The Mycobacterium tuberculosis GroEL1 Chaperone Is a Substrate of Ser/Thr Protein Kinases

Self Cite

Background:The catabolite control protein A (CcpA) plays an important role in Staphylococcus aureus virulence, biofilm formation, and central metabolism. Results: Phosphorylation of CcpA negatively affects its DNA binding activity and thus the expression of its target genes. Conclusion: CcpA activity is modulated by Stk1 phosphorylation. Significance: This study highlights that phosphorylation of CcpA represents a novel regulatory control mechanism for this major transcriptional factor.

Section: Resultsmentioning

confidence: 91%

A Novel Mode of Regulation of the Staphylococcus aureus Catabolite Control Protein A (CcpA) Mediated by Stk1 Protein Phosphorylation

Leiba

Hartmann

Cluzel

et al. 2012

Self Cite

“…25 and Thr 54 , were originally identified by mass spectrometry (39). Whereas a significant decrease of the phosphorylation signal was observed with the GroEL_T25A mutant, phosphorylation of the GroEL_T54A mutant was not affected, suggesting that Thr 25 and Thr 54 were not equivalent with respect to phosphorylation by PknF, as Thr 25 appears to be the primary phosphorylation site (39).…”

Section: Discussionmentioning

confidence: 99%

“…MabA Is Phosphorylated in Vitro on Three Threonine Residues-Mass spectrometry was used to identify the number and nature of the phosphorylation sites on MabA, a method that has been successfully used to elucidate the phosphoacceptors in a sequence-specific fashion for several other M. tuberculosis STPK substrates (32,39). Briefly, MabA was incubated with cold ATP in the presence of PknB (one of the most active kinases for MabA, Fig.…”

Section: Maba Is Phosphorylated In Vitro By Multiple Ser/thrmentioning

confidence: 99%

Phosphorylation of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA Regulates Mycolic Acid Biosynthesis

Veyron‐Churlet

Zanella-Cléon

Cohen‐Gonsaud

et al. 2010

Self Cite

Mycolic acids are key cell wall components for the survival, pathogenicity, and antibiotic resistance of the human tubercle bacillus. Although it was thought that Mycobacterium tuberculosis tightly regulates their production to adapt to prevailing environmental conditions, the molecular mechanisms governing mycolic acid biosynthesis remained extremely obscure. Meromycolic acids, the direct precursors of mycolic acids, are synthesized by a type II fatty acid synthase from acyl carrier protein-bound substrates that are extended iteratively, with a reductive cycle in each round of extension, the second step of which is catalyzed by the essential ␤-ketoacylacyl carrier protein reductase, MabA. In this study, we investigated whether post-translational modifications of MabA might represent a strategy employed by M. tuberculosis to regulate mycolic acid biosynthesis. Indeed, we show here that MabA was efficiently phosphorylated in vitro by several M. tuberculosis Ser/Thr protein kinases, including PknB, as well as in vivo in mycobacteria. Mass spectrometric analyses using LC-ESI/MS/MS and site-directed mutagenesis identified three phosphothreonines, with Thr 191 being the primary phosphor-acceptor. A MabA_T191D mutant, designed to mimic constitutive phosphorylation, exhibited markedly decreased ketoacyl reductase activity compared with the wild-type protein, as well as impaired binding of the NADPH cofactor, as demonstrated by fluorescence spectroscopy. The hypothesis that phosphorylation of Thr 191 alters the enzymatic activity of MabA, and subsequently mycolic acid biosynthesis, was further supported by the fact that constitutive overexpression of the mabA_T191D allele in Mycobacterium bovis BCG strongly impaired mycobacterial growth. Importantly, conditional expression of the phosphomimetic MabA_T191D led to a significant inhibition of de novo biosynthesis of mycolic acids. This study provides the first information on the molecular mechanism(s) involved in mycolic acid regulation through Ser/Thr protein kinase-dependent phosphorylation of a type II fatty acid synthase enzyme.

“…This newly synthesized TMM would then be targeted for conversion to TDM by antigen-85 followed by closely coupled processing into FM by Msmeg_1529. The recent report of phosphorylation of GroEL1 provides a possible mechanism for activation of its role during biofilm growth (50).…”

Section: Discussionmentioning

confidence: 99%

Enzymatic Hydrolysis of Trehalose Dimycolate Releases Free Mycolic Acids during Mycobacterial Growth in Biofilms

Ojha

Trivelli

Guérardel

et al. 2010

Self Cite

123

137

Mycobacterial species, like other microbes, spontaneously form multicellular drug-tolerant biofilms when grown in vitro in detergent-free liquid media. The structure of Mycobacterium tuberculosis biofilms is formed through genetically programmed pathways and is built upon a large abundance of novel extracellular free mycolic acids (FM), although the mechanism of FM synthesis remained unclear. Here we show that the FM in Mycobacterium smegmatis biofilms is produced through the enzymatic release from constitutively present mycolyl derivatives. One of the precursors for FM is newly synthesized trehalose dimycolate (TDM), which is cleaved by a novel TDM-specific serine esterase, Msmeg_1529. Disruption of Msmeg_1529 leads to undetectable hydrolytic activity, reduced levels of FM in the mutant, and retarded biofilm growth. Furthermore, enzymatic hydrolysis of TDM remains conserved in M. tuberculosis, suggesting the presence of a TDM-specific esterase in this pathogen. Overall, this study provides the first evidence for an enzymatic release of free mycolic acids from cell envelope mycolates during mycobacterial growth.