MycP1 was shown to be not only essential for its secretion, but also necessary for the controlled secretion of CFP-10/ESAT-6 [18]. While the knock-out of MycP1 was observed to completely abolish secretion, the presence of a mutant MycP1 without any protease activity was shown to increase the secretion of CFP-10/ESAT-6 and accumulation of EspB in the periplasm-like space [18]. These observations indicate a probable regulatory role of this serine protease (MycP1) in the periplasm-like space. Furthermore, in our analysis, the identified MycP1 cleavage motif within the N- terminal ubiquitin-like domain of Rv3877 indicates that, the N- terminal domain of Rv3877 probably acts as a gate, the cleavage of which by MycP1 may be necessary for the opening of the inner membrane channel and initiating translocation. Mycomembrane translocation: The subsequent step in the secretion mechanism is the translocation of proteins through the mycomembrane. Currently, the exact components of the ESX-1 secretion machinery involved in the transport across mycomem- brane have not been identified. Only three outer membrane pore proteins (OmpA, Rv1698 and Rv1973) have been experimentally characterized in mycobacterial species and none of them is reported to be linked to the ESX-1 secretion pathway [45–47]. Thus, one of the goals of our analysis was to identify probable candidates that could aid in the transport of substrates across the mycomembrane. The hypothetical protein, Rv3869 has been previously shown to be an essential component of the ESX-1 secretion machinery, as disruption of this gene was observed to result in the loss of CFP- 10/ESAT-6 secretion [22]. Our analysis predicted that both Rv3869 and Rv3882c contain long C terminal domains protruding into the periplasm-like space. These observations, along with the detection of a series of glycosylation sites in this region of Rv3869 suggests the probable association of both Rv3869 and Rv3882c with the periplasm-like space and/or the mycomembrane. These results also corroborate with those obtained by Mah et al. (2010), which considered several other properties like presence of signal peptides, beta content, etc. and predicted both these proteins as outer (or myco) membrane pore forming proteins (OMP). Based on these observations, we hypothesize that, Rv3869 and Rv3882c could act as probable mycomembrane translocon components of the ESX-1 secretion machinery, similar to the outer membrane components of other secretion systems. Other than Rv3869 and Rv3882c, our analysis (using phylogenetic profiles as well as protein-protein association networks) suggests four proteins (Mce1B, Mce1C, Mce1F and Rv0177) corresponding to MCE cluster 1 to have probable functional linkage to the ESX-1 secretion pathway. Additionally, these proteins were also predicted earlier as probable outer membrane pore forming proteins [45,46,48]. Thus, we hypothe- size that, these proteins could act as accessory components of the ESX-1 secretion machinery, to aid in the transport of secretory proteins across the mycomembrane. Our analysis thus suggests that the probable candidates which may constitute the mycomembrane transolocon machinery are Rv3869, Rv3882c, Mce1B, Mce1C, Mce1F and Rv0177. These proteins, either independently or in some combination, probably form the mycomembrane translocon machinery of the ESX-1 secretion system. Additional components: Apart from the components described above, genes encoding two additional proteins of PE and PPE family (PE35 and PPE68) are located inside the ESX-1 gene cluster. While PE35 has been reported to be secreted [8], no such data is available for PPE68. PE35 has also been shown to affect the expression of CFP-10/ESAT-6 [22]. An experimental study Model of Type VII Secretion System reported PPE68 as a cell envelope associated protein [10]. Similarly, another study suggested PPE68 to function as a gating component for the controlled secretion of effector molecules [38]. These results corroborate with the results from our analysis which predicts PPE68 to be associated with the mycomembrane and/or the periplasm-like space (based on the series of glycosylation sites predicted in this protein). The PE/PPE pair was earlier shown to adopt a four-helix- bundle structure with one helix-turn-helix being contributed by the PE protein and the other contributed by the PPE protein [27]. The tip of the structure (formed solely by the PPE counterpart) contains an elongated solvent accessible hydrophobic patch which can provide a probable interaction site for proteins containing similar patches. These observations suggest that the PE/PPE pair is likely to regulate the secretion of effector molecules by interacting with the ESX-1 components forming the mycomem- brane translocon. Proposed model of Type VII secretion system Combining the results of our analysis with those known from literature, we propose the following model for secretion of proteins through ESX-1 secretion machinery. The overall model is depicted in Figure 2. Secretion of CFP-10, ESAT-6, EspA and EspC The initiation event for secretion of virulence factors through ESX-1 secretion pathway may depend on phagosomal signals, which are sensed by WhiB6. The WhiB6 in turn, may directly or indirectly bring about the transcriptional initiation of the machinery. Subsequently, PhoP and Rv3866 facilitate the expression of the pairs EspA/EspC and CFP-10/ESAT-6, respectively. Once the two pairs (CFP-10/ESAT-6 and EspA/ EspC) are expressed, Rv3868 interacts with both the pairs and the TPR domain of Rv3868 helps in assembly of CFP-10/ESAT-6/ EspA/EspC into a multimer. Rv3871, a FtsK family protein, then recognizes the multimeric protein assembly of CFP-10/ESAT-6/ EspA/EspC through its specific interaction with the C-terminal end of CFP-10. Rv3871 interacts with another FtsK family protein Rv3870 and forms a translocase pore (as observed for other Ftsk family of proteins). Once recognized by Rv3871, the multimeric protein assembly (CFP-10/ESAT-6/EspA/EspC) is likely to pass through this translocase pore. This translocase pore not only recruits the substrates to the inner membrane pore protein (Rv3877), but also facilitates the ATP-dependent translocation of the substrates through the inner membrane. The interaction (and possibly the cleavage) of the N-terminal gate-like domain of Rv3877 by MycP1 opens the pore, thus enabling translocation of the substrate assembly from cytoplasm to the periplasm-like space. Subsequently, the substrate assembly crosses the mycomembrane through a channel formed by Rv3869, Rv3882c and the four proteins of MCE cluster 1 (Mce1B, Mce1C, Mce1F and Rv0177), either exclusively or in a collaborative manner. Secretion of EspB EspB pairs up with Rv3879c in a manner similar to the two pairs of secretory proteins namely, CFP-10/ESAT-6 and EspA/ EspC. Adopting a strategy similar to that described in the previous section, Rv3871 then recognizes and recruits this pair to the inner membrane pore formed by Rv3877. Using this pore, the EspB/ Rv3879c pair moves to the periplasm-like space in a manner similar to that of CFP-10/ESAT-6/EspA/EspC. Subsequently, MycP1 cleaves EspB in the periplasm-like space, which disrupts the EspB/Rv3879c pair, resulting in the secretion of EspB. The translocation of EspB through the mycomembrane probably PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e27980