AUTHOR=De Siena Barbara , Campolattano Nicoletta , D’Abrosca Gianluca , Russo Luigi , Cantillon Daire , Marasco Rosangela , Muscariello Lidia , Waddell Simon J. , Sacco Margherita 

TITLE=Characterization of the Mycobacterial MSMEG-3762/63 Efflux Pump in Mycobacterium smegmatis Drug Efflux

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.575828

DOI=10.3389/fmicb.2020.575828

ISSN=1664-302X

ABSTRACT=<p>Multi-drug resistant tuberculosis (MDR-TB) represents a major health problem worldwide. Drug efflux and the activity of efflux transporters likely play important roles in the development of drug-tolerant and drug-resistant mycobacterial phenotypes. This study is focused on the action of a mycobacterial efflux pump as a mechanism of drug resistance. Previous studies demonstrated up-regulation of the TetR-like transcriptional regulator <italic>MSMEG_3765</italic> in <italic>Mycobacterium smegmatis</italic> and its ortholog <italic>Rv1685c</italic> in <italic>Mycobacterium tuberculosis</italic> (<italic>Mtb</italic>) in acid-nitrosative stress conditions. MSMEG-3765 regulates the expression of the <italic>MSMEG_3762/63/65</italic> operon, and of the orthologous region in <italic>Mtb</italic> (<italic>Rv1687c/86c/85c</italic>). MSMEG-3762 and Rv1687c are annotated as ATP-binding proteins, while MSMEG-3763 and Rv1686c are annotated as trans-membrane polypeptides, defining an ABC efflux pump in both <italic>M. smegmatis</italic> and <italic>Mtb</italic>. The two putative efflux systems share a high percentage of identity. To examine the role of the putative efflux system MSMEG-3762/63, we constructed and characterized a <italic>MSMEG-3763</italic> deletion mutant in <italic>M. smegmatis</italic> (∆<italic>MSMEG_3763</italic>). By comparative analysis of wild type, knockout, and complemented strains, together with structural modeling and molecular docking bioinformatics analyses of the MSMEG-3763 trans-membrane protein, we define the protein complex MSMEG-3762/63 as an efflux pump. Moreover, we demonstrate involvement of this pump in biofilm development and in the extrusion of rifampicin and ciprofloxacin (CIP), antimicrobial drugs used in first- and second-line anti-TB therapies.</p>