Membrane Insertion of Mycobacterium tuberculosis EsxA in Cultured Lung Epithelial Cells

EsxA has long been recognized as an important virulence factor of Mycobacterium tuberculosis (Mtb) that plays an essential role in Mtb cytosolic translocation presumably by penetrating phagosomal membranes with its acidic pH-dependent membrane permeabilizing activity (MPA). However, current data suggest that the observed cytolytic activity of EsxA at neutral pH is due to contamination of ASB-14, a detergent used in EsxA protein purification, and the role of EsxA MPA in Mtb cytosolic translocation is also questionable. Here, we have obtained evidence that it is ASB-14, not EsxA that causes cytolysis at neutral pH. Quantitative liquid chromatography and mass spectrometry showed that even after gel filtration, dialysis, or passing through detergent removal column, the remaining ASB-14 in the EsxA protein solution was still at a concentration enough to kill cultured lung epithelial cells. When treated with trypsin or proteinase K, the digested EsxA protein solution with ASB-14 was still cytotoxic. Interestingly, however, we have found that the exogenously added EsxA is endocytosed into lung epithelial cells and inserts into the host membranes within acidic subcellular compartments, which can be blocked by cytochalasin D and bafilomycin A. It is for the first time EsxA is found to insert into the host membranes within acidic subcellular compartments. ImportanceEsxA has long been recognized as an important virulence factor of Mycobacterium tuberculosis (Mtb) that plays an essential role in Mtb virulence. However, current data regarding to its role in Mtb virulence are controversial. Here, we have obtained evidence showing that the cytolytic activity of EsxA at neutral pH is due to contamination of ASB-14, a detergent used in EsxA preparation. Moreover, it is for the first time we have found that EsxA protein is endocytosed into lung epithelial cells and inserts into the host membranes within acidic subcellular compartments, implicating an important role of the acidic pH-dependent membrane permeabilizing activity of EsxA in Mtb virulence.