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 ...