Rv1288, a Two Domain, Cell Wall Anchored, Nutrient Stress Inducible Carboxyl-Esterase of Mycobacterium tuberculosis, Modulates Cell Wall Lipid

Abstract

Rv1288, a conserved hypothetical protein of M. tuberculosis (M.tb), was recently characterized as two-domain esterase enzyme by in silico study. In the present study, Rv1288 and its domains (Est and Lyt) were cloned individually from M.tb into E. coli for expression and purification. The purified rRv1288 and rEst proteins exhibited lipolytic activity with medium chain length esters as optimum substrates, while Lyt domain did not show enzymatic activity. However, presence of Lyt domain resulted in enhanced rate of protein aggregation at higher temperature. Both rRv1288 and rEst followed the similar patterns of substrate specificity, temperature and pH activity. Site directed mutagenesis confirmed the Ser-294, Asp-391 and His-425 as catalytic site residues. Rv1288 was found to be present in cell wall fraction of M.tb H37Ra. Peptidoglycan binding activity of Rv1288 and its domains demonstrated that the Lyt domain is essential for anchoring protein to the cell wall. Expression of rv1288 was up regulated in M.tb under nutrient starved condition. Over expression of rv1288 in surrogate host M. smegmatis led to change in colony morphology, enhanced pellicle and aggregate formation that might be linked with the changed lipid composition of bacterial cell wall. Cell wall of M. smegmatis expressing rv1288 had higher amount of lipids, with a significant increase in trehalose dimycolate content. Rv1288 also leads to increase in drug resistance of M. smegmatis. Rv1288 also enhanced the intracellular survival of M. smegmatis in Raw264.7 cell line. Overall, this study suggested that Rv1288, a cell wall localized carboxyl hydrolase with mycolyl-transferase activity, modulated the cell wall lipids to favor the survival of bacteria under stress condition.

Rv3091, An Extracellular Patatin-Like Phospholipase in Mycobacterium tuberculosis, Prolongs Intracellular Survival of Recombinant Mycolicibacterium smegmatis by Mediating Phagosomal Escape

“…Rv3091 protein overexpression allow recombinant Ms3091 to use lipids as a major carbon source, which further supports the role of this protein in bacterial intracellular survival, which agrees with the findings of previous studies (Zambrano and Kolter, 2005;Wang et al, 2008;Santucci et al, 2018a). These results were consistent with previous studies (Kumar et al, 2017;Maan et al, 2018;Kaur and Kaur, 2019).…”

Mycobacterium tuberculosis Small RNA MTS1338 Confers Pathogenic Properties to Non-Pathogenic Mycobacterium smegmatis

“…Therefore, in this study, we examined the functional effects of MTS1338 by expressing it in non-pathogenic Mycolicibacterium smegmatis (basonym of Mycobacterium smegmatis , MSmeg), which shares about 79% nucleotide sequence identity with MTb and is very similar to it in terms of cell wall composition and metabolism, but lacks the MTS1338 gene [ 16 , 17 , 18 , 19 ]. Our results indicate that MTS1338 upregulates the enzymes responsible for the synthesis of the mycobacterial cell envelope and promotes the survival of MSmeg in macrophages, in which the MTS1338-expressing MSmeg induces phagosome maturation arrest and modulates the expression of pro-inflammatory cytokines.…”

Persistence of Mycobacterium tuberculosis in response to infection burden and host-induced stressors

“…Since esterases/lipases are involved in lipid degradation, this provides M. tuberculosis with an efficient source of energy and carbon for intracellular persistence (Maurya et al, 2018;Nazarova et al, 2019), as shown using recombinant strains (Lun and Bishai, 2007). Esterases/lipases are furthermore involved in remodelling of cell wall lipids, which have been shown to alter colony morphology, aggregation, and pellicle formation, thereby enhancing M. tuberculosis antibiotic tolerance and persistence (Singh et al, 2016;Kumar et al, 2017;Maan et al, 2018).…”