Serine/Threonine Protein Phosphatase-Mediated Control of the Peptidoglycan Cross-Linking l , d - Transpeptidase Pathway in Enterococcus faecium

Abstract

The last step of peptidoglycan polymerization involves two families of unrelated transpeptidases that are the essential targets of β-lactam antibiotics. d,d-transpeptidases of the penicillin-binding protein (PBP) family are active-site serine enzymes that use pentapeptide precursors and are the main or exclusive cross-linking enzymes in nearly all bacteria. However, peptidoglycan cross-linking is performed mainly by active-site cysteine l,d-transpeptidases that use tetrapeptides in Mycobacterium tuberculosis, Clostridium difficile, and β-lactam-resistant mutants of Enterococcus faecium. We have investigated reprogramming of the E. faecium peptidoglycan assembly pathway by a switch from pentapeptide to tetrapeptide precursors and bypass of PBPs by l,d-transpeptidase Ldtfm. Mutational alterations of two signal transduction systems were necessary and sufficient for activation of the l,d-transpeptidation pathway, which is essentially cryptic in wild-type strains. The first one is a classical two-component regulatory system, DdcRS, that controls the activity of Ldtfm at the substrate level. As previously described, loss of DdcS phosphatase activity leads to production of the d,d-carboxypeptidase DdcY and conversion of the pentapeptide into the tetrapeptide substrate of Ldtfm. Here we show that full bypass of PBPs by Ldtfm also requires increased Ser/Thr protein phosphorylation resulting from impaired activity of phosphoprotein phosphatase StpA. This enzyme negatively controlled the level of protein phosphorylation both by direct dephosphorylation of target proteins and by dephosphorylation of its cognate kinase Stk. In combination with production of DdcY, increased protein phosphorylation by this eukaryotic-enzyme-like Ser/Thr protein kinase was sufficient for activation of the l,d-transpeptidation pathway in the absence of mutational alteration of peptidoglycan synthesis enzymes.

Mutation Landscape of Acquired Cross-Resistance to Glycopeptide and β-Lactam Antibiotics in Enterococcus faecium

“…Recombinant StpA containing a C-terminal 6-histidine tag was produced in Escherichia coli BL21(DE3) and purified by nickel affinity and size exclusion chromatography methods, as previously described (11). Hydrolysis of para-nitrophenyl-phosphate (Sigma) by StpA was determined at 37°C in 50 mM Tris-HCl (pH 8.0).…”

“…A second mutation was detected in the protein phosphatase gene stpA of mutant M1 located upstream from the Ser/Thr protein kinase gene stk (11). The stpA mutation of M1 impairs the phosphatase activity of StpA, leading to hyperphosphorylation of Stk and several uniden- tified proteins (11). Genetic analyses showed that impaired phosphatase activities of DdcS and StpA are both required and sufficient for high-level ampicillin resistance (11 (12).…”

“…1). A second mutation was detected in the protein phosphatase gene stpA of mutant M1 located upstream from the Ser/Thr protein kinase gene stk (11). The stpA mutation of M1 impairs the phosphatase activity of StpA, leading to hyperphosphorylation of Stk and several uniden- tified proteins (11).…”

“…The stpA mutation of M1 impairs the phosphatase activity of StpA, leading to hyperphosphorylation of Stk and several uniden- tified proteins (11). Genetic analyses showed that impaired phosphatase activities of DdcS and StpA are both required and sufficient for high-level ampicillin resistance (11 (12). Four additional selection steps on increasing concentrations of vancomycin or teicoplanin were required to obtain mutant M9 (12), which was coresistant to ampicillin and glycopeptides (MICs of all three drugs, Ͼ1,000) (Fig.…”

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“…Mutation in the PBP gene, however, translates into a slightly different but functional protein which prevents the binding of β-lactam antibiotics such as penicillin [18][19][20]. Examples of microbes harboring this ability are drug-resistant strains of Clostridium difficile, Enterococcus faecium and Streptococcus pneumoniae [21][22]. In addition, microbial resistance towards ciprofloxacin, which interferes with cellular division through interaction between the DNA gyrase and topoisomerase, is also categorized under this mechanism [23][24].…”