Abstract
Tuberculosis is a world widespread disease, caused by Mycobacterium tuberculosis (M.tb). Although considered an obligate aerobe, this organism can resist life-limiting conditions such as microaerophily mainly due to its set of enzymes responsible for energy production and coenzyme restoration under these conditions. One of these enzymes is fumarate reductase, an heterotetrameric complex composed of a catalytic (FrdA), an iron-sulfur cluster (FrdB) and two transmembrane (FrdC and FrdD) subunits involved in anaerobic respiration and important for the maintenance of membrane potential. In this work, aiming to further characterize this enzyme function in mycobacteria, we analyzed the expression of FrdB-containing proteins in M.tb and Mycobacterium bovis Bacillus Calmette–Guérin (BCG) Moreau, the Brazilian vaccine strain against tuberculosis. We identified three isoforms in both mycobacteria, two of them corresponding to the predicted encoded polypeptides of M.tb (27 kDa) and BCG Moreau (40 kDa) frd sequences, as due to an insertion on the latter’s operon a fused FrdBC protein is expected. The third 52 kDa band can be explained by a transcriptional slippage event, typically occurring when mutation arises in a repetitive region within a coding sequence, thought to reduce its impact allowing the production of both native and variant forms. Comparative modeling of the M.tb and BCG Moreau predicted protein complexes allowed the detection of subtle overall differences, showing a high degree of structure and maybe functional resemblance among them. Axenic growth and macrophage infection assays show that the frd locus is important for proper bacterial development in both scenarios, and that both M.tb’s and BCG Moreau’s alleles can partially revert the hampered phenotype of the knockout strain. Altogether, our results show that the frdABCD operon of Mycobacteria may have evolved to possess other yet non-described functions, such as those necessary during aerobic logarithmic growth and early stage steps of infection.
Highlights
Tuberculosis (TB) remains as an important health issue and is among the 10 diseases in death numbers worldwide
Analysis of other species belonging to the M. tuberculosis complex shows that this mutation is only present in the M. bovis lineage, and the M.tb H37Rv pattern is observed throughout M.tb lineages 1 to 4, the
When looking to amino acids interacting with known coenzymes and inhibitors of fumarate reductase (FRD), we observe local changes in the intermolecular interactions, that are all related to rotations in the residues side chain (Figure 7)
Summary
Tuberculosis (TB) remains as an important health issue and is among the 10 diseases in death numbers worldwide. Despite the availability of early diagnostic tools and well-established treatment protocols, TB remains a global health emergency, as declared in 1993 by the World Health Organization (WHO) (Dolin et al, 1993). Since 2015, this scenario has aggravated and TB surpassed the human acquired immunodeficiency syndrome, reaching the first place in death numbers caused by an infectious contagious disease (World Health Organization, 2019). In the early infection phase, bacteria are taken-up by macrophages and other immune cells, where they encounter life-limiting conditions such as low oxygen tension and nutrient availability that can lead M.tb to a non-replicative state (Salgame et al, 2015). It is known that M.tb displays a high capacity to adapt to these different environments, a feature represented by its repertoire of enzymes involved in energy metabolism
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