Abstract
Tuberculosis still remains a concerning health problem worldwide. Its etiologic agent, Mycobacterium tuberculosis, continues to be the focus of research to unravel new prophylactic and therapeutic strategies against this disease. The only vaccine in use against tuberculosis is based on the in vitro attenuated strain, M. bovis BCG. Dodecin is a dodecameric complex important for flavin homeostasis in Archea and Eubacteria, and the M. tuberculosis protein is described as thermo- and halostable. M. bovis BCG Moreau, the Brazilian vaccine strain, has a single nucleotide polymorphism in the dodecin start codon, leading to a predicted loss of seven amino acids at the protein N-terminal end. In this work we aimed to characterize the effect of this mutation in the BCG Moreau protein features. Our recombinant protein assays show that the predicted BCG homolog is less thermostable than M.tb’s but maintains its dodecamerization ability, although with a lower riboflavin-binding capacity. These data are corroborated by structural analysis after comparative modeling, showing that the predicted BCG dodecin complex has a lower interaction energy among its monomers and also a distinct electrostatic surface near the flavin binding pocket. However, western blotting assays with the native proteins were unable to detect significant differences between the BCG Moreau and M.tb orthologs, indicating that other factors may be modulating protein structure/function in the bacterial context.
Highlights
Dodecin plays an important role in bacterial physiology, mainly due to its predicted function as a storage protein for cofactors needed by these organisms throughout their life cycles, such as flavins and coenzyme A (Meissner et al, 2007)
Due to its importance in flavin homeostasis, already described for other microorganisms (Grininger et al, 2009), here we report the initial characterization of Bacillus Calmette-Guérin (BCG) Moreau dodecin, comparing it with the known M.tb homolog, aiming to understand the possible functional impacts of this mutation
The BCG Moreau dodecin coding gene (BCG_M1530c) carries the same single nucleotide polymorphism (SNP) described for the M. bovis AF2122-97 and BCG Pasteur orthologs to M. tuberculosis H37Rv rv1498A
Summary
Dodecin plays an important role in bacterial physiology, mainly due to its predicted function as a storage protein for cofactors needed by these organisms throughout their life cycles, such as flavins and coenzyme A (Meissner et al, 2007). In Archaea, like Halobacterium salinarum, this protein is suggested as a key player on flavin homeostasis, functioning as a buffer for this kind of molecule (Grininger et al, 2009), and acting as a source of active flavins (FADH2 and FMN) or riboflavin for production of the former when required. It may prevent harmful effects of flavin light-induced reactions on the bacterial cytosol, similar to eukaryotic flavin-binding proteins (Kelley et al, 2017). Dispersal of antibioticresistant strains and the increase in HIV coinfection cases (Lai et al, 2016) justify the urgency for new prophylactic and therapeutic approaches, warranting the need for a better understanding of mycobacterial metabolism and physiology
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