ABSTRACT
Avermectins are macrocyclic lactones with anthelmintic activity. Recently, they were found to be effective against Mycobacterium tuberculosis, which accounts for one third of the worldwide deaths from antimicrobial resistance. However, their anti-mycobacterial mode of action remains to be elucidated. The activity of selamectin was determined against a panel of M. tuberculosis mutants. Two strains carrying mutations in DprE1, the decaprenylphosphoryl-ß-D-ribose oxidase involved in the synthesis of mycobacterial arabinogalactan, were more susceptible to selamectin. Biochemical assays against the Mycobacterium smegmatis DprE1 protein confirmed this finding, and docking studies predicted a binding site in a loop that included Leu275. Sequence alignment revealed variants in this position among mycobacterial species, with the size and hydrophobicity of the residue correlating with their MIC values; M. smegmatis DprE1 variants carrying these point mutations validated the docking predictions. However, the correlation was not confirmed when M. smegmatis mutant strains were constructed and MIC phenotypic assays performed. Likewise, metabolic labeling of selamectin-treated M. smegmatis and M. tuberculosis cells with 14C-labeled acetate did not reveal the expected lipid profile associated with DprE1 inhibition. Together, our results confirm the in vitro interactions of selamectin and DprE1 but suggest that selamectin could be a multi-target anti-mycobacterial compound.
Subject(s)
Alcohol Oxidoreductases/antagonists & inhibitors , Antiparasitic Agents/pharmacology , Antitubercular Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Ivermectin/analogs & derivatives , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/enzymology , Alcohol Oxidoreductases/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , Binding Sites , Dose-Response Relationship, Drug , Drug Discovery , Ivermectin/pharmacology , Microbial Sensitivity Tests , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation , Structure-Activity RelationshipABSTRACT
Ferric and ferrous iron is an essential transition metal for growth of many bacterial species including mycobacteria. The genomic region msmeg_0234 to msmeg_0252 from Mycobacterium smegmatis is putatively involved in iron/heme metabolism. We investigate the genes encoding the presumed two component system MSMEG_0244/MSMEG_0246, the neighboring gene msmeg_0243 and their involvement in this process. We show that purified MSMEG_0243 indeed is a heme binding protein. Deletion of msmeg_0243/msmeg_0244/msmeg_0246 in Mycobacterium smegmatis leads to a defect in biofilm formation and colony growth on solid agar, however, this phenotype is independent of the supplied iron source. Further, analysis of the corresponding mutant and its lipids reveals that changes in morphology and biofilm formation correlate with altered acylation patterns of phosphatidylinositol mannosides (PIMs). We provide the first evidence that msmeg_0244/msmeg_0246 work in concert in cellular lipid homeostasis, especially in the maintenance of PIMs, with the heme-binding protein MSMEG_0243 as potential partner.
