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Mycobacterial Membrane Transporter – An Entry Way for Therapeutic Intervention

To date, approximately one-third of the world’s population is harboring Mycobacterium tuberculosis (M. tuberculosis, Mtb), the causative pathogen of Tuberculosis (TB), a disease recognized by the World Health Organization as a world health threat.1 Two major hurdles in TB drug development are the impermeability of the mycobacterial cell wall to small molecules and the action of bacterial efflux pumps that actively transport drugs out of the cell.1 The acknowledged approach of engaging membrane transporters in directed drug uptake or as drug targets is very promising to overcome the thick and waxy cell membrane as a major hurdle in drug development. This strategy however will certainly require a more detailed understanding of the transporters and their dedicated substrates present in mycobacteria. We will contribute to this field in characterizing membrane transporters in various mycobacterial species.

To date, identification of membrane transporters and assignment of substrate molecules has been based mainly on in silico analysis followed by uptake assays using radioisotope-labeled probes and transporter knockout mutants, or by screening of mutant libraries and subsequent whole genome sequencing, searching for clones, which are not able to grow on the substrate of interest.2 Recently, we have established a simple and straightforward workflow to identify ABC-transporters of a given substrate using a label-free proteomics protocol.3 Our protocol relies on the induced expression of ABC-transporters by their respective substrates and the isolation of lipoproteins, as substrate binding units of classical mycobacterial ABC-transporters (Figure 1 A). Our protocol focuses so far on bacterial carbohydrate ABC-transporters, however we have strong evidence that it is also applicable to other potential substrates, such as vitamins or amino acids and to study permeases e.g. from the major-facilitator type.

Figure 1: ABC-Transporter in mycobacteria. A) Representative sketch of a classical importer-type ABC-transporter. SBP: substrate binding protein; TDM: transmembrane domain; ABC: ATP binding cassette. B) Differential expression analysis of membrane transporters expressed under various culturing conditions.

In this project, we want to determine uptake transporters for a subset of nutrient molecules and analyze their differential expression in relevant mycobacterial species (Figure 1 B, Mycobacterium smegmatis, Mycobacterium abscessus subsp. abscessus, Mycobacterium bovis BCG and Mycoabcterium tuberculosis H37Ra). Comparing these four species is especially interesting, as they comprise fast and slow-growing strains, which originate from different habitats; hence pose different demands in terms of required energy und carbon sources. After identifying potential transport proteins, we are planning follow up experiments using transporter knockout or knockdown clones that will be generated by double crossover events4 or CRSIPRi3 technique. Follow up studies will include, but are not limited to, structural and functional studies to determine the substrate scope of the proteins, as well as evaluation of their potential to function as drug transporter or drug target.

 

References

[1] Koch, A.; Cox, H.; Mizrahi, V. Drug-Resistant Tuberculosis: Challenges and Opportunities for Diagnosis and Treatment. Curr. Opin. Pharmacol. 2018, 42, 7–15. https://doi.org/10.1016/j.coph.2018.05.013.
[2] Braibant, M.; Gilot, P.; Content, J. The ATP Binding Cassette (ABC) Transport Systems of Mycobacterium Tuberculosis. FEMS Microbiol Rev 2000, 24 (4), 449–467.
[3] Dutta, A. K.; Choudhary, E.; Wang, X.; Záhorszka, M.; Forbak, M.; Lohner, P.; Jessen, H. J.; Agarwal, N.; Korduláková, J.; Jessen-Trefzer, C. Trehalose Conjugation Enhances Phototoxicity of Photosensitizers against Mycobacteria. ACS Cent. Sci. 2019, DOI: 10.1021/acscentsci.8b00962.
[4] Li, M.; Müller, C.; Fröhlich, K.; Gorka, O.; Zhang, L.; Groß, O.; Schilling, O.; Einsle, O.; Jessen-Trefzer, C. Detection and Characterization of a Mycobacterial L-Arabinofuranose ABC-Transporter Identified with a Rapid Lipoproteomics Protocol. Cell Chem Biol 2019, DOI: 10.1016/j.chembiol.2019.03.002.

 

Contact

Dr. Claudia Jessen-Trefzer

Department of Pharmaceutical Biology and Biotechnology
Stefan-Meier-Str. 19 (VF)
79104 Freiburg

Phone: +49 (761) 203 8381
Fax: +49 (761) 203 8383