The Serine Protease EspC from Enteropathogenic Escherichia coli Regulates Pore Formation and Cytotoxicity Mediated by the Type III Secretion System

RESEARCH ARTICLE The Serine Protease EspC from Enteropathogenic Escherichia coli Regulates Pore Formation and Cytotoxicity Mediated by the Type III Secretion System Julie Guignot1,2,3,4, Audrey Segura1,2,3,4, Guy Tran Van Nhieu1,2,3,4* 1 Equipe Communication Intercellulaire et Infections Microbiennes, Centre de Recherche Interdisciplinaire en Biologie (CIRB), Collège de France, Paris, France, 2 Institut National de la Santé et de la Recherche Médicale U1050, Paris, France, 3 Centre National de la Recherche Scientifique UMR7241, Paris, France, 4 MEMOLIFE Laboratory of Excellence and Paris Science Lettre, Paris, France * Abstract OPEN ACCESS Citation: Guignot J, Segura A, Tran Van Nhieu G (2015) The Serine Protease EspC from Enteropathogenic Escherichia coli Regulates Pore Formation and Cytotoxicity Mediated by the Type III Secretion System. PLoS Pathog 11(7): e1005013. doi:10.1371/journal.ppat.1005013 Editor: Tomoko Kubori, Osaka University, JAPAN Received: February 16, 2015 Accepted: June 8, 2015 Published: July 1, 2015 Copyright: © 2015 Guignot et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by grants from the Inserm, the CNRS, the Collège de France and the LABEX MEMOLIFE Laboratory of Excellence and Paris Sciences et Lettres, Paris 75005. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Type III secretion systems (T3SSs) are specialized macromolecular machines critical for bacterial virulence, and allowing the injection of bacterial effectors into host cells. The T3SS-dependent injection process requires the prior insertion of a protein complex, the translocon, into host cell membranes consisting of two-T3SS hydrophobic proteins, associated with pore-forming activity. In all described T3SS to date, a hydrophilic protein connects one hydrophobic component to the T3SS needle, presumably insuring the continuum between the hollow needle and the translocon. In the case of Enteropathogenic Escherichia coli (EPEC), the hydrophilic component EspA polymerizes into a filament connecting the T3SS needle to the translocon composed of the EspB and EspD hydrophobic proteins. Here, we identify EspA and EspD as targets of EspC, a serine protease autotransporter of Enterobacteriaceae (SPATE). We found that in vitro, EspC preferentially targets EspA associated with EspD, but was less efficient at proteolyzing EspA alone. Consistently, we found that EspC did not regulate EspA filaments at the surface of primed bacteria that was devoid of EspD, but controlled the levels of EspD and EspA secreted in vitro or upon cell contact. While still proficient for T3SS-mediated injection of bacterial effectors and cytoskeletal reorganization, an espC mutant showed increased levels of cell-associated EspA and EspD, as well as increased pore formation activity associated with cytotoxicity. EspP from enterohaemorrhagic E. coli (EHEC) also targeted translocator components and its activity was interchangeable with that of EspC, suggesting a common and important function of these SPATEs. These findings reveal a novel regulatory mechanism of T3SS-mediated pore formation and cytotoxicity control during EPEC/EHEC infection. Competing Interests: The authors have declared that no competing interests exist. PLOS Pathogens | DOI:10.1371/journal.ppat.1005013 July 1, 2015 1 / 23 EspC Regulates Type III Secretion Induced Pores Author Summary Enteropathogenic Escherichia coli (EPEC) is an important diarrheal pathogen responsible for infant diarrhoea associated with significant morbidity and mortality rates in developing countries. Upon ingestion EPEC colonizes the intestinal mucosa, causing characteristic lesions on enterocytes. Using a type III secretion system (T3SS) acting as a molecular syringe, EPEC injects numerous bacterial proteins into host cells that disrupt the intestinal epithelium homeostasis. Injection of T3SS proteins requires the insertion into the host cell plasma membrane of bacterial protein complex, called the "translocon", associated with pore-forming activity. In addition to the T3SS, EPEC also secretes other bacterial toxins involved in virulence. Among these, the EspC is a protease reported to degrade various host proteins. In this paper, we have characterized an "unsuspected role" for EspC. We show that EspC degrades the T3SS translocon components following cell contact and regulates T3SS-dependent pore formation in epithelial cells. The EspC control of pore formation limits cytotoxicity and thus, is expected to limit the emission of danger signals, which would otherwise favour bacterial clearance at the onset of infection. This work describes a novel regulatory mechanism of pore formation mediated by the T3SS, that are likely to be relevant for other extracellular pathogens. Introduction EPEC and EHEC are related pathogens causing severe diarrhoeal diseases. EPEC and EHEC form Attaching and Effacing (A/E) lesions on the mucosal intestinal surface, corresponding to the destruction of enterocyte microvilli and the intimate bacterial adherence to the host cell plasma membrane onto an actin-rich pedestal structure [1]. A/E pathogens carry the Locus of Enterocyte Effacement (LEE) encoding a type III secretion apparatus (T3SA) that allows the delivery of bacterial effector proteins directly from the bacterial cytoplasm into the cytoplasm of eukaryotic cells [2]. The translocator proteins EspA, B and D are required for the injection of type III effectors. Upon cell contact, EspB and EspD insert into the host cell plasma membrane and associate into a pore-forming “translocon” complex. The hydrophilic translocator protein EspA polymerizes into a hollow filamentous structure connecting the T3SA needle to the translocon [3]. SPATEs are serine protease autotransporters that are widely spread among Enterobacteriaceae. SPATEs have been reported to cleave host proteins implicated in diverse functions [4,5]. EspC has been described to cleave focal adhesion proteins, following cellular internalization, as well as other eukaryotic proteins such as haemoglobin, pepsin and human coagulation factor V [5,6,7]. Epidemiological studies indicated that EspC was predominantly found in t-EPEC strains and that EPEC strains carrying EspC and the OI-122 pathogenicity island were associated with high virulence [8,9]. These observations suggest that EspC could contribute to bacterial virulence by regulating the action of virulence factors. Although secretion of EspC occurs through a T3SS-independent mechanism, several intriguing features link EspC and the T3SS. The expression of EspC is coupled to that of t (...truncated)