Biophysical characterization and analysis of different mycobacterial WXG100 proteins to study possible inter-loci complex formation

Abstract

Tuberculosis has remained a global public health concern for the past 30 years. The causative agent Mycobacterium tuberculosis has developed several strategies to evade the host immune system and for intracellular survival. Extensive genomic and proteomic studies have revealed hundreds of genes involved in the bacterium’s resistance and survival. The first known secreted virulence factors, EsxA and EsxB, are the paragons of a group of heterologous proteins, termed the “WXG100 family”. M. tuberculosis has 23 WXG100 proteins (EsxA-EsxW), five of such protein pairs are found within a genomic cluster making up a type-VII-secretion system (ESX-1 to ESX-5). Mycobacterial WXG100 proteins appear to share a similar secondary structure and form complexes with neighboring co-expressed proteins. The question arises as to whether Esx proteins from different genomic loci, i.e. not within the same operon, can form complexes. If this were the case, even only for some, the recombination of individual complex partners could greatly increase the versatility of the bacterium in reacting to the numerous host defense mechanisms. In this work, ten different mycobacterial Esx proteins were recombinantly expressed and purified in order to evaluate the possibility of inter-loci complex formation and their structural properties. The results suggest that inter-loci complex formation between several Esx proteins is possible. Special focus was put on the complex formation between EsxB and EsxT, which was evaluated with chemical crosslinking studies and single-molecule FRET experiments. Kinetic evaluations suggest a rather low binding constant between EsxB and EsxT (mM range), compared to the high binding constant between the native partners EsxB and EsxA (nM range). The proteins EsxF and EsxE were found to be highly heat resistant, both in complex and monomeric form, with a high structural recovery of over 90 % after heating to 96 °C. In addition, evidence of homo-dimer or homo-oligomer formation was found for EsxF and EsxU, which has not been reported previously.