Identification of N-formylated Peptides with Neutrophilic Chemotactic Activity in Mycobacterium tuberculosis

Neutrophil infiltration of the lungs is associated with granuloma formation and the severity of tuberculosis infection. Although several cytokines and chemokines are known to contribute to lung neutrophil infiltration, the neutrophilic chemotactic factors of Mycobacterium tuberculosis (Mtb) remain unexplored. Therefore, we performed Transwell based chemotactic assays using neutrophils from human peripheral blood and mouse bone marrow to probe the chemotactic activity of the culture filtrates (CF) of Mtb H37Rv. CF of H37Rv induced chemotaxis of both human and mouse neutrophils, and this was also confirmed with CF of 9 clinical isolates and Erdman strain of Mtb with neutrophil chemotactic activity. Sulfasalazine, an N-formyl-Met-Leu-Phe (fMLF) receptor inhibitor, blocked the chemotaxis of neutrophils induced by CF of Mtb, thus indicating the involvement of the fMLF receptor in Mtb CF induced chemotaxis of neutrophils. Mass spectrometry analysis of CF of H37Rv identified three candidate N-formylated heptapeptides. The chemotactic activity of the identified peptides was confirmed with their synthetic mimetics that they induced neutrophil chemotaxis in a manner dependent on N-terminal formylation. For all formylated peptides and CF of Mtb, the induced Ca2+ influx in neutrophils was suppressed by sulfasalazine. Thus, we identified novel formylated Mtb peptides with neutrophil chemotactic activity.