roehrich et al, 2017). We anticipate that the novel receptor role revealed here and analyzed in detail for CesAB might be universal for translocator chaperones, even those structurally distinct from CesAB [e.g., the CesD and CesD2 class III chaperones of EspB and D (Wainwright & Kaper, 1998;Neves et al, 2003)].…”

Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

“…S. flexneri 5a M90T, along with S. flexneri 2a, is one of the most commonly employed laboratory reference strain for S. flexneri in many independent research groups across the globe [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]. Indeed, much of our knowledge of the molecular mechanisms of Shigella pathogenesis has been obtained using S. flexneri M90T as a model.…”