br Materials and methods br Results
Materials and methods
Discussion Tuberculosis is caused by the mycobacterium M. tuberculosis, a pathogen able to survive in the hostile conditions through sophisticated defence mechanisms. In an attempt to clarify some aspects of the M. tuberculosis defence mechanisms, we have investigated the effect of methylation stress caused by sub-lethal concentration of MMS in clinal strains of M. tuberculosis and other tubercular species. Most of mycobacteria appeared sensitive to MMS incubation showing a clear decrease in cell viability, including the strains that had developed isoniazid resistant capabilities. Recently we demonstrated that treatment of E. coli with small amounts of MMS caused a strong decrease in biofilm formation (18). A global investigation of mycobacteria response to alkylating agents at the molecular level was then performed using M. smegmatis, a non pathogenic mycobacteria strain. Surprisingly, contrary to E. coli, when treated with a sublethal amount of MMS, M. smegmatis showed a clear increase in biofilm formation. Comparative proteomics allowed us to establish that MMS treatment induces upregulation of several proteins involved in ghrelin receptor antagonist biosynthesis and biofilm formation. Among these, molecular chaperones DnaK and GroEL1 have recently been associated to biofilm formation and stress resistance mechanisms. Chemical inhibition of DnaK was reported to reduce biofilm production in E. coli (40) and decreased biofilm formation and adhesion properties in S. aureus were observed in the presence of a DnaK non-functional mutation (52). The GroEL1 protein affects the bio-synthesis of mycolates, crucial components of mycobacterial cell wall, specifically during production of biofilm. Moreover, inactivation of the groEL1 gene prevented the formation of mature biofilms in M. smegmatis (41). We speculated that the increase in biofilm formation induced by alkylation stress might be part of a more general strategy adopted by mycobacterium to defend itself when submitted to different stress conditions. For instance, biofilm formation and glmS expression are increased in E. coli by exposure to PEG, which mimics an osmotic stress (53). Clustering analysis of proteomics data showed that several up-regulated proteins gathered within the metabolic pathway leading to peptidoglycan biosynthesis. Among these, we focused our attention on the bifunctional enzyme GlmU whose expression was largely increased under conditions inducing biofilm formation. GlmU is involved in the biosynthesis of UDP-N-acetylglucosamine (UDP-GlcNAc) an essential precursor of β-1,6-N-acetyl-D-glucosamine polysaccharide adhesin needed for biofilm production in E. coli and S. epidermidis (43). Moreover, GlmU was reported to play a role in biofilm formation in P. aeruginosa, K. pneumoniae and E. foecalis 19, 21, 51. On this ground, we pursued a detailed investigation on the putative role of GlmU in biofilm production in M. smegmatis. We developed a GlmU conditional deletion mutant in which the glmU promoter was under the control of Atc (MsΔglmU) and an overexpressing mutant where the glmU gene was under the control of IVN (Ms.pNit-glmU). The MsΔglmU mutant showed normal biofilm formation under methylation stress conditions in the absence of ATc. However, a decrease in biofilm production was detected when GlmU was depleted at early stage of cell growth by ATc treatment. On the contrary, an increase in biofilm production was detected when the Ms.pNit-glmU mutant was stimulated with IVN, suggesting that increasing amount of GlmU positively affected biofilm production. However, since the GlmU enzyme is essential for bacterial growth, experiments with M. smegmatis mutants might be questionable as the increase/reduction in biofilm formation might also be related to increase/decrease rate of replication of M. smegmatis and not directly to GlmU activity. Therefore, to further define whether or not GlmU is involved in biofilm production in M. smegmatis, we examined the effect enzymatic inhibitors able to interfere with the GlmU acetyltransferase domain on biofilm formation 19, 20, 46, 47, 48, 49, 50. In the presence of either IAA or GlcNAc-1P, well known inhibitors of the GlmU acetyltransferase activity, biofilm formation was diminished decreasing to a level very similar to control cells, whereas both cell viability and bacterial growth were only slightly affected.