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

Abstract

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 ofMycobacterium 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) ofMtbH37Rv. 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 ofMtbwith neutrophil chemotactic activity. Sulfasalazine, an N-formyl-Met-Leu-Phe (fMLF) receptor inhibitor, blocked the chemotaxis of neutrophils induced by CF ofMtb, thus indicating the involvement of the fMLF receptor inMtbCF 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 ofMtb,the induced Ca2+influx in neutrophils was suppressed by sulfasalazine. Thus, we identified novel formylatedMtbpeptides with neutrophil chemotactic activity.