Functional and structural characterization of the Mycobacterium tuberculosis transporter Rv2563/Rv2564

• Grant number: 24/02428-2
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: September 01, 2024
• End date: January 31, 2028
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Andrea Balan Fernandes
• Grantee: Nelson Alejandro Sierra Cortés
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Mycobacterium tuberculosis (Mtb) is the infectious agent that causes tuberculosis, one of the main causes of death and health problems in the world. Within the bacillus's survival and infection processes, the ability to remodel the cell envelope after entering the macrophage, escape from the immune system and resistance to antimicrobial agents stands out. Among many relevant proteins in these processes, ABC-type transporters (ATP-Binding Cassette) are highlighted as they belong to one of the largest families of membrane proteins, conserved in all organisms and because they are related to cellular transport, virulence , pathogenesis and the phenomenon of multiple drug resistance. The M. tuberculosis Rv2563/Rv2564 transporter was previously characterized by our group as a transporter similar to MacB transporters (involved in drug transport), consisting of two ATPase domains and two transmembrane domains that extend to the periplasm, whose expression is increased during the macrophage infection process. In this project we intend to carry out the functional characterization of the transporter using molecular biology and biophysical and structural techniques using X-ray crystallography and cryo-electron microscopy. Protocols for expression and purification of the transporter and its separate domains have already been established by the student, as well as the production of the periplasmic (Rv2563per) and ATP-binding (Rv2564) domains. The Rv2563per domain was used to produce antibodies that will be used in future trials and produced on a large scale for Nuclear Magnetic Resonance trials to be carried out this month. The ATPase was produced and subjected to crystallization tests at Robolab in Campinas and its activity will be measured. The conservation of this transporter and a paralogue only in species of the M. tuberculosis complex (MTBC) suggests that the transporter has an important role in virulence and pathogenesis processes in vivo and stands out as a target for the development of inhibitors.