Characterisation of a putative AraC transcriptional regulator from Mycobacterium smegmatis

MSMEG_0307 is annotated as a transcriptional regulator belonging to the AraC protein family and is located adjacent to the arylamine N-acetyltransferase (nat) gene in Mycobacterium smegmatis, in a gene cluster, conserved in most environmental mycobacterial species. In order to elucidate the function of the AraC protein from the nat operon in M. smegmatis, two conserved palindromic DNA motifs were identified using bioinformatics and tested for protein binding using electrophoretic mobility shift assays with a recombinant form of the AraC protein. We identified the formation of a DNA:AraC protein complex with one of the motifs as well as the presence of this motif in 20 loci across the whole genome of M. smegmatis, supporting the existence of an AraC controlled regulon. To characterise the effects of AraC in the regulation of the nat operon genes, as well as to gain further insight into its function, we generated a ΔaraC mutant strain where the araC gene was replaced by a hygromycin resistance marker. The level of expression of the nat and MSMEG_0308 genes was down-regulated in the ΔaraC strain when compared to the wild type strain indicating an activator effect of the AraC protein on the expression of the nat operon genes

Cholesteroid nature of free mycolic acids from M. tuberculosis

Mycolic acids (MAs) are a major component of the cell walls of Mycobacterium tuberculosis and related organisms. These alpha-alkyl beta-hydroxy long fatty acids have been the subject of numerous studies for their immunological properties. We previously reported that an interaction between cholesterol and mycolic acids could be responsible for the low accuracy in the serodiagnosis of TB when using free mycolic acid in an ELISA assay. The aim of this work was to investigate if this interaction could be due to a similarity in the structural properties between mycolic acids and cholesterol. The investigation revealed that patient sera cross-reacted with mycolic acids and cholesterol in an ELISA experiment suggesting that both molecules may present related functionality in a similar structural orientation. This relation was further supported by the interaction of mycolic acids with Amphotericin B (AmB), a known binding agent to ergosterol and cholesterol. Using a resonant mirror biosensor, we observed that AmB recognised both cholesterol and rnycolic acids. In addition, a specific attraction was observed between mycolic acid and cholesterol by the accumulation of cholesterol from liposomes ill Suspension onto immobilized mycolic acids containing liposomes, detected with a biosensor technique. Combined, these results suggest that mycolic acids call assume a three-dimensional conformation similar to a sterol. This requires that mycolic acid exposes its hydroxyl group and assumes rigidity in its chain structure to generate a hydrophobic surface topology matching that of cholesterol. A particular folded conformation Would be required for this, of which a few different types have already been proven to exist in monolayers of mycolic acids