The mycobacterial cell envelope is a lipid-rich complex structure that surrounds the bacillus and is thought to play a critical role in the pathogenicity of Mycobacterium tuberculosis. Nearly 2.5% of the M. tuberculosis H37Rv proteome is predicted to consist of lipoproteins . A large number of these mycobacterial lipoproteins have been suggested to be important components for the synthesis of the mycobacterial cell envelope, as well as for sensing processes, protection from stressful factors and host-pathogen interactions; nevertheless, the function and localization of a considerable number of putative lipoproteins remains yet unknown .
Lipoproteins are translocated across the cytoplasmic membrane and then anchored to either the periplasm or the outer membrane and have been suggested to play important roles related to virulence because they are predicted to participate in intracellular transport, cell-wall metabolism, cell adhesion, signaling and protein degradation . Rv0679c was initially classified as a hypothetical membrane protein of M. tuberculosis  and was later suggested to be a putative lipoprotein . It is a 165-amino-acid-long protein with a theoretical molecular mass of 16.6 kDa, whose function has not been fully characterized yet.
In this study, PCR and RT-PCR techniques were used to examine the distribution of the Rv0679c gene in the MTC, as well as in mycobacteria other than tuberculosis (which included saprophytic and environmental species), with the aim of establishing a preliminary relationship between the presence of the protein encoding gene in a particular mycobacterial species and its virulence, considering that to develop a subunit antituberculous vaccine, it would be better to select peptides (more specifically HABPs) from M. tuberculosis proteins involved in host cell invasion that are exclusively present in MTC or in mycobacterium species related to invasive processes or causing disease, such as Rv0679c. The results of this study indicate that the gene encoding Rv0679c is present in the MTC, as shown by the PCR amplification of a 346-bp band from genomic DNA of M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. africanum, M. bovis, M. bovis BCG and M. microti; but no amplification was detected in Mycobacterium spp. strains outside the complex. Nevertheless, it is worth noting that Rv0679c homologues have been recently reported in different Mycobacterium genomes (e.g. M. smegmatis, M. marinum and M. avium), which indicates that such primers are specific for the MTC strains assessed in this study. Furthermore, reverse transcription assays indicate that the gene is actively transcribed in M. tuberculosis H37Rv, M. tuberculosis H37Ra and M. africanum. Intriguingly, although expression of Rv0679c homologous protein in M. bovis BCG was described by Matsuba et al. , gene transcription was not detected in M. bovis nor in M. bovis BCG in this study under normal culture conditions.
Once the presence and transcription of Rv0679c was determined in the MTC, the next step consisted in evaluating protein expression by Western blot analysis of M. tuberculosis H37Rv sonicate. Goat anti-Rv0679c peptide serum detected two bands of about 18 and 20 kDa, which differ from the theoretical molecular mass of 16.6 kDa predicted based on its amino acid composition. This slight difference could be caused by the post-translational modifications that lipoproteins undergo before reaching their destination as mature proteins, considering that pro-lipoproteins tend to be 2-3 kDa larger than mature lipoproteins .
According to bioinformatics predictions, Rv0679c lacks of transmembrane regions and contains an N-terminal signal sequence as well as a SPAse II cleavage site between residues 32-33, as indicated by the presence of a "lipobox" motif [LAGC] between amino acids 30-33. The presence of a signal peptide detected by using SignalP suggests that this protein is secreted via the Sec-dependent pathway, and is probably targeted by the lipobox motif to membrane surface where it remains attached by hydrophobic interactions. Briefly, after Rv0679c is translocated across the cytoplasmic membrane, the Cys residue of the lipobox motif is linked to a diacylglyceryl moiety. Then, a signal II peptidase cleaves off the signal peptide and the protein is anchored to the mycobacterial membrane via the diacylglyceryl moiety . These computational predictions are in agreement with the cellular localization observed in IEM studies in which the protein was detected on the surface of M. tuberculosis H37Rv bacilli.
To determine whether the peptides comprising Rv0679c established ligand-receptor interactions with M. tuberculosis susceptible human host cells, binding assays were performed with the U937 phagocytic and A549 epithelial cell lines. HABPs 30985 to 30987 comprising amino acids 121-165 showed higher binding activities to receptors on the surface of epithelial cells, whereas their binding activities to the phagocytic line were lower. Such differential binding behavior may be caused by differences between the surface receptors expressed by each cell line or their distinct physiological functions.
Interestingly, Rv0679c HABPs 30985, 30986 and 30987 are consecutively positioned within the protein's C-terminus, suggesting that the region formed by these three HABPs is implicated in binding of M. tuberculosis to target cells. Also, the Hill analysis showed high binding affinity interactions with a large number of receptor molecules on the surface of U937 cells, as indicated by their dissociation constant within the nanomolar range. Moreover, the formation of ligand-receptor complexes appears to facilitate binding of more HABPs, as shown by the positive Hill coefficient.
All HABPs tested in invasion inhibition assays prevented cell invasion by M. tuberculosis by a larger or comparable percentage, compared to the colchicine and Cytochalasin D controls. Regarding HABP 30986, an inhibitory effect similar to the one shown by HABPs 30985 and 30987 was observed on A549 cells at all concentrations used in this assay. Moreover, HABP 30987 showed larger inhibitory effect at the smaller concentration tested in this assay. HABP 30979 inhibited invasion of both cell types by a larger or even higher percentage than the ones shown by the colchicine and Cytochalasin controls. This HABP showed a dose-dependent inhibitory effect on both cells, achieving the highest inhibitory percentage at 200 μM.
The ability of Rv0679c peptides to inhibit M. tuberculosis invasion of target cells suggests that active and specific binding to cell surface receptors prevents entry of M. tuberculosis through this invasion pathway. Such notion is further supported by the results of internalization assays carried out with peptide-coated latex beads and epithelial cells, where peptide-coated beads were more actively internalized than uncoated beads. Particularly HABP 30979, which was the strongest invasion inhibitor, displayed the highest internalization percentages.
On the other hand, the large inhibition percentages obtained with phagocytic cells in comparison to the ones obtained with epithelial cells might be explained by the cooperativity phenomenon observed in saturation assays with the phagocytic cell line, since the amount of peptide that binds to surface receptors is proportional to the probability of forming more stable ligand-receptor complexes and thereby of restricting mycobacterial entrance. Furthermore, since some HABPs showed high binding activity to one cell type but low binding activity to the other one, it could be suggested that peptide binding activity depends on specific receptor molecules expressed on each cell type. Consequently, binding of Rv0679c HABPs with high activity to both cell lines could be due to the presence of the same receptor on both cell types or to different receptors with similar characteristics.
To date, no structural model has been reported for this protein. Therefore, CD assays were conducted in order to determine whether there was a relationship between the secondary structure of Rv0679c peptides and their binding ability or in their ability to inhibit mycobacterial invasion. CD spectrum data suggested that the secondary structure of HABP 30979 and 30985 was formed by α-helix and random coil elements, while peptides 30982 to 30984 and HABPs 30986 and 30987 showed undefined structural features. The results indicate that there is not a direct relationship between the structure of HABPs and their ability to binding to target cells.
Interestingly, the results obtained in this study showed that the HABPs that inhibited mycobacterial invasion to target cells more efficiently were also the ones that showed the larger internalization percentages, therefore suggesting that Rv0679c HABPs promote entry of pathogenic M. tuberculosis into host cells. Specifically, the binding region formed by HABPs 30985-30987 at the protein's C-terminal region appears to have a key role during M. tuberculosis invasion.
The confirmation of Rv0679c's location in mycobacterial surface, together with the identification of a binding region formed by HABPs 30985-30987, suggest that this protein may be related to adhesion and/or invasion processes. In addition, such surface localization could be facilitating contact between the bacilli and its host cell, thereby leading to triggering the host's immune response via interaction with host cell surface receptors .