ESAT-6 fromMycobacterium tuberculosisDissociates from Its Putative Chaperone CFP-10 under Acidic Conditions and Exhibits Membrane-Lysing Activity

Abstract

The 6-kDa early secreted antigenic target ESAT-6 and the 10-kDa culture filtrate protein CFP-10 of Mycobacterium tuberculosis are secreted by the ESX-1 system into the host cell and thereby contribute to pathogenicity. Although different studies performed at the organismal and cellular levels have helped to explain ESX-1-associated phenomena, not much is known about how ESAT-6 and CFP-10 contribute to pathogenesis at the molecular level. In this study we describe the interaction of both proteins with lipid bilayers, using biologically relevant liposomal preparations containing dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol, and cholesterol. Using floatation gradient centrifugation, we demonstrate that ESAT-6 showed strong association with liposomes, and in particular with preparations containing DMPC and cholesterol, whereas the interaction of CFP-10 with membranes appeared to be weaker and less specific. Most importantly, binding to the biomembranes no longer occurred when the proteins were present as a 1:1 ESAT-6 ⅐ CFP-10 complex. However, lowering of the pH resulted in dissociation of the protein complex and subsequent protein-liposome interaction. Finally, cryoelectron microscopy revealed that ESAT-6 destabilized and lysed liposomes, whereas CFP-10 did not. In conclusion, we propose that one of the main features of ESAT-6 in the infection process of M. tuberculosis is the interaction with biomembranes that occurs after dissociation from its putative chaperone CFP-10 under acidic conditions typically encountered in the phagosome.Mycobacterium tuberculosis is one of the most successful human pathogens, infecting nearly one-third of the world's population. Among the various factors that contribute, it is certainly the bacterium's ability to multiply and persist within professional phagocytic cells that is of primary importance (30).The extended RD1 region of M. tuberculosis encodes ESX-1, a novel protein secretion system involved in the immunogenicity and pathogenicity. The system, which is absent from the attenuated vaccines Mycobacterium bovis BCG and Mycobacterium microti (4, 6, 24), is responsible for the export of the 6-kDa early secreted antigenic target ESAT-6 and the 10-kDa culture filtrate protein CFP-10. ESX-1 is present in the saprophyte Mycobacterium smegmatis (9), where it acts in DNA uptake (11), which suggests that pathogenic mycobacteria might have adapted an ancestral conjugation system for protein secretion, which is required for survival and multiplication in the host cell.The importance of ESX-1 proteins for pathogenicity was shown by reintroduction of the extended RD1 region into BCG (34), deletion of RD1 from M. tuberculosis (23), signaturetagged and insertional mutagenesis (8,40,46), and targeted gene deletions (5,15,18). Several effects related to pathogenicity have been found to be associated with the expression of ESX-1 in M. tuberculosis and/or Mycobacterium marinum, a fish pathogen that harbors an ESX-1 system similar to that of M. tuberculosis (14,27). These ...

Mycobacterium tuberculosis ESAT-6 Exhibits a Unique Membrane-interacting Activity That Is Not Found in Its Ortholog from Non-pathogenic Mycobacterium smegmatis

“…Consistent with our results, an earlier circular dichroism study showed that the complex formed by MtbESAT-6 and MtbCFP-10 was too stable to dissociate at low pH (25). In contrast, a published study using the native MtbESAT-6 extracted from the culture filtrate of M. tuberculosis suggested that MtbESAT-6 was dissociated from MtbCFP-10 at low pH (19). One possible explanation for this discrepancy is that the native proteins possess FIGURE 6.…”

“…lysis activity (11,19). However, the molecular mechanism of MtbESAT-6 membrane interaction has not been defined.…”

The innate immune response in human tuberculosis

“…The ESX‐1 type VII secretion system (T7SS) that is lacking from most of the non‐pathogenic mycobacterial strains (Abdallah et al ., 2007) is required for Mtb localization in the cytosol. As part of the T7SS, ESAT‐6 protein is believed to make pores on cellular membranes (Hsu et al ., 2003; Jonge et al ., 2007; Wong and Jacobs, 2011). However, mechanistically, how ESAT‐6 lyses the phagosomal membrane in host cells is still unknown.…”

ESX-1 dependent impairment of autophagic flux byMycobacterium tuberculosisin human dendritic cells

“…As the extreme C terminus of ESAT-6 represents a floppy, structurally not welldefined region of the protein, 35 it is likely that this region is free to interact with host proteins or might help the interaction of ESAT-6 with the lipid bilayers of the subcellular organelles or the cell membrane. 27,36 As an alternative to a direct interaction of ESAT-6 with the autophagic machinery, ESX-1 proteins could allow other Mtb-encoded factors to translocate into the cytosol to inhibit the autophagy flux. In this regard, a potential candidate for the antiautophagic activity of Mtb could be the phosphatidylinositol 3-phosphate phosphatase SapM, which has been described to block BCG-containing phagosome maturation.…”