Characterization of differential pore‐forming activities of ESAT‐6 proteins from Mycobacterium tuberculosis and Mycobacterium smegmatis

Abstract

Mycobacterium tuberculosis ESAT-6 (MtbESAT-6) plays essential roles in pathogenesis. MtbESAT-6 exhibits a unique pore-forming activity (PFA) that is not found in its ortholog from non-pathogenic Mycobacterium smegmatis (MsESAT-6). Here we characterized the differential PFAs and found that exchange of I25-H26/T25-A26 between two proteins reciprocally affected their PFAs. MtbESAT-6(IH/TA) had ~40% reduction, while MsESAT-6(TA/IH) fully acquired its activity similar to MtbESAT-6. Mutations of A17E, K38T, N67L or R74Q on MtbESAT-6(IH/TA) further reduced the activity, with MtbESAT-6(IH/TA-17) being the lowest. This study suggests I25-H26 as the pH-sensor essential for MsESAT-6 to fully acquire the activity, while multiple residues contributed to MtbESAT-6 PFA.

Mycobacterial ESX-1 secretion system mediates host cell lysis through bacterium contact-dependent gross membrane disruptions

“…Rather, we find that the various lytic activities attributed to ESAT-6 at neutral pH are the result of a widely used protocol to isolate ESAT-6, which leaves residual detergent in the preparations. We find that detergent-free ESAT-6 preparations can disrupt liposomes under acidic conditions, as previously observed (25,27,28). However, this intrinsic low pHdependent activity of ESAT-6 is not responsible for mycobacterial phagosomal permeabilization in host macrophages.…”

“…Prior work had suggested that ESAT-6 mediated ESX-1 membrane lytic activity through pore-forming activity (15,18,28,34). If so, mycobacterial culture supernatants should produce hemolysis.…”

Esx Systems and the Mycobacterial Cell Envelope: What's the Connection?

“…These initial biophysical data were extended by several independent reports, including those demonstrating EsxA-dependent lysis of physiologically relevant liposome membranes (177), characterizing pore formation in sheep red blood cell (sRBC) membranes by EsxA (53), and the lysis of type 1 and type 2 pneumocytes by either purified EsxA or EsxA applied to the surface of ESX-1-deficient mycobacterial strains (167). Several more recent studies have focused on the mechanism of EsxA membrane lysis by investigating changes to membrane lysis as a function of pH (177), by comparing the activities of EsxA proteins from pathogenic and nonpathogenic mycobacterial species (178,179), and by generating specific point mutations which disrupt the membranolytic activity of EsxA (180). For a recent comprehensive review of the evidence of EsxA pore-forming activity, please see the study by Peng and Sun (181).…”

“…Coupled with evidence that ESX-1 substrates are present in the capsule and on the cell surface, discovered by our group and several others, direct translocation of ESX-1 substrates into the host macrophage may not occur. Rather, the presence of ESX-1 substrates on the cell surface may be sufficient for promoting phagosomal lysis (134,(166)(167)(168)(169)(170)(171)(172)(173)(174)(175)(176)(177)(178)(179)(180)(181)(182)(183)(184)(185). Alternatively, contact with membranes may induce ESX-1 secretion through an unknown signal transduction mechanism.…”

Interplay of human macrophages and Mycobacterium tuberculosis phenotypes

“…This discrepancy between native and recombinant proteins could be explained by mycobacterium-specific post-translational modifications on ESAT6 [52]. Both recombinant and native ESAT6 lyse artificial membranes [59,[61][62][63][64] due to a hypothetical conformational change of ESAT6 that also facilitated membrane interaction [61]. The helix-turn-helix structure of ESAT6 was shown to insert into membranes upon acidification and to form a membrane-spanning structure, which could lead to membrane lysis [65].…”