Production of recombinant urease from Cryptococcus neoformans in Pichia pastoris eukaryotic model

• Grant number: 23/11169-8
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Effective date (Start): March 01, 2024
• Effective date (End): February 28, 2025
• Field of knowledge: Biological Sciences - Microbiology - Biology and Physiology of Microorganisms
• Principal Investigator: Kelly Ishida
• Grantee: Thayná Lopes Barreto
• Supervisor: Angela Gelli
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Research place: University of California, Davis (UC Davis), United States
• Associated to the scholarship: 20/04229-6 - Investigation of the action mechanism and the antifungal effect of urease inhibitors on Cryptococcus spp., BP.DR

Abstract

Cryptococcosis is a mycosis caused by Cryptococcus spp. with high incidence in HIV/AIDS patients leading to high mortality and morbidity rates. The current therapy of the severe clinical form, cryptococcal meningitis, is based on the combination of amphotericin B with fluconazole and flucytosine. However, factors such as high toxicity, unfavorable pharmacokinetics, high cost and unavailability further reduce the therapeutic options for cryptococcosis. Thus, new antifungal molecules have been researched in recent years, addressing new targets of action, and the urease enzyme may be an interesting candidate. The urease produced by Cryptococcus spp. catalyzes the hydrolysis of urea into ammonia, which weakens the endothelial wall of the blood-brain barrier, facilitating the entry of the pathogen into the central nervous system, an important step in establishing cryptococcal meningitis. In addition, urea hydrolysis is also important for the survival of fungi within macrophages. Considering that urease is naturally absent in humans, enzymatic inhibition may be an interesting strategy in the treatment of cryptococcal meningitis, mainly in association with conventional antifungals. However, the three-dimensional structure of this enzyme is not yet known. In the doctorate project we will investigate synthetic molecules of new chemical classes for the control of cryptococcosis targeting the urease enzyme inhibition. Thus, in this proposal we will produce the recombinant urease from C. neoformans using the eukaryotic expression system with Pichia pastoris, characterize the enzyme by biochemical and biophysical analysis for future structural studies of enzyme alone or when inhibited with leader molecules previously selected, aiming to extend the knowledge at the molecular level of this enzyme and thus propose a therapeutic alternative for the treatment of mycoses caused by the urease-dependent fungus. (AU)