Mycobacterial surface-associated ESX-1 virulence factors play a role in mycobacterial adherence and invasion into lung epithelial cells

Abstract

EsxA has 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). Currently, the reported cytolytic activity of EsxA at neutral pH is controversial. In this study we have obtained direct evidence that it is the ASB-14, a detergent used in EsxA purification, but not EsxA that causes cytolysis at neutral pH. 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 (MmEsxA: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.

The C terminus of the mycobacterium ESX-1 secretion system substrate ESAT-6 is required for phagosomal membrane damage and virulence

“…However, we and others found that many of the pore-forming activities ascribed to ESAT-6, such as RBC lysis, was due to residual detergent contamination in ESAT-6 preparations made using standard, widely distributed protocols (8,23,24). Moreover, we found that true ESX-1-mediated RBC lysis was contact dependent and caused gross membrane disruptions as opposed to distinct pores (8).…”

A Small Protein but with Diverse Roles: A Review of EsxA in Mycobacterium–Host Interaction

“…This protocol had been used in several studies [ 28 , 50 , 187 ]. Our recent data show that even after several detergent removal procedures, the concentration of the residual ASB-14 in the purified EsxA still ranges from 12 to 32 μg/mL, which is enough to cause cell lysis [ 188 ]. In fact, instead of causing cytolysis, the exogenously added EsxA protein without ASB-14 is internalized into the lung epithelial cells and traffics to acidic subcellular compartments, where it inserts into the membranes [ 188 ].…”

“…The Mm strain with the deletion of esxBA has an increased adherence to the murine macrophages [ 44 ]. Most recently, our preliminary data have shown that the Mm strain with EsxA deletion has an increased adherence but a decreased invasion in WI-26 [ 188 ]. Recent studies have shown that the secreted EsxAB remains associated with the mycobacterial cell wall, rather than released into the medium, and the cell wall-associated EsxAB correlates with bacterial virulence [ 85 , 198 , 204 ].…”

“…Upon acidification, EsxA inserts into the liposomal membrane and forms a transmembrane structure [ 61 ], and the exogenously added EsxA with a NBD label is internalized into the lung epithelial cells and inserts into the host membranes within the acidic subcellular compartment [ 188 ]. Most recently, molecular dynamic simulation shows that EsxA inserts into lipid membranes in the form of a C4 oligomer [ 205 ].…”

The C terminus of the mycobacterium ESX-1 secretion system substrate ESAT-6 is required for phagosomal membrane damage and virulence

“…Purified ESAT-6, but not its co-secreted partner CFP-10, lysed artificial lipid bilayers, liposomes, and red blood cells (RBCs), leading to the conclusion that ESAT-6 functions as a classical pore-forming toxin (15,21,22). However, we and others found that many of the pore-forming activities ascribed to ESAT-6, such as RBC lysis, was due to residual detergent contamination in ESAT-6 preparations made using standard, widely distributed protocols (8,23,24). Moreover, we found that true ESX-1-mediated RBC lysis was contact-dependent and caused gross membrane disruptions as opposed to distinct pores (8).…”