Functional analysis of ESAT-6 and EspB, two virulence proteins secreted by the ESX-1 system in Mycobacterium marinum

Abstract

Mycobacterium tuberculosis (Mtb) and Mycobacterium marinum (Mm) are able to persist inside host cell macrophages by modulating the phagosome environment. ESX-1 is a specialized secretion system that is required for virulence. Two of the proteins secreted by ESX-1 are ESAT-6 and EspB. They are codependent for secretion and are important virulence effectors, though their specific functions are not known. Mm is able to escape from the phagosome into the host cell cytosol where it can initiate actin-based motility. Mm escape is dependent on a functional ESX-1 system. I show that the ESAT-6 protein is able to form pores in host cell membranes which may play a role in Mm escape from the phagosome. I also dissect the Mm EspB protein and show that cleavage of EspB is required for growth inside RAW cells, virulence in zebrafish, and for modulating ESAT-6 secretion. The resulting C-terminal 11 kDa fragment is sufficient for the codependent secretion of ESAT-6; while the 50 kDa N-terminal fragment seems to be somewhat dispensable for ESAT-6 secretion but is definitely required for virulence. When EspB is expressed as a full-length protein, the highly conserved WXG motif in the N-terminal fragment is involved in the codependent secretion of the two proteins since secretion is reduced when this motif is mutated. Interestingly, when the N-terminal fragment is expressed without the C-terminal fragment it can secrete independent of the ESX-1 system, indicating that the C-terminus confers specificity for EspB secretion through ESX-1. I show that the virulence function of the EspB N-terminal fragment is dependent on the secretion of ESAT-6, and EspB must be expressed in its full-length form in order to be fully functional. These results indicate that EspB function is dependent on a close association with ESAT-6. It is possible that the N-terminus is translocated into the host cell cytosol through the ESAT-6 formed pore.