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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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Functional Characterization of the Γ-Aminobutyric Acid Transporter from Mycobacterium Smegmatis MC2 155 Reveals Sodium-Driven GABA Transport This is a repository copy of Functional Characterization of the γ-Aminobutyric Acid Transporter from Mycobacterium smegmatis MC2 155 Reveals Sodium-Driven GABA Transport. White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/168970/ Version: Accepted Version Article: Pavić, A, Ji, Y, Serafini, A et al. (8 more authors) (2020) Functional Characterization of the γ-Aminobutyric Acid Transporter from Mycobacterium smegmatis MC2 155 Reveals Sodium-Driven GABA Transport. Journal of Bacteriology. ISSN 0021-9193 https://doi.org/10.1128/jb.00642-20 © 2020 Pavić et al. This is an author produced version of an article published in Journal of Bacteriology. Uploaded in accordance with the publisher's self-archiving policy. Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ 1 Functional characterization of the γ-aminobutyric acid transporter from 2 Mycobacterium smegmatis MC2 155 reveals sodium-driven GABA transport 3 4 Ana Pavića,b, Yurui Jia,c*, Agnese Serafinid, Acely Garza-Garciad, Martin J. McPhilliee, 5 Alexandra O. M. Holmesa, Luiz Pedro Sório de Carvalhod, Yingying Wangc, Mark Bartlamf#, 6 Adrian Goldmana,f,g#, Vincent L. G. Postisa,b#. 7 aAstbury Centre for Structural Molecular Biology, School of Biomedical Sciences, University 8 of Leeds, Leeds, UK 9 bBiomedicine Research Group, Faculty of Health and Social Sciences, Leeds Beckett 10 University, Leeds, UK 11 cCollege of Environmental Sciences & Engineering, Nankai University, Tianjin, China 12 dMycobacterial Metabolism and Antibiotic Research Laboratory, The Francis Crick Institute, 13 London, UK 14 eSchool of Chemistry, Faculty of Engineering & Physical Sciences, University of Leeds, Leeds, 15 UK 16 fCollege of Life Sciences, Nankai University, Tianjin, China 17 gMolecular and Integrative Biosciences, University of Helsinki, Helsinki, Finland 18 19 #Address correspondence to Adrian Goldman, [email protected], Vincent Postis, 20 [email protected] and Mark Bartlam, [email protected]. 21 22 *Present address: New Energy Department, Tianjin Sino-German University of Applied 23 Sciences, Tianjin, China. 24 25 Ana Pavić and Yurui Ji contributed equally to this work. Author order was determined on the 26 basis of the length of the first names. 27 28 Running title: Sodium ions drive transport in M. smegmatis GabP 1 29 ABSTRACT 30 Characterizing the mycobacterial transporters involved in the uptake and/or catabolism of host- 31 derived nutrients required by mycobacteria may identify novel drug targets against 32 tuberculosis. Here, we identify and characterize a member of the amino acid-polyamine- 33 organocation superfamily, a potential γ-aminobutyric acid transport protein, GabP, from 34 Mycobacterium smegmatis. The protein was expressed to a level allowing its purification to 35 homogeneity and Size Exclusion Chromatography-Multi Angle Laser Light Scattering analysis 36 of the purified protein showed that it was dimeric. We showed that GabP transported γ- 37 aminobutyric acid in vitro and when over-expressed in E. coli. Additionally, transport was 38 greatly reduced in the presence of β-alanine, suggesting that it could be either substrate or 39 inhibitor of GabP. Using GabP reconstituted into proteoliposomes, we demonstrated that γ- 40 aminobutyric acid uptake is driven by the sodium gradient and is stimulated by membrane 41 potential. Molecular docking showed that γ-aminobutyric acid binds MsGabP, another 42 Mycobacterium smegmatis putative GabP and the Mycobacterium tuberculosis homologue in 43 the same manner. This study represents the first expression, purification and characterization 44 of an active γ-aminobutyric acid transport protein from mycobacteria. 45 2 46 IMPORTANCE 47 The spread of multidrug resistant tuberculosis increases its global health impact in humans. As 48 there is transmission both to and from animals, the spread of the disease also increases its 49 effects in a broad range of animal species. Identifying new mycobacterial transporters will 50 enhance our understanding of mycobacterial physiology and furthermore provides new drug 51 targets. Our target protein is the gene product of msmeg_6196, annotated as GABA permease, 52 from Mycobacterium smegmatis strain MC2 155. Our current study demonstrates that it is a 53 sodium-dependent GABA transporter that may also transport β-alanine. As GABA may well 54 be an essential nutrient for mycobacterial metabolism inside the host, this could be an attractive 55 target for the development of new drugs against tuberculosis. 56 3 57 INTRODUCTION 58 Tuberculosis (TB), one of the oldest and deadliest human diseases, is caused by Mycobacterium 59 tuberculosis (Mtb). Mtb is a leading infectious killer, claiming the lives of about 1.2 million 60 people annually (1). It is a re-emerging pathogen, due to the development of multiple-drug 61 resistant (MDR) and extensively-drug resistant (XDR) strains (2). In addition, animal 62 tuberculosis is a globally distributed zoonotic chronic disease, posing a significant impact on 63 the costs in global agricultural losses (3). Since transmission between humans and animals has 64 been demonstrated in both directions, TB has been described as a One Health issue, having 65 similar consequences for humans and animals (4), and causing huge socioeconomic impact 66 both in terms of human lives and resources. 67 The unique cell wall composition of Mtb is believed to be the major determinant of its 68 resistance to a large range of antibiotics (5), leading to a continuing need to discover new ones. 69 A new approach to developing therapeutics against TB is through comprehensive discovery 70 and characterization of metabolic pathways, their impact on key features of Mtb pathogenesis 71 (6) and their contribution to drug resistance. Because Mtb has a reduced genome (7), it has 72 neither extended de novo synthesis pathways nor duplicated transporters – the essential 73 transmembrane proteins that mediate the nutrient uptake and metabolite efflux (8), meaning 74 that there are many essential metabolites. The transporters, required for the uptake of essential 75 nutrients, enable mycobacteria to persist in the harsh intracellular host environments by 76 scavenging nutrients. They thus represent potential new drug targets. Characterizing unique 77 mycobacterial transporters provides another route to discover novel therapeutics to treat TB 78 (9). 79 Despite considerable progress in the development of genetic methods for mycobacteria the 80 function of many transporters is still unknown. Nonetheless, AnsP1, an aspartate importer, was 4 81 identified by targeted mass-spectrometry based metabolomics. This method targets a pre- 82 defined group of compounds and aims to determine which one is transported. A mixture of 83 biomolecules is introduced into the mass spectrometer either directly or following a separation 84 procedure. In particular, liquid chromatography-mass spectrometry-based targeted 85 metabolomics achieves high level sensitivity and accuracy (10). AnsP1 was shown to be 86 involved in nitrogen metabolism and essential for mycobacterial infection and survival (11- 87 13). This provides evidence that Mtb relies on amino acid uptake and degradation pathways to 88 thrive inside the host and confirms a strong link between nutrition and pathogenicity in Mtb. 89 To identify the substrates of other orphan transporters from mycobacteria, we adopted a similar 90 approach for five homologous genes of AnsP1 from three different mycobacterial species. 91 From this initial screen, the annotated GABA permease from Mycobacterium smegmatis MC2 92 155, (MsGabP hereafter), proved promising for further studies. M. smegmatis is widely used 93 as a convenient model system to study Mtb biology, cell structure and persistence under 94 conditions of nutrient starvation (14) because it has a faster growth rate and lower biosafety 95 level than M. tuberculosis. Based on its sequence, this putative permease belongs to the group 96 of amino acid-polyamine-organocation (APC) superfamily of transport proteins (15), which is 97 widely found in all living organisms. To date, no in vivo or in vitro information is available for 98 Mycobacterium smegmatis GabP despite it being annotated as a probable GABA permease 99 based on its sequence identity to E. coli GabP (16). The hydropathic profile produced by 100 TMHMM2 predicted that MsGabP has twelve transmembrane-spanning helices (17). 101 Homology modelling of the protein shows that it adopts an arrangement known as the LeuT 102 fold (18) (Fig. S1). 103 In eukaryotes, besides its primary function as an inhibitory neurotransmitter (19), GABA may 104 have a role in the modulation of immune responses (20) but it is unclear whether and how 5 105 GABAergic signalling regulates antimicrobial host defences during infections. GABA does, 106 however, act as a specific cytotoxicity
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