Functional characterization of Mycobacterium tuberculosis Rv2969c membrane protein

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Abstract

Identifying Mycobacterium tuberculosis membrane proteins involved in binding to and invasion of host cells is important in designing subunit-based anti-tuberculosis vaccines. The Rv2969c gene sequence was identified by PCR in M. tuberculosis complex strains, being transcribed in M. tuberculosis H37Rv, M. tuberculosis H37Ra, and M. bovis BCG. Rabbits immunized with synthetic peptides from highly specific conserved regions of this protein produced antibodies recognizing 27 and 29 kDa bands in M. tuberculosis lysate, which is consistent with the molecular weight of the Rv2969c gene product in M. tuberculosis H37Rv. Immunoelectron microscopy revealed the protein was localized on the bacillus surface. Four and three specific high activity binding peptides (HABPs) to the A549 alveolar epithelial and U937 monocyte cell lines were found, respectively. Two of the HABPs found inhibited M. tuberculosis invasion of A549 cells, suggesting that these peptides might be good candidates to be included in a multiepitopic, subunit-based anti-tuberculosis vaccine.

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Materials and methods

Bioinformatics analysis. Mycobacterium tuberculosis Rv2969c protein sequences were obtained from the Tuberculist Webserver (http://www.genolist.pasteur.fr/TubercuList/). Protein alignments were done with BLAST and protein features and GRAVY scores were calculated by using the PROTPARAM tool (http://www.us.expasy.org/tools/protparam.html) [10]. Signal sequences, non-classical secretion, transmembrane domains, and O-glycosylation sites were predicted

Mycobacterial species and strains. The ATCC and

Molecular analysis of the Rv2969c gene

The presence of Rv2969c in MTC (M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis, M. bovis BCG, M. africanum, and M. microti) strains was confirmed by the amplification of a 587 bp region from this gene (Fig. 1A). A similar amplification band was also seen in 10 M. tuberculosis isolates from patients suffering pulmonary and extra-pulmonary tuberculosis (data not shown). Sequence analysis of all clinical isolates PCR amplicons revealed no sequence variation suggesting that this protein was

Acknowledgments

This research has been supported by COLCIENCIAS contract RC-2008. Nora Martinez translated this manuscript.

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    It has been reported that a considerable number of secreted proteins are protective antigens, and therefore have been considered as attractive candidates to develop subunit vaccines [3,33,36], or proteins that bind to the host cell and that can be used in designing diagnostic methods [62] Moreover, they are hypothesized to mediate mycobacterial entry into the host cell [59]. The methodology used here for identifying binding fragments from the selected proteins has been previously applied in P. falciparum studies [4,28,30,31,44,55,68,69], Plasmodium vivax [48,67] and M. tuberculosis [12,18,26,49,50,53,58,70]. In the case of P. falciparum, identified sequences have been the base to the rational design of subunit-based, multiantigenic, multistage chemically synthesized vaccines, and a universal methodology has been proposed, that might be applied to prevent different infectious diseases [13,51,52].

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