Cryo‐ EM Structural characterization of the M. tuberculosis ESX ‐1 secreted virulence factor EspB

Mycobacterium tuberculosis (Mtb) is the top infectious disease killer worldwide. The threat posed by this bacterium is higher than ever, with the recent discovery of multi and extended‐drug resistant (MDR, XDR) strains with improved virulence. Virulent mycobacteria infect their host cells through a Trojan Horse strategy: they get ingested by the normal phagocytosis route but instead of being digested through acidification and maturation of the phagosome to phagolysosome, these bacteria enter the host's cell cytosol where they replicate freely. Controlled death of the host cell will allow the spreading of the descendants in the infected organism. A key step in the infectious life cycle of the virulent mycobacteria is their escape from the phagosome compartment [1]. This phenomenon is mediated by the release of different proteins (EsxA, EsxB, EspB) with cytolytic activities, that will perforate the phagosome membrane. These factors are secreted by the specialized machinery ESX‐1 of the recently discovered Type 7 Secretion System family [2]. The ESX‐1 machinery is a multi‐protein complex thought to span the entire mycobacterial cell envelope. It is constituted by a core of structural proteins (EccAC ab BDE 1 ) and the assembly and/or functioning of the machinery seems to require the recruitment of additional proteins, including substrates. Among the different ESX‐1 substrates, the 48 kDa protein EspB seems to carry multiple roles: this secreted protein carries cytolytic activity on its own; it is co‐secreted with EsxA (the main virulence factor) and this secretion is mutually dependent; EspB may also play a structural role suggested by its ability to oligomerize as well as its proteolytic maturation by MycP 1 during the translocation process. Recently, the structure of the N‐terminus of EspB was published [3] showing the N‐terminal be composed of a bundle of α‐helices, similar to other ESX substrates; the C‐terminal is predicted to be unfolded. A heptameric structure was modelled computationally based on the monomeric X‐ray data in combination with negative‐stain electron microscopy 2D classes. Here, we report structural characterization of EspB using transmission electron microscopy data. We determined the structure of the N‐terminal helix bundle, the mature form as well as the pre‐protein EspB at sub‐nanometer resolution. Moreover, we were able to identify several discrete oligomeric states of the proteins that were not previously observed: these data will aid to a better understanding of ESX‐specific substrates.