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Functional characterization of the γ-aminobutyric acid transporter from Mycobacterium smegmatis MC2 155 reveals sodium-driven GABA transport.

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posted on 29.01.2021, 09:33 by Ana Pavić, Yurui Ji, Agnese Serafini, Acely Garza-Garcia, Martin J McPhillie, Alexandra OM Holmes, Luiz Pedro Sório de Carvalho, Yingying Wang, Mark Bartlam, Adrian Goldman, Vincent LG Postis
Characterizing the mycobacterial transporters involved in the uptake and/or catabolism of host-derived nutrients required by mycobacteria may identify novel drug targets against tuberculosis. Here, we identify and characterize a member of the amino acid-polyamine- organocation superfamily, a potential γ-aminobutyric acid transport protein, GabP, from Mycobacterium smegmatis The protein was expressed to a level allowing its purification to homogeneity and Size Exclusion Chromatography-Multi Angle Laser Light Scattering analysis of the purified protein showed that it was dimeric. We showed that GabP transported γ-aminobutyric acid in vitro and when over-expressed in E. coli Additionally, transport was greatly reduced in the presence of β-alanine, suggesting that it could be either substrate or inhibitor of GabP. Using GabP reconstituted into proteoliposomes, we demonstrated that γ-aminobutyric acid uptake is driven by the sodium gradient and is stimulated by membrane potential. Molecular docking showed that γ-aminobutyric acid binds MsGabP, another Mycobacterium smegmatis putative GabP and the Mycobacterium tuberculosis homologue in the same manner. This study represents the first expression, purification and characterization of an active γ-aminobutyric acid transport protein from mycobacteria.IMPORTANCE The spread of multidrug resistant tuberculosis increases its global health impact in humans. As there is transmission both to and from animals, the spread of the disease also increases its effects in a broad range of animal species. Identifying new mycobacterial transporters will enhance our understanding of mycobacterial physiology and furthermore provides new drug targets. Our target protein is the gene product of msmeg_6196, annotated as GABA permease, from Mycobacterium smegmatis strain MC2 155. Our current study demonstrates that it is a sodium-dependent GABA transporter that may also transport β-alanine. As GABA may well be an essential nutrient for mycobacterial metabolism inside the host, this could be an attractive target for the development of new drugs against tuberculosis.

Funding

Crick (Grant ID: 10060, Grant title: Carvalho FC001060) Wellcome Trust (Grant ID: 104785/B/14/Z, Grant title: WT 104785/B/14/Z)

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