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Identifying putative Mycobacterium tuberculosis Rv2004c protein sequences that bind specifically to U937 macrophages and A549 epithelial cells

¹ Fundación Instituto de Inmunología de Colombia, Bogotá 020304, Colombia
² CORPOICA-CEISA, Bogotá 020304, Colombia
³ Universidad Nacional de Colombia, Bogotá 020304, Colombia

(RECEIVED June 1, 2005; FINAL REVISION August 6, 2005; ACCEPTED August 15, 2005)

Virulence and immunity are still poorly understood in Mycobacterium tuberculosis. The H37Rv M. tuberculosis laboratory strain genome has been completely sequenced, and this along with proteomic technology represent powerful tools contributing toward studying the biology of target cell interaction with a facultative bacillus and designing new strategies for controlling tuberculosis. Rv2004c is a putative M. tuberculosis protein that could have specific mycobacterial functions. This study has revealed that the encoding gene is present in all mycobacterium species belonging to the M. tuberculosis complex. Rv2004c gene transcription was observed in all of this complex’s strains except Mycobacterium bovis and Mycobacterium microti. Rv2004c protein expression was confirmed by using antibodies able to recognize a 54-kDa molecule by immunoblotting, and its location was detected on the M. tuberculosis surface by transmission electron microscopy, suggesting that it is a mycobacterial surface protein. Binding assays led to recognizing high activity binding peptides (HABP); five HABPs specifically bound to U937 cells, and six specifically bound to A549 cells. HABP circular dichroism suggested that they had an -helical structure. HABP–target cell interaction was determined to be specific and saturable; some of them also displayed greater affinity for A549 cells than U937 cells. The critical amino acids directly involved in their interaction with U937 cells were also determined. Two probable receptor molecules were found on U937 cells and five on A549 for the two HABPs analyzed. These observations have important biological significance for studying bacillus–target cell interactions and implications for developing strategies for controlling this disease.

Keywords: Mycobacterium tuberculosis; Rv2004c protein; high activity binding peptides; U937 cells; A549 cells

Abbreviations: HABP, high activity binding peptide • ¹²⁵I-HABP, 125- iodine radiolabeled HABP • PBS, phosphate buffer saline • CD, circular dichroism • TMC, Trudeau Mycobacterial Collection • RP-HPLC, reverse-phase, high-performance liquid chromatography • BS₃, bis (sulfosuccinimidyl suberate) • ATCC, American Tissue Culture Collection.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051592505.

Reprint requests to: Álvaro Puentes, Fundación Instituto de Inmunología de Colombia, Carrera 50 No. 26-00, Bogotá 020304, Colombia; e-mail: puentesalvaro{at}hotmail.com; fax: 571-4815269.

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