Mycobacterial surface-associated ESX-1 virulence factors play a role in mycobacterial adherence and invasion into 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 by penetrating phagosomal membranes with its acidic pH-dependent membrane permeabilizing activity (MPA). Since the reported cytolytic activity of EsxA at neutral pH is controversial, in the present study we have obtained direct evidence that it is the residual ASB-14, a detergent used in EsxA purification, but not EsxA that causes cytolysis at neutral pH. Besides, we have also found that the exogenously added EsxA was internalized into lung epithelial cells (WI-26) and inserted into the host membranes, and these processes could be blocked by cytochalasin D and bafilomycin A. This indicates that EsxA is bound by host surface receptors and internalized into acidic endosomal compartments. This observation has intrigued us to investigate the role of EsxA in mycobacterial adherence and invasion in host cells. Interestingly, compared to the Mycobacterium marinum (Mm) wild type strain, the Mm strain with deletion of the esxBA operon (Mm{Delta}EsxA:B) had a lower adherence but a higher invasion in WI-26 cells. More interestingly, either inducible knockdown of EsxAB or removal of the bacterial surface-associated EsxAB by Tween-80 exhibited opposite results compared to gene knockout. Finally, the surface-associated EsxA is correlated to mycobacterial virulence. Together, the present study has shown for the first time that EsxA is internalized into the host cells and inserts into the host membranes, and mycobacterial surface-associated EsxAB plays an important role in mycobacterial adherence and invasion in host cells, which warrants further investigation.