Computational Analysis of the ESX-1 Region of Mycobacterium tuberculosis : Insights into the Mechanism of Type VII Secretion System Chandrani Das., Tarini Shankar Ghosh . , Sharmila S Mande* Bio-sciences R& D Division, Tata Consultancy ServicesInnovation Labs, Tata Consultancy Services Ltd, Hyderabad, Andhra Pradesh, India Abstract Type VII secretion system (T7SS) is a recent discovery in bacterial secretion systems. First identified in Mycobacterium tuberculosis, this secretion system has later been reported in organisms belonging to the Actinomycetales order and even to distant phyla like Firmicutes. The genome of M. tuberculosis H37Rv contains five gene clusters that have evolved through gene duplication events and include components of the T7SS secretion machinery. These clusters are called ESAT-6 secretion system (ESX) 1 through 5. Out of these, ESX-1 has been the most widely studied region because of its pathological importance. In spite of this, the overall mechanism of protein translocation through ESX-1 secretion machinery is not clearly understood. Specifically, the structural components contributing to the translocation through the mycomembrane have not been characterized yet. In this study, we have carried out a comprehensive in silico analysis of the genes known to be involved in ESX-1 secretion pathway and identified putative proteins having high probability to be associated with this particular pathway. Our study includes analysis of phylogenetic profiles, identification of domains, transmembrane helices, 3D folds, signal peptides and prediction of protein-protein associations. Based on our analysis, we could assign probable novel functions to a few of the ESX-1 components. Additionally, we have identified a few proteins with probable role in the initial activation and formation of mycomembrane translocon of ESX-1 secretion machinery. We also propose a probable working model of T7SS involving ESX-1 secretion pathway. Citation: Das C, Ghosh TS, Mande SS (2011) Computational Analysis of the ESX-1 Region of Mycobacterium tuberculosis: Insights into the Mechanism of Type VII Secretion System. PLoS ONE 6(11): e27980. doi:10.1371/journal.pone.0027980 Editor: Niyaz Ahmed, University of Hyderabad, India Received August 16, 2011; Accepted October 28, 2011; Published November 30, 2011 Copyright: 2011 Das 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. Funding: No current external funding sources for this study. Competing Interests: Although all the authors are employed by a commercial company, (Tata Consultancy Services Ltd.), this does not alter their adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: sharmila@atc.tcs.com . These authors contributed equally to this work. Introduction Bacterial secretion systems are responsible for the export of virulence factors either to the extracellular environment or directly into the host cell and thus, play a crucial role in the virulence of a pathogen [1]. Currently, seven categories of secretion systems (Type I to Type VII) have been identified in bacteria [1–3]. These secretion systems not only differ in terms of the secreted effector molecules, but also in their structural components. While Type I, II, III, V and VI have been found to be typically associated with Gram- negative bacteria, Type IV is found in both Gram-positive as well as Gram-negative bacteria. The most recently categorized Type VII secretion system (T7SS) is observed to be present in the Gram- positive species, mostly belonging to the Actinomycetales order [4]. A few components related to the T7SS have also been identified in some species belonging to the phylum Firmicutes [4–6]. The T7SS was first identified in the pathogenic organism Mycobacterium tuberculosis H37Rv and the corresponding gene clusters were later referred to as the ESX (ESAT-6 Secretion System) regions [2–4,7]. The T7SS has been shown to secrete proteins lacking classical signal peptides in contrast to that observed in Type II, IV and V secretion systems. Furthermore, most of the proteins secreted by T7SS follow a pairwise dependency, both for secretion and function [8]. The first ESX region (ESX-1) was discovered during the comparative genomic analysis of the attenuated strain Mycobacte- rium bovis Bacille Calmette-Guerin (BCG) and other pathogenic mycobacterial species [9]. It was observed that the genome of the M. bovis BCG had ten different regions of deletion (RD1-RD10) as compared to that of M. tuberculosis. Out of these, the region RD1, containing a total of nine genes including the genes encoding the secreted antigens CFP-10 and ESAT-6, was observed to be especially responsible for virulence [10,11]. Restoration of this region not only enabled the secretion of ESAT-6, but also led to increased virulence in M. bovis BCG [10], implicating the role of the genes in RD1 region in the virulence of the bacteria. Concurrently, several computational studies have attempted to predict the functional role of the genes encoded in the RD1 region [12,13]. It was predicted that, this region contained genes encoding ATP dependent motors, a number of transmembrane proteins, a protease and secretory proteins [13]. Furthermore, most of the genes encoded in this region lacked significant similarity to previously characterized proteins. Based on these observations, Pitius et al. (2001) hypothesized that, the RD1 region (ESX-1) in the Mycobacterium genus encodes components of a novel secretion system. Apart from ESX-1, four other gene clusters (ESX-2 to ESX-5) were subsequently discovered in M. tuberculosis PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27980