Periodic Reporting for period 1 - CryoMyco (Atomic dissection of type VII secretion systems from pathogenic mycobacteria)
Berichtszeitraum: 2021-04-01 bis 2023-03-31
Mycobacterium tuberculosis is the single, deadliest bacterial pathogen worldwide as the etiological agent of tuberculosis (TB). In 2022 alone, over 10 million new cases appeared, and 1.6 million people died from TB, while approximately one quarter of the world’s population was estimated to carry the disease asymptomatically. However, tuberculosis is not the only mycobacterial disease, with a suite of other mycobacterial pathogens causing diseases such as leprosy, Buruli ulcer and others.
Despite the world scale commitment, via the End TB strategy, TB remains a global threat. As outlined in the strategy, the TB global incidence should have already decreased at a rate more than twice, while the funds necessary for care, prevention and R&D face a gap of approximately 3 billion euros. To make matters worse, the Covid-19 pandemic has reversed the last ~5 to 7 years of progress in the fight against the disease.
Type VII secretion systems (T7SSs) are specialized secretion systems used by mycobacteria to export proteins and virulence factors across their very specific and impermeable, diderm cell envelope. They play central roles in mycobacteria, ranging from virulence to uptake of iron or nutrients to conjugation of DNA. The aim of this project was to uncover the structural basis of T7SS protein transport across the mycobacterial cell envelope, focusing on elucidating the fully assembled T7SS inner-membrane translocation machinery and identifying the components responsible for protein translocation across the mycobacterial outer-membrane.
Despite the world scale commitment, via the End TB strategy, TB remains a global threat. As outlined in the strategy, the TB global incidence should have already decreased at a rate more than twice, while the funds necessary for care, prevention and R&D face a gap of approximately 3 billion euros. To make matters worse, the Covid-19 pandemic has reversed the last ~5 to 7 years of progress in the fight against the disease.
Type VII secretion systems (T7SSs) are specialized secretion systems used by mycobacteria to export proteins and virulence factors across their very specific and impermeable, diderm cell envelope. They play central roles in mycobacteria, ranging from virulence to uptake of iron or nutrients to conjugation of DNA. The aim of this project was to uncover the structural basis of T7SS protein transport across the mycobacterial cell envelope, focusing on elucidating the fully assembled T7SS inner-membrane translocation machinery and identifying the components responsible for protein translocation across the mycobacterial outer-membrane.
Despite several technical and scientific challenges, the project progressed well, with good contingency measures put in place. We cloned the esx-5 locus of M. tuberculosis and expressed it in the fast growing and non-pathogenic species M. smegmatis. This allowed us to purify large amounts of fully assembled inner membrane complexes which contained all five conserved membrane components, i.e. EccB5, EccC5, EccD5, EccE5 and MycP5. Further stabilization with amphipols and the transition state analogue nucleotide ADP-AlF3 led to homogenous and stable protein preparations that could be visualized by cryo-electron microscopy. Analysis of these particles showed two separate populations, one with an apparent six-fold symmetry composed of EccB5/C5/D5/E5 and one with a three-fold symmetry in which also the protease MycP5, was attached at the periplasmic side. Further analysis evidenced that MycP5 induced a conformational change on the periplasmic assembly formed by EccB5 and that MycP5 had a stabilizing role on the membrane complex. We also observed a large central pore that was gated by the EccC5 motor protein which, in turn, displayed two conformational states for its large cytosolic domain.
Beyond the inner membrane, nothing was known with regards to secretion through the mycobacterial T7SSs. To this end, we identified specific substrates, in the form of PPE proteins, that were required for secretion of Esx heterodimers as well as other PPE substrates. Tagging the specific PPEs allowed us to show their localization in the mycobacterial outer-membrane by means of subcellular fractionation, flow cytometry and solubilization trials.
Beyond the inner membrane, nothing was known with regards to secretion through the mycobacterial T7SSs. To this end, we identified specific substrates, in the form of PPE proteins, that were required for secretion of Esx heterodimers as well as other PPE substrates. Tagging the specific PPEs allowed us to show their localization in the mycobacterial outer-membrane by means of subcellular fractionation, flow cytometry and solubilization trials.
The project pushed further our understanding of bacterial specialized secretion systems. The most important discovery highlighted a T7SS inner-membrane complex that was a massive 2.32 MDa and contained a staggering 165 transmembrane domains with a gated central pore that would be large enough to allow the secretion of folded proteins. The finding that specific PPE proteins could be responsible for protein translocation across the mycobacterial outer-membrane has important implications both from a fundamental and an applied perspective. The results generated here will be important for understanding folded protein transport across biological membranes and the structural analysis will highlight potential drug binding sites that inhibit the function of T7SSs, thus preventing the propagation of infection.