Abstract
Phosphodiesterase enzymes, involved in cAMP hydrolysis reaction, are present throughout phylogeny and their phosphorylation mediated regulation remains elusive in prokaryotes. In this context, we focused on this enzyme from Mycobacterium tuberculosis. The gene encoded by Rv0805 was PCR amplified and expressed as a histidine-tagged protein (mPDE) utilizing Escherichia coli based expression system. In kinase assays, upon incubation with mycobacterial Clade I eukaryotic-type Ser/Thr kinases (PknA, PknB, and PknL), Ni-NTA purified mPDE protein exhibited transphosphorylation ability albeit with varying degree. When mPDE was co-expressed one at a time with these kinases in E. coli, it was also recognized by an anti-phosphothreonine antibody, which further indicates its phosphorylating ability. Mass spectrometric analysis identified Thr-309 of mPDE as a phosphosite. In concordance with this observation, anti-phosphothreonine antibody marginally recognized mPDE-T309A mutant protein; however, such alteration did not affect the enzymatic activity. Interestingly, mPDE expressed in Mycobacterium smegmatis yielded a phosphorylated protein that preferentially localized to cell wall. In contrast, mPDE-T309A, the phosphoablative variant of mPDE, did not show such behavior. On the other hand, phosphomimics of mPDE (T309D or T309E), exhibited similar cell wall anchorage as was observed with the wild-type. Thus, our results provide credence to the fact that eukaryotic-type Ser/Thr kinase mediated phosphorylation of mPDE renders negative charge to the protein, promoting its localization on cell wall. Furthermore, multiple sequence alignment revealed that Thr-309 is conserved among mPDE orthologs of M. tuberculosis complex, which presumably emphasizes evolutionary significance of phosphorylation at this residue.
Highlights
Cells respond to various environmental changes by an orchestra of events and transfer information through the process of signal transduction (McDonough and Rodriguez, 2012)
It is pertinent to mention that phosphodiesterases in Mycobacterium avium and M. smegmatis (MSMEG_2647 and MSMEG_6343), are distantly placed from M. tuberculosis (Figure 1)
Our findings clearly demonstrated that mutation of phosphorylating residue (Thr309) to alanine affected the localization of protein to cell wall (Figure 7B, compare lanes 3 and 6 of upper panel), whereas the presence of other non-specific bands obtained in Figure 7A were not seen with M. tuberculosis phosphodiesterase (mPDE) specific antibody
Summary
Cells respond to various environmental changes by an orchestra of events and transfer information through the process of signal transduction (McDonough and Rodriguez, 2012). M. tuberculosis is considered to be one of the most successful pathogen as it is capable of withstanding host defense mechanism(s) for its survival (Chevalier et al, 2014; Dey and Bishai, 2014; Orme, 2014) Such compliance might be attributed to its ability to camouflage within the micro-environment of the host and exhibit atypical or unique signal transduction mechanisms. In this context, signaling involving small molecules such as 3′, 5′-cyclic AMP, cyclic di-AMP, cyclic di-GMP etc., are less explored (Hong et al, 2013; Zaveri and Visweswariah, 2013; Manikandan et al, 2014). These cyclic nucleotide levels are controlled by different phosphodiesterases by promoting their hydrolytic degradation and play a crucial role in the process (Shenoy et al, 2005; Hong et al, 2013; Yang et al, 2014)
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