various effector proteins by the Type VII secretion system. The proposed model provides a hypothesis based framework for the further characterization of the T7SS. Functional assignments of ESX-1 components The proteins associated with the ESX-1 secretion pathway can be broadly classified into three groups – (A) secreted proteins, (B) regulatory proteins and (C) structural proteins. Details of these proteins and their probable mechanisms of action/regulation are described in the following sub-sections. (A) Secreted proteins. The experimentally identified substrates of ESX-1 secretion pathway include CFP-10, ESAT-6, EspA, EspC and EspB [3,7,8,14,18,21]. CFP-10 and ESAT-6 are known to form pairs, which adopt stable four-helix-bundle structures [8,16,21,22]. Similarly, EspA and EspC have been shown to be dependent on each other and on the CFP-10/ESAT- 6 pair for their stability and secretion [8,21]. Interestingly, in the present study, the prediction of helix-turn-helix structures (for both EspA and EspC) and the detection of conserved WXG motifs linking the helices (in EspA) indicates that, EspC and EspA probably interact with each other, forming a four-helix-bundle (similar to that formed by the CFP-10/ESAT-6 pair). EspB, another secretory protein, was previously reported to interact with Rv3879c and Rv3871 for its secretion and was also shown to regulate the secretion of CFP-10/ESAT-6 [15]. While Rv3879c was predicted to remain within the cell, EspB was shown to be secreted out. However, a separate study identified Rv3879c as a potential T-cell antigen [34]. The observation that Rv3879c is able to induce T-cell response, suggests that, Rv3879c is either secreted or is partially anchored to the mycomembrane (i.e. it undergoes at least an inner membrane translocation). Our analysis predicts both EspB and Rv3879c to contain helical structures with conserved WXG motifs (similar to CFP-10/ESAT-6), connecting the two helices. Thus, the results obtained from previous studies (EspB interacting with Rv3879c) and from our 3D fold analysis suggest that, EspB and Rv3879c probably pair up to form a four- helix-bundle like CFP-10/ESAT-6 or EspA/EspC, and cross the inner cell membrane. Finally, the results of the 3D fold analysis seem to suggest an interesting aspect of the Type VII secretion system, which is its preference for substrates having four-helix-bundle structures. The probable reason for this is explained in later sections. (B) Regulatory proteins. The results obtained from the current analysis and those known from previously reported studies have indicated a list of probable regulatory components of ESX-1 secretion pathway (Table 1). These regulatory components can be divided into two functional categories: (i) Initial activation and transcription of secreted proteins and (ii) Negative regulation of the ESX-1 secretion machinery. Initial activation and transcription of secreted proteins: Our analysis showed that the phylogenetic profile of WhiB6 is identical Model of Type VII Secretion System to Rv3866 (and similar to the phylogenetic profiles of a majority of ESX-1 components). WhiB6 is a member of WhiB family of transcriptional regulators. WhiB family of proteins are unique to Actinomycetes and seven proteins belonging to the WhiB family (WhiB1-7) have been identified in M. tuberculosis H37Rv [35]. The proof for their regulatory role was established from a study where WhiB3 was shown to bind the sigma factor of RNA polymerase [36]. WhiB6 has been reported as a sensor of phagosomal signals [37]. It has also been shown to be up- regulated under several stress conditions which are typically observed inside the phagosome [35]. Interestingly, previous studies have also suggested the ESX-1 components to be involved in macrophage escape and cell to cell spread of the bacteria [7,38]. Thus, we hypothesize that WhiB6 probably acts as a receptor of phagosomal signals and participates in initial activation of ESX-1 secretion machinery. Our analysis identified Rv3866 to have a DNA binding domain. The protein is also reported to be involved in the synthesis and/or stability of ESAT-6 [38]. Thus, Rv3866 probably functions as a transcriptional regulator of ESAT-6. Another protein located outside ESX-1 region, PhoP (Rv0757) (not included in our study) was previously shown to regulate the operon Rv3612c-Rv3616c (including the genes corresponding to EspA and EspC), which in turn affects secretion of ESAT-6 [39]. The above observations thus suggest that, WhiB6 probably acts as an initial receptor of phagosomal signals and brings about the initial activation of the ESX-1 secretion machinery either directly or indirectly. The indirect activation probably involves other transcriptional regulators like Rv3866 and PhoP. Negative Regulation of secretion: Our analysis identified Rv3876 to contain a FlhG domain and a three dimensional fold similar to that adopted by ParA family of proteins. The ParA family of proteins as well as proteins containing the FlhG domain are known to negatively regulate the processes of chromosome partitioning and flagellar biogenesis respectively [24,26]. These observations suggest that, Rv3876 may have a role in negative regulation of ESX-1 region. (C) Structural Proteins. The translocation process can be divided into various stages. Based on probable roles/functions derived from the present study as well as from literature, we have divided the structural components into functional categories, depending on the stages of protein translocation process to which they contribute. Details of these stages and the participating structural proteins have been discussed below and are further summarized in Table 2. Multimeric assembly of secretory proteins: The TPR domain identified (in the present study) in the N-terminal region of Rv3868 suggests its probable involvement in the assembly of multi-protein complexes. This is analogous to that predicted for the protein VCA0119 in the Type VI Secretion System [40]. Furthermore, Rv3868 has also been experimentally shown to interact with CFP- Table 1. Functional assignments of regulatory components involved in ESX-1 secretion pathway. Componentname Rv3866 Rv3862 (WhiB6) Rv3876 doi:10.1371/journal.pone.0027980.t001 Known function (from literature) Cytoplasmic protein involved in expression of ESAT-6 Transcriptional regulatory protein Unknown Probable function (obtained from present study) Probably involved in transcriptional activation of ESAT-6 Initial activation of the ESX-1 secretion machinery Negative Regulation of secretion Function derived from Domain analysis, literature Phylogenetic profile analysis, literature Domain analysis, 3D fold analysis, literature PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e27980