CFP-10 from Mycobacterium tuberculosis Selectively Activates Human Neutrophils through a Pertussis Toxin-Sensitive Chemotactic Receptor

Abstract

cUpon infection with Mycobacterium tuberculosis, neutrophils are massively recruited to the lungs, but the role of these cells in combating the infection is poorly understood. Through a type VII secretion system, M. tuberculosis releases a heterodimeric protein complex, containing a 6-kDa early secreted antigenic target (ESAT-6) and a 10-kDa culture filtrate protein (CFP-10), that is essential for virulence. Whereas the ESAT-6 component possesses multiple virulence-related activities, no direct biological activity of CFP-10 has been shown, and CFP-10 has been described as a chaperone protein for ESAT-6. We here show that the ESAT-6:CFP-10 complex induces a transient release of Ca 2؉ from intracellular stores in human neutrophils. Surprisingly, CFP-10 rather than ESAT-6 was responsible for triggering the Ca 2؉ response, in a pertussis toxin-sensitive manner, suggesting the involvement of a G-protein-coupled receptor. In line with this, the response was accompanied by neutrophil chemotaxis and activation of the superoxide-producing NADPH-oxidase. Neutrophils were unique among leukocytes in responding to CFP-10, as monocytes and lymphocytes failed to produce a Ca 2؉ signal upon stimulation with the M. tuberculosis protein. Hence, CFP-10 may contribute specifically to neutrophil recruitment and activation during M. tuberculosis infection, representing a novel biological role for CFP-10 in the ESAT-6:CFP-10 complex, beyond the previously described chaperone function.

Functional selective ATP receptor signaling controlled by the free fatty acid receptor 2 through a novel allosteric modulation mechanism

“…A change in intracellular Ca 2+ was also measured using flow cytometry with mixed leukocytes as previously described (24). In short, mixed leukocyte populations were loaded with the Ca 2+ indicator dyes Fluo-3-AM (fluorescence increases upon Ca 2+ binding; Thermo Fisher Scientific) and FuraRed-AM (fluorescence decreases upon Ca 2+ binding; Thermo Fisher Scientific).…”

P2Y2 receptor signaling in neutrophils is regulated from inside by a novel cytoskeleton-dependent mechanism

“…The fluorescence change was followed using excitation wavelengths of 340 nm and 380 nm and an emission wavelength of 510 nm and the [Ca 2 þ ] i was calculated as described earlier [17,31]. In some experiments Fura-2 was replaced with the Ca 2 þ indicator dyes Fluo-3 and FuraRed (Molecular Probes) and Ca 2 þ mobilization from intracellular stores was measured using an Accuri C6 flow cytometer (BD Accuri) [32].…”

The Neutrophil Response Induced by an Agonist for Free Fatty Acid Receptor 2 (GPR43) Is Primed by Tumor Necrosis Factor Alpha and by Receptor Uncoupling from the Cytoskeleton but Attenuated by Tissue Recruitment

“…A change in intracellular Ca 2ϩ was also measured using flow cytometry with mixed leukocytes as described previously (27). In short, mixed leukocyte populations were loaded with the Ca 2ϩ indicator dyes Fluo-3 (Molecular Probes; fluorescence increases upon Ca 2ϩ binding) and FuraRed (Molecular Probes; fluorescence decreases upon Ca 2ϩ binding).…”