Characterization of the function of RV1268c, an ATP binding cassette transporter in Mycobacterium tuberculosis.

Abstract

Tuberculosis (TB) is an epidemic disease that is caused by a bacterium called Mycobacterium tuberculosis (Mtb). This disease infects and kills millions of people globally. Anti-Tuberculosis (anti-TB) drugs such as isoniazid, ethambutol, pyrazinamide, and fluoroquinolones have been discovered and produced for TB treatment but regardless, TB persists because of the resistance to these drugs, leading to the development of multidrug-resistant (MDR) Mtb and extensively-drug resistant (XDR) Mtb. One of the key areas to focus on for the development of new effective anti- TB drugs are efflux systems, because they transport molecules outside cells and have a role in the resistance against TB treatment. This study aimed to identify the biological function of the Mtb protein, Rv1268c. RV1268c was one of the identified proteins in a study that was done by Chiliza et al., 2019 where a few protein biomarkers that are recognized by both active TB and latent TB patient antibodies. Some of these biomarkers that were studied are TreY, Bfr, and TrpG, which are biomarkers that are specific to ATP and play an important role in pathogenesis. Other biomarkers included MoaE, PonA1, and NarG, which are specific to latent TB and play a role in dormancy. The Rv1268c protein of Mtb is classified as a hypothetical membrane protein of the cell envelope and its associated proteins are Rv1267c and RV1269c, which are regulatory protein and a conserved putatively exported protein, respectively. The Rv1268c protein is hypothesised to be an ATP-binding cassette (ABC) transporter. The nucleotide and protein sequences of Rv1268c were v downloaded from the database of Mtb H37Rv using Mycobrowser. To create a knock down strain, annealed oligos were ligated to PLJR965 plasmid and transformed into XL10-Gold ultracompetent cells and grown on kanamycin-containing plates. Extracted plasmids were electroporated into Mtb, and after 4 weeks of incubation, the colonies were screened to check if they carried the knock down plasmid . DNA was then extracted to characterize the function of the Rv1268c Mtb protein. The results showed that Rv1268c had no effect on the in vitro growth of Mtb, while the Ethidium bromide (EtBr) assay displayed a difference on the extrusion of EtBr as the knock down and the knock down with anhydrotetracycline (aTc) had lower fluorescence as compared to the wild type which implies that Rv1268c is not an ABC transporter. The statistical analysis showed that the was no significant difference on the drug susceptibility between the wild type, knock down, and the knock down with aTc strains . The growth of neither the wild type or knock down strains was completely inhibited by either of the drugs tested. Keywords: