CesT is a bivalent enteropathogenicEscherichia colichaperone required for translocation of both Tir and Map

Abstract

SummaryMap is an enteropathogenic Escherichia coli (EPEC) protein that is translocated into eukaryotic cells by a type III secretion system. Although not required for the induction of attaching and effacing (A/E) lesion formation characteristic of EPEC infection, translocated Map is suggested to disrupt mitochondrial membrane potential, which may impact upon subsequent functions of the organelle such as control of cell death. Before secretion, many effector proteins are maintained in the bacterial cytosol by association with a specific chaperone. In EPEC, chaperones have been identified for the effector proteins translocated intimin receptor (Tir) and EspF, and for the translocator proteins EspB and EspD. In this study, we present evidence that the Tir-specific chaperone, CesT, also performs a chaperone function for Map. Using a combination of biochemical approaches, we demonstrate specific interaction between CesT and Map. Similar to other chaperone-effector pairings, binding is apparent at the amino-terminus of Map and is indicated to proceed by a similar mechanism to CesT:Tir interaction. Map secretion from a cesT mutant strain (SE884) is shown to be reduced and, importantly, its translocation from this strain after infection of HEp-2 cells is almost totally abrogated. Although other chaperones are reported to have a bivalent binding specificity, CesT is the first member of its family that chaperones more than one protein for translocation.

Hierarchical Delivery of an Essential Host Colonization Factor in Enteropathogenic Escherichia coli

“…1). Indeed, it has been reported that the CesT-interacting type III effector Map is expressed at very low levels within EPEC (32), which may explain why it was not detected in our analyses. Nonetheless, from our observations and those from other reports (32-34), CesT physically interacts or is involved in the secretion of every LEE-encoded effector described to date, further strengthening its central role in EPEC pathogenesis.…”

“…30 -34). Recently, we demonstrated that the efficient secretion of many other EPEC type III effector proteins also required CesT (32,33), suggesting a possible interaction of these proteins with CesT. To identify new CesT interacting type III effector proteins, we took advantage of a ⌬sepD strain of EPEC that is known to hypersecrete type III effector proteins but not translocator proteins (38).…”

“…In the case of EPEC, the LEE encoded type III secretion chaperone CesT was initially shown to bind and stabilize Tir within the bacterial cell (30,31); however, additional studies have demonstrated interactions with the effectors Map, EspF, and NleA (32)(33)(34). CesT is also required for the efficient in vitro type III secretion of other LEE and non-LEE type III effectors (33), suggesting that other chaperone-effector interactions may occur within the bacterium.…”

Nck adaptors, besides promoting N-WASP mediated actin-nucleation activity at pedestals, influence the cellular levels of enteropathogenicEscherichia coliTir effector

“…6 The chaperone CesT binds and enhances the translocation of Tir, Map and other LEE-effectors (EspH, EspJ, EspZ) and non-LEE (Nle) effectors (A, B1, B2, C, G, H1 and H2). 7,42 With the aim of analyzing the levels of Map, another CesT-dependent effector, we infected Nck-deficient cells with EPEC which carried a plasmid expressing Map fused in frame, to enable WB detection, with haemagglutinin (HA) and T7 epitope tags (WTCT7-map-HA).…”

Dual temporal transcription activation mechanisms control cesT expression in enteropathogenic Escherichia coli

“…The cesT gene encodes a multicargo type III secretion chaperone protein. CesT was initially discovered to be the chaperone for E. coli secreted protein Tir (Abe et al, 1999;Elliott et al, 1999), although subsequent studies implicated CesT roles for other type III effectors that are encoded within and outside of the LEE (Creasey et al, 2003;Thomas et al, 2005). LEE5 gene regulation is multifaceted, with at least two known promoter elements (Sánchez-SanMartín et al, 2001).…”