Abstract
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 MSMEG_3765 in Mycobacterium smegmatis and its ortholog Rv1685c in Mycobacterium tuberculosis (Mtb) in acid-nitrosative stress conditions. MSMEG-3765 regulates the expression of the MSMEG_3762/63/65 operon, and of the orthologous region in Mtb (Rv1687c/86c/85c). 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 M. smegmatis and Mtb. 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 MSMEG-3763 deletion mutant in M. smegmatis (∆MSMEG_3763). 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.
Highlights
Tuberculosis (TB) is still considered a public health alert worldwide, with 10 million people falling ill and an estimated 1.4 million deaths in 2018 (Global Tuberculosis Report 2019)
ethidium bromide (EtBr) accumulation was higher for the mutant compared to the wild type and the complemented strains (Figure 1B), demonstrating that loss of MSMEG-3763 reduced the ability of mycobacteria to remove EtBr, and suggesting that this complex possessed efflux pump activity
Multi-drug resistance of an increasing number of pathogens represents a growing health problem worldwide. This is urgent for diseases like tuberculosis, mainly caused by Mycobacterium tuberculosis (Mtb), or nosocomial infections caused by opportunistic pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii (Dheda et al, 2017; Botelho et al, 2019; Guo et al, 2020; Moubareck and Halat, 2020)
Summary
Tuberculosis (TB) is still considered a public health alert worldwide, with 10 million people falling ill and an estimated 1.4 million deaths in 2018 (Global Tuberculosis Report 2019). Other factors are associated with more severe TB, like renal disease, air pollution, tobacco consumption, alcohol abuse, and an aging population (Hameed et al, 2018). In this scenario, multi-drug resistance (MDR-TB) represents a significant risk to global public health, and is considered one of the most urgent problems to address (Ayabina et al, 2016; Dheda et al, 2017; Singh et al, 2019). Efflux pumps may be important for bacterial survival within macrophages, by detoxifying intracellular bacilli from acidification, toxic metal accumulation and the effects of nitrogen and oxygen reactive intermediates (Ehrt and Schnappinger, 2009)
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