Insights from the analysis of predicted Rv0679c protein peptide from Mycobacterium tuberculosis with Toll like Receptors in host

Peptides of Rv0679c a membrane protein of the cell envelope (16.6 KDa) of Mycobacterium tuberculosis (M. tb), inhibited entry of live bacilli into epithelial (A549) and macrophage (U937) cell lines in vitro, suggesting a possible role in invasion. Receptors associated with Rv0679c antigen entry into cell lines were not characterized. We are reporting that Rv0679c peptides could bind to Toll like receptors (TLRs), the principal class of pathogen recognition receptors on host cells (PRR) by docking studies. Peptide structures were predicted using PEP FOLD and docking of truncated peptides with TLR’s was performed using Cluspro 2.0. Docked complexes were analyzed using Swiss-PDB Viewer. Nine peptides of Rv0679c protein assessed were able to bind to TLR2-1 and TLR 4-MD2; however the binding energy was better with TLR 4-MD2. Peptide 30985 (-866.4 kcal/mol) has better binding energy with TLR2-1, in contrast peptide 30982 showed a better binding energy to TLR 4-MD2 dimer with a score of -1291.7 kcal/mol. Interactive residue analysis revealed that GLU 173 and SER 454 of TLR 1; ARG 447 and ARG 486 of TLR2; ARG 264 of TLR 4 and SER 120, LYS 122 and GLU 92 of MD2 region are predominant residues interacting with peptides of Rv0679c protein. Our study suggests that predominant residues and receptors of TLR2 and TLR4 are important for Rv0679c protein binding, which could further lead to invasion of M. tb into the host cell.


Background:
Tuberculosis caused by Mycobacterium tuberculosis (M. tb) is a major concern as new cases are diagnosed and many die because of the disease [1]. Emergence of acquired immune deficiency syndrome and development of multidrug-resistant mycobacteria has increased the risk of disease [2]. Hence, it is important to understand the host -pathogen interaction and find ways to stop the entry / invasion in host cells either in new individuals or individuals who are already diseased.
Mycobacterium tuberculosis whole genome was sequenced [3], which led to deciphering the biology of the bacterium and predicted around 99 lipoproteins genes could be possible and Rv0679c gene was one of them [4]. Emphasis was on these envelope proteins and only few of them were experimentally characterized so far indicating their probable role in invasion [5]. However, Rv0679c gene / protein are not yet well characterized so far. Rv0679c protein (16.6 KDa) of M.tb was classified as an envelope protein consisting of 165 amino acids with a putative N-terminal signal sequence and a consensus lipoprotein-processing motif using E. coli expression system [3]. Study using globomycin treatment, Triton X-144 separation and mass spectrometry analysis revealed Rv0679c, a lipoprotein and exists as a tight complex with Lipoarbinomanan (LAM), one of the major components of cell envelope involved in pro-inflammatory and anti-inflammatory responses in M. tb/M. bovis BCG [6].
The functional role of either the Rv0679c gene or protein of M. tb in pathogen-host interaction is not well known / characterized.
Recently, Nakajima C et al. 2013 [7] reported clinical significance of Rv0679c gene in demarcating the lineages of M.tb strains. They identified a unique SNP at position 426 where C/G only in Beijing genotype family of M.tb and developed a multiplex PCR assay for diagnostic purpose. We assessed the significance of Rv0679c gene of M. tb in Indian clinical isolates and reported that Rv0679c gene is highly conserved and SNP C426G was not observed in Indian clinical isolates indicating lineages are different except in one isolate which might be of Beijing origin [28]. Cifuentes et al. [8] demonstrated for the first time that fragmented peptides of Rv0679c protein could selectively inhibit the entry of live M. tb bacilli into epithelial (A549) and macrophage (U937) cell lines in vitro indicating a probable role of Rv0679c protein in invasion of the host. However, they have not assessed the receptors associated with entry/invasion or no reports are available in the literature regarding Rv0679c peptides/protein and its interaction with either TLRs or any other receptors on the host cells as crystallography structure of Rv0679c protein is not known. In view of this, we thought of assessing whether peptides of Rv0679c protein could bind to TLR receptors using Insilco approach.  Amino acids in bold are interactive residues of TLR-2 and TLR-4 and normal ones are interactive residues of TLR-1 and MD2 with peptides respectively. A representation of two peptides was depicted and the rest of the peptides were analyzed in a similar way. Residues underlined in peptides are interacting with TLR, which is depicted by arrows.
Toll-like receptors (TLRs) play a major role among the pattern recognition receptors and in infectious diseases, inflammatory diseases and cancer [9]. TLRs constitute a family of trans-membrane proteins expressed on many cells of the immune system and help in recognizing many microbial components like lipopeptides by TLR- Messenger RNA expression of TLRs 1-9 has been shown in human lungs, indicating a major site for TLR activity [20]. The identification of Rv0679c peptides ability to inhibit target cell invasion by M.tb in cell lines suggests further studies related to host-cell receptors and their interactions for better understanding. Pulmonary TB being associated with lungs, we were interested in assessing the interaction of peptides of Rv0679c protein with Toll like receptors (TLRs) using Insilco approach.

Methodology: Selection of Protein Sequence of Rv0679c and Structure Prediction of Peptides
The protein sequence of Rv0679c was retrieved from TubercuList database (http://tuberculist.epfl.ch). The sequence was truncated into 9 peptides (20 mer

Analysis of Interacting residues of the TLR-Peptide Complex
The docked complexes of truncated peptides with TLR's, and the interacting residues were analyzed using Swiss-PdbViewer (SPDBV) (Guex,N) as given in Table 2. SPDBV provides a user friendly interface allowing analyzing several proteins at the same time. The software allows proteins to be superimposed in order to deduce structural alignments and compare their active sites or any other relevant parts. The interacting residues of TLRs with peptides fall within the TLR binding pockets which was further verified and confirmed using CASTP software. showed that tri-acylated lipopeptide Pam3CSK4 and di-acylated lipopeptide Pam2CSK4 even though bind to TLR-2, a proper heterodimer formation was induced only with Pam3CSK4 but not with Pam2CSK4.

Results and Discussion
Peptide 30985 has better binding ability to TLR-2-1 heterodimer out of the nine peptides assessed compared to TLR4-MD2 dimer where the peptide stand at eight position, indicating its preference towards TLR-2. Noteworthy is the peptide 30985 which was binding better energy to TLR-2 in our study correlates well with the findings of Cifuentes et al. [8] who showed that at low concentration it is efficient in inhibition compared to high concentrations where the inhibition is maximum and leading to cytotoxicity on A549 and U 937 cell lines. In other words this peptide failed to respond in a dose dependent manner in their study. Peptide 30982 has better binding affinity to TLR-4 in our study. The same peptide could not inhibit the invasion/entry of M. tb into the peptide pretreated cell lines indicating lack of inhibition. Hence this peptide was used as a negative control in their study which turns out to be a better TLR-4 binder suggests that TLR-4 might not be the receptor involved in invasion.

Assessment of Interactive residues of TLRs with Peptides of Rv0679c antigen in docked complexes:
Peptides of Rv0679c antigen bound to TLR2-1 and TLR4-MD-2 regions. A representative illustration showing the interactive residues of TLR 2-1 and the peptide 30986 is given in Figure 3A.
The interacting amino acid residues of TLR 2-1 hetero-dimer and TLR 4-MD2 dimer with two peptides are represented in Figure 3B, even though all peptides are assessed in the same way and the results are summarized in the text.

Conclusion:
Peptides of Rv0679c protein bind to TLR-2 and TLR-4 however peptides 30979, 30987 and 30982, 30985 preferred binding to TLR2 and TLR4 respectively. One would assume route of entry or invasion of the pathogen into the host depends on interacting residues at that given point of time which could determine the outcome. Studies based on invasion of M. tb using it's envelop antigens into the host using TLRs [24, 25], Cifuentes et al. [8] invasion studies using Rv0679c peptides and our own findings we speculate that Rv0679c antigen prefers TLR-2 receptor over TLR-4 for invasion of M. tb pathogen into the host cell. Within the Rv0679c protein, peptides 30979 and 30987 are being preferably associated with TLR2; interception of these interactions by intervening strategies could be beneficial in a disease scenario.