Structural and functional role of urease from Mycobacteria

• Grant number: 25/14069-0
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: August 01, 2025
• End date: July 31, 2027
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal Investigator: Marcio Vinicius Bertacine Dias
• Grantee: Henrique Iglesias Neves
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 21/10577-0 - Biology of Bacteria and Bacteriophages Research Center, AP.CEPID

Abstract

Tuberculosis (TB) remains a serious problem for public health. This infectious disease is one of the significant causes of death by a single pathogenic microorganism in humankind. Although the number of infected people has declined recently, during 2020-2021, there was a regression because of the COVID-19 pandemic. Also, other serious problems are associated with this disease, which include the number of cases of co-infection with HIV and the multi-resistance to all first-line drugs used in the treatment. Based on this, understanding the pathogenesis of the disease and exploring novel therapeutic strategies are essential for controlling it. One of the most critical aspects of tuberculosis is its ability to survive intracellularly in the macrophage and arrest its function. In recent years, several protein systems have been identified as contributing to the modulation of macrophage function by Mycobacterium tuberculosis or other mycobacteria. Among the virulence factors of M. tuberculosis that contribute to evading macrophage function is urease. Urease is a large protein composed of three different subunits, formed by UreA, UreB, and UreC, with the latter being the catalytic domain. Urease is a nickel-dependent metalloenzyme that catalyzes the hydrolysis of urea to ammonia and carbamate, which is further converted to ammonia and carbon dioxide. Urease is a virulence factor of several bacteria and modulates the pH environment, particularly in the urinary and gastrointestinal tracts. In tuberculosis, it is thought that it could play an essential role in pH modulation in lung tissue or the phagolysosome. Still, only a few studies have been conducted so far on the role and characterization of this enzyme in M. tuberculosis infection. Recently, UreC was confirmed to be involved in modulating host cellular function by suppressing DNA repair and increasing intracellular survival through interaction with RuvB-like protein 2 (RUVBL2). Additionally, the inhibition of UreC was enough to cause the clearance of bacteria from a murine model. Based on these results, this project aims to obtain the structure of the urease from Mycobacterium smegmatis as a surrogate model and investigate its role in macrophage infection. On the other hand, we also aim to obtain the recombinant UreC subunit and screen using binding techniques, such as ligand libraries and urease inhibitors. Finally, we also expect to obtain the structure of UreC in complex with ligands and the protein-protein complex with RUVBL2. To achieve our goals, we will employ a range of biophysical techniques and strategies for structure determination, including protein crystallography and cryo-electron microscopy. We also expect to obtain a triple-mutant ¿ureA¿ureB¿ureC in M. smegmatis and test its viability and intracellular survival in macrophage cell culture. Gathering all this information, we will provide significant insights into the function of urease in mycobacteria and contribute to the development of new therapies.