EspB. In this study, CesAB (Orf3 of the LEE) was identified as a chaperone for
EspA and EspB. Specific ... dimer and bind a discrete region in the N-terminal
part.
Microbiology (2003), 149, 3639–3647
DOI 10.1099/mic.0.26735-0
CesAB is an enteropathogenic Escherichia coli chaperone for the type-III translocator proteins EspA and EspB Elizabeth A. Creasey,1 Devorah Friedberg,2 Robert K. Shaw,3 Tatiana Umanski,2 Stuart Knutton,3 Ilan Rosenshine2 and Gad Frankel1 Correspondence Gadi Frankel
[email protected]
1
Centre for Molecular Microbiology and Infection, Department of Biological Sciences, Imperial College, London SW7 2AZ
2
Departments of Molecular Genetics and Biotechnology, The Hebrew University, Faculty of Medicine, POB 12272, Jerusalem 91120, Israel
3
Institute of Child Health, University of Birmingham, Birmingham B4 6NH, UK
Received 27 August 2003 Revised
13 October 2003
Accepted 15 October 2003
Enteropathogenic Escherichia coli (EPEC) are extracellular pathogens that colonize mucosal surfaces of the intestine via formation of attaching and effacing (A/E) lesions. The genes responsible for induction of the A/E lesions are located on a pathogenicity island, termed the locus of enterocyte effacement (LEE), which encodes the adhesin intimin and the type III secretion system needle complex, translocator and effector proteins. One of the major EPEC translocator proteins, EspA, forms a filamentous conduit along which secreted proteins travel before they arrive at the translocation pore in the plasma membrane of the host cell, which is composed of EspB and EspD. Prior to secretion, many type III proteins, including translocators, are maintained in the bacterial cytoplasm by association with a specific chaperone. In EPEC, chaperones have been identified for the effector proteins Tir, Map and EspF, and the translocator proteins EspD and EspB. In this study, CesAB (Orf3 of the LEE) was identified as a chaperone for EspA and EspB. Specific CesAB–EspA and CesAB–EspB protein interactions are demonstrated. CesAB was essential for stability of EspA within the bacterial cell prior to secretion. Furthermore, a cesAB mutant failed to secrete EspA, as well as EspB, to assemble EspA filaments, to induce A/E lesion following infection of HEp-2 cells and to adhere to, or cause haemolysis of, erythrocytes.
INTRODUCTION Enteropathogenic Escherichia coli (EPEC) is the archetype of a group of pathogens that adhere tightly to host enterocytes and cause extensive host cell cytoskeletal rearrangements resulting in the formation of a pedestal beneath the adherent bacteria and the localized destruction of microvilli, a phenomenon known as the attaching and effacing (A/E) lesion (reviewed by Frankel et al., 1998). The genes encoding the A/E phenotype are located in the locus of enterocyte effacement (LEE), a pathogenicity island present in the chromosome of all A/E pathogens (McDaniel et al., 1995). Characterization of this locus revealed that it encodes a type III secretion system (TTSS; Jarvis et al., 1995), translocator and effector proteins, their chaperones and the adhesin intimin (Elliott et al., 1998). TTSSs are utilized by many Gram-negative pathogens to deliver virulence factors or effectors, directly into host cells Abbreviations: A/E, attaching and effacing; EPEC, enteropathogenic Escherichia coli; FAS, fluorescence actin staining; LEE, locus of enterocyte effacement; RBC, red blood cells; TTSS, type III secretion system.
0002-6735 G 2003 SGM
(reviewed by Hueck, 1998). Components of the delivery apparatus are highly conserved among TTSSs and secretion complexes have very similar organization (Kubori et al., 1998; Tamano et al., 2000; Blocker et al., 2001; Sekiya et al., 2001; Daniell et al., 2001). The secretion apparatus spans both bacterial membranes and extends from the bacterial cell via a needle-like projection. In order to translocate proteins across the host cell’s membrane, a pore is formed by type III secreted proteins known as translocators (reviewed by Buttner & Bonas, 2002). In addition to these common structures, the LEE-encoded TTSS possesses a long filamentous structure that connects the distal end of the needle structure to the host cell membrane (Knutton et al., 1998; Daniell et al., 2001; Sekiya et al., 2001; Wilson et al., 2001). The filament is composed of many copies of EspA forming a helical bundle with a central pore through which secreted proteins may pass (Daniell et al., 2003). Secretion of many type III substrates requires the presence of cytoplasmic chaperones. There is little or no sequence similarity between TTSS chaperones; however, they have some common characteristics, i.e. low molecular mass
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