Date Published: August 17, 2018
Publisher: Public Library of Science
Author(s): Dustin J. Little, Brian K. Coombes, Isabelle Derré.
Enteropathogenic Escherichia coli (EPEC) use a needle-like injection apparatus known as the type III secretion system (T3SS) to deliver protein effectors into host cells. Effector translocation is highly stratified in EPEC with the translocated intimin receptor (Tir) being the first effector delivered into the host. CesT is a multi-cargo chaperone that is required for the secretion of Tir and at least 9 other effectors. However, the structural and mechanistic basis for differential effector recognition by CesT remains unclear. Here, we delineated the minimal CesT-binding region on Tir to residues 35–77 and determined the 2.74 Å structure of CesT bound to an N-terminal fragment of Tir. Our structure revealed that the CesT-binding region in the N-terminus of Tir contains an additional conserved sequence, distinct from the known chaperone-binding β-motif, that we termed the CesT-extension motif because it extends the β-sheet core of CesT. This motif is also present in the C-terminus of Tir that we confirmed to be a unique second CesT-binding region. Point mutations that disrupt CesT-binding to the N- or C-terminus of Tir revealed that the newly identified carboxy-terminal CesT-binding region was required for efficient Tir translocation into HeLa cells and pedestal formation. Furthermore, the CesT-extension motif was identified in the N-terminal region of NleH1, NleH2, and EspZ, and mutations that disrupt this motif reduced translocation of these effectors, and in some cases, overall effector stability, thus validating the universality of this CesT-extension motif. The presence of two CesT-binding regions in Tir, along with the presence of the CesT-extension motif in other highly translocated effectors, may contribute to differential cargo recognition by CesT.
Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) cause acute gastroenteritis in humans and are a common source of outbreaks . EPEC is a significant pathogen in the pediatric population, especially in areas with limited access to healthcare and clean water, whereas EHEC is a common food- or water-borne contaminant in industrialized nations . EPEC and EHEC contain a genomic island called the locus of enterocyte effacement (LEE) that encodes a type III secretion system (T3SS)  necessary for the formation of attaching and effacing (A/E) lesions on epithelial cells . The T3SS is a needle-like protein injectisome used by Gram-negative bacteria to deliver effector proteins into host cells directly from the bacterial cytosol , where they target specific host processes to allow for attachment, survival, and propagation of the bacteria [5, 6]. Enteric pathogens that use a T3SS for host attachment, infection, and/or colonization are significantly attenuated when lacking their encoded T3SS , identifying it as a key mediator of host-pathogen interactions.
Tir drives the committal step of intimate attachment between EPEC and the host cell through an extracellular interaction with intimin on the bacterial cell surface [41, 42]. Thus, despite at least 12 effectors having full or partial dependence on CesT for translocation into host cells, Tir is the first effector to be released [22, 23]. This ultimately leads to attaching and effacing lesions from Tir-dependent signaling cascades that cause actin polymerization at the site of attachment, followed by Tir-independent effects on the host cell resulting from the release of secondary effectors . Despite almost two decades of work on Tir and CesT, the mechanism that discriminates Tir secretion over that of other effectors remains unclear. Early transcriptional activation of tir is one possible mechanism to ensure Tir is available first for secretion. However, this does not seem to be the driving mechanism because LEE5 (which contains tir, cesT, and eae) is activated concurrently with LEE2, LEE3, LEE4, and LEE7 approximately 70 minutes after exposure to T3SS inducing conditions . Furthermore, Tir secretion occurs approximately 30 minutes after transcriptional activation , suggesting a post-translational mechanism might drive preferential Tir secretion, as a number of other LEE-encoded effectors would also be present in the cytosol that would require discrimination within the cell. The nature in which Tir interacts with CesT is a possible mechanism by which this discrimination occurs. In this work we identified a second CesT-binding region in the C-terminal domain of Tir, and identify a CesT-extension motif, distinct from the known chaperone-binding β-motif, that is present in the CesT-binding regions of Tir and other highly translocated effectors. Our data raise the possibility that the presence of these features contribute to cytosolic discrimination by CesT, however formal assessment of this hypothesis in the context of the bacterial cell remains to be tested.