ESAT-6 from Mycobacterium tuberculosis dissociates 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 flotation 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.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.

FIG. 1.

Binding of purified recombinant, native, or culture filtrate-derived (C.F.) ESAT-6 and CFP-10 to liposomes studied by liposome floatation analysis. (A and B) Protein samples were incubated for 10 min at 37°C in the presence (+lipo.) or absence (−lipo.) of liposomes and subsequently applied to a sucrose density gradient. (C) Equal quantities of rESAT-6 and rCFP-10 were incubated for 5 min at room temperature and subsequently for 10 min in the presence or absence of liposomes. Gradients were collected in five fractions from the top (1 to 5), trichloroacetic acid precipitated, and analyzed by immunoblotting using a monoclonal antibody for ESAT-6 (anti-ESAT-6) or affinity-purified polyclonal CFP-10 antibodies (anti-CFP-10).

FIG. 2.

Liposome floatation analysis using BCG::RD1-derived culture filtrates in buffers with different pHs. Culture filtrates of BCG::RD1 were incubated with liposomes in the appropriate buffers for 10 min at 37°C, applied to sucrose density gradients, and processed for immunoblotting.

FIG. 3.

Determination of the effect of acidic pH on the stability of the CFP-10·ESAT-6 complex by surface plasmon resonance (SPR). nESAT-6 was injected at pH 6.5 on a CFP-10 surface from time t₀ to t₁. The complex was allowed to dissociate spontaneously at pH 6.5 until time t₂, when 1-minute pulses at different pHs were applied (until t₃), leading to a partial or total disruption of the complexes. RU, resonance units (1 RU ≈ 1 pg·mm⁻²).

FIG. 4.

Liposome floatation analysis with liposomes containing different lipids. To analyze the specificity of lipid interaction, rESAT-6 or rCFP-10 was incubated with DMPC-DMPG (4:1), DMPC-cholesterol (4:1), or DMPG-cholesterol (1:1) liposomes; applied to sucrose gradients; and processed for immunoblotting.

FIG. 5.

Cryoelectron microscopy analysis of liposomes with or without ESAT-6 or CFP-10. Typical electron cryomicrographs of liposomes are shown. (A and B) Liposomes alone (A) and liposomes incubated with rCFP-10 (B), where no lysis was observed. (C to F) Liposomes incubated with nESAT-6 (C, E, and F) or rESAT-6 (D), all showing rigidification of the membranes and lysis of the liposomes. Bars, 100 nm (A, B, and D) and 50 nm (C, E, and F).