Structural and functional characterization of ABC transporter of glutamine from Mycobacterium tuberculosis.

Author(s)

Marcelo de Cássio Barreto de Oliveira

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2021-11-08

Examining board members

Andrea Balan Fernandes; Cristiane Rodrigues Guzzo Carvalho; Andre Kipnis; Gustavo Fernando Mercaldi

• Advisor: Andrea Balan Fernandes

Abstract

Tuberculosis is the main infectious disease worldwide, being responsible for thousands of deaths. Caused by the Mycobacterium tuberculosis bacillus (MTB), it is a disease that is difficult to diagnose and has limited treatment options. ABC transporters, ATP-Binding Cassette, represent the main class of transport proteins in MTB, and although they are highly relevant in the process of drug resistance and survival in the macrophage, little is known about their specific function. The function of most of the systems described, or their essentiality during infection, has been attributed by genomic or proteomic approaches with putative functions. Since the first review on the subject, in 2000, more specific studies of such transporters have revealed an enormous functional and structural diversity, increasing interest in the area. Among these systems, the study target of this work stands out, the Rv2563-Rv2564 transporter, whose putative function is not yet known. Based on bioinformatics analysis, we identified that the transporter presents domains and structural characteristics of transporters from the MacB superfamily, whose main representatives are related to drug efflux and recycling of membrane components, and that together with another M. tuberculosis transporter (Rv0072-Rv0073), with which it shares high sequential/structural similarity, are present only in M. tuberculosis complex bacteria, indicating that there may be duplication of functions. From different constructs, the periplasmic domains of the permease Rv2563 (Rv2563per), the complete permease and the ATP binding domain (ATPase) were obtained. Although we were unable to stably obtain the complex, permease crystals were obtained which diffracted at 4.95 Å resolution. The periplasmic region of the membrane protein Rv2563 was characterized in circular dichroism assays and used to obtain antibodies in C57BL/6 mice. Western blot, pull-down, mass spectrometry and RT-PCR assays, revealed that the transporter is constitutively transcribed under the conditions analyzed and was not regulated by the presence of antibiotics in the culture medium. Data suggest that their participation may be related to cellular stress conditions. The data set obtained in this work consists of the first compilation of studies on this transporter that can be an excellent target for the development of new drugs, diagnostic tests, and immunogens. (AU)

• FAPESP's process: 15/17476-3 - Functional and structural characterization of the glutamine transporter from Mycobacterium tuberculosis
• Grantee: Marcelo de Cássio Barreto de Oliveira
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)