Fisiologia e inibição de um complexo membranar crucial para a produção de sulfureto pela microbiota intestinal, associado a cancro e doenças inflamatórias intestinais

A Western diet rich in fat and animal protein is associated with increased levels of sulfide in the gut, which is strongly associated with diseases like IBD and CRC, and is mostly produced by taurine-utilizing members of the microbiota through the Dsr pathway. The main objective of this project is to find new inhibitors for these gut bacteria, to be used as therapeutics with the aim of controlling the build-up of gut sulfide and thus promote a healthier microbiota. We will take a novel approach by targeting a key membrane complex, DsrMKJOP, which is involved in the last step of the Dsr pathway and allows the bacteria to get energy from reducing sulfite to sulfide. We were pioneers in identifying the involvement of the DsrMKJOP complex in the Dsr pathway [TR3] and showing that it is responsible for the last step of sulfide production [TR5]. Recently, we were successful in obtaining the first Cryo-EM structure of this complex in collaboration with Dr. B. Murphy (see SI). So, we are now in an ideal position to target this complex to inhibit sulfide production by the Dsr pathway. This approach has not been tested so far. This project has the following objectives: O1 – Elucidate the functional mechanism of the DsrMKJOP complex; O2 – Reveal how DsrMKJOP is involved in energy conservation, supporting bacterial growth; O3 – Identify and design new inhibitors for this complex; To reach these objectives, we designed a workplan where we will: i) Set up expression systems for DsrMKJOP to produce high amounts of protein and variants for in vivo physiological studies and in vitro biochemical studies; ii) Reconstitute the DsrMKJOP complex and variants in liposomes to study inhibition and investigate the mechanism of proton pumping; iii) Characterize the variants produced, and possible catalytic intermediates, using kinetics, Cryo-EM and spectroscopic studies (EPR and MCD); iv) - Develop inhibitors of the DsrMKJOP complex targeting the quinone-binding site(s); We will set up expression systems for the DsrMKJOP complex from two organisms: D. vulgaris and A. fulgidus. The D. vulgaris system is targeted for in vivo growth and inhibition studies, and the A. fulgidus system is targeted for protein production for in vitro studies, including enzyme assays involving the DsrC-T (since we have well established production systems for DsrAB and DsrC from this organism, required to produce DsrC-T [TR2, TR5]), spectroscopic, Cryo-EM and inhibition studies. The proposed workplan will enable us to answer the following questions: Q1 – What is the mechanism of the DsrMKJOP complex? How does it interact with the DsrC-T? How is the DsrC-T reduced by the deeply buried active site? What is the function of the different redox centres? Q2 – Is DsrMKJOP involved in proton pumping and energy conservation? What is the function of the DsrJOP module? Are there 2 quinone binding sites? How are protons and electrons transferred through the complex? Q3 - What compounds inhibit the function of DsrMKJOP? Which quinone analogues inhibit the complex? Q4 – How efficient are the inhibitors in preventing growth of sulfidogenic bacteria? This ambitious project will strengthen a recent line of research in the lab studying gut sulfidogenic bacteria and their interplay with disease. It will bring important advances in understanding sulfur metabolism and controlling excessive sulfide in the human gut, which can have a huge impact in promoting a healthy microbiota and preventing IBD and CRC.