Dataset Information

Characterization of the bifunctional glycosyltransferase/acyltransferase penicillin-binding protein 4 of Listeria monocytogenes.

ABSTRACT: Multimodular penicillin-binding proteins (PBPs) are essential enzymes responsible for bacterial cell wall peptidoglycan (PG) assembly. Their glycosyltransferase activity catalyzes glycan chain elongation from lipid II substrate (undecaprenyl-pyrophosphoryl-N-acetylglucosamine-N-acetylmuramic acid-pentapeptide), and their transpeptidase activity catalyzes cross-linking between peptides carried by two adjacent glycan chains. Listeria monocytogenes is a food-borne pathogen which exerts its virulence through secreted and cell wall PG-associated virulence factors. This bacterium has five PBPs, including two bifunctional glycosyltransferase/transpeptidase class A PBPs, namely, PBP1 and PBP4. We have expressed and purified the latter and have shown that it binds penicillin and catalyzes in vitro glycan chain polymerization with an efficiency of 1,400 M(-1) s(-1) from Escherichia coli lipid II substrate. PBP4 also catalyzes the aminolysis (d-Ala as acceptor) and hydrolysis of the thiolester donor substrate benzoyl-Gly-thioglycolate, indicating that PBP4 possesses both transpeptidase and carboxypeptidase activities. Disruption of the gene lmo2229 encoding PBP4 in L. monocytogenes EGD did not have any significant effect on growth rate, peptidoglycan composition, cell morphology, or sensitivity to beta-lactam antibiotics but did increase the resistance of the mutant to moenomycin.

SUBMITTER: Zawadzka-Skomial J

PROVIDER: S-EPMC1426562 | biostudies-literature | 2006 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Characterization of the bifunctional glycosyltransferase/acyltransferase penicillin-binding protein 4 of Listeria monocytogenes.

Zawadzka-Skomial Joanna J Markiewicz Zdzislaw Z Nguyen-Distèche Martine M Devreese Bart B Frère Jean-Marie JM Terrak Mohammed M

Journal of bacteriology 20060301 5

Multimodular penicillin-binding proteins (PBPs) are essential enzymes responsible for bacterial cell wall peptidoglycan (PG) assembly. Their glycosyltransferase activity catalyzes glycan chain elongation from lipid II substrate (undecaprenyl-pyrophosphoryl-N-acetylglucosamine-N-acetylmuramic acid-pentapeptide), and their transpeptidase activity catalyzes cross-linking between peptides carried by two adjacent glycan chains. Listeria monocytogenes is a food-borne pathogen which exerts its virulenc ...[more]

PMID: 16484198

Similar Datasets

Project description:BACKGROUND: Bacterial penicillin-binding proteins (PBPs) can be visualized by their ability to bind radiolabeled or fluorescent ?-lactam derivatives both whole cells and membrane/cell enriched fractions. Analysis of the Listeria monocytogenes genome sequence predicted ten genes coding for putative PBPs, but not all of their products have been detected in studies using radiolabeled antibiotics, thus hindering their characterization. Here we report the positive identification of the full set of L. monocytogenes PBPs and the characteristics of the hitherto undescribed PBPD2 (Lmo2812). RESULTS: Eight L. monocytogenes PBPs were identified by the binding of fluorescent ?-lactam antibiotic derivatives Boc-FL, Boc-650 and Amp-Alexa430 to proteins in whole cells or membrane/cell wall extracts. The gene encoding a ninth PBP (Lmo2812) was cloned and expressed in Escherichia coli as a His-tagged protein. The affinity purified recombinant protein had DD-carboxypeptidase activity and preferentially degraded low-molecular-weight substrates. L. monocytogenes mutants lacking the functional Lmo2812 enzyme were constructed and, compared to the wild-type, the cells were longer and slightly curved with bent ends.Protein Lmo1855, previously designated PBPD3, did not bind any of the antibiotic derivatives tested, similarly to the homologous enterococcal protein VanY. CONCLUSIONS: Nine out of the ten putative L. monocytogenes PBP genes were shown to encode proteins that bind derivatives of ?-lactam antibiotics, thus enabling their positive identification. PBPD2 (Lmo2812) was not visualized in whole cell extracts, most probably due to its low abundance, but it was shown to bind Boc-FL after recombinant overexpression and purification. Mutants lacking Lmo2812 and another low molecular mass (LMM) PBP, PBP5 (PBPD1)--both with DD-carboxypeptidase activity--displayed only slight morphological alterations, demonstrating that they are dispensable for cell survival and probably participate in the latter stages of peptidoglycan synthesis. Since Lmo2812 preferentially degrades low-molecular- mass substrates, this may indicate a role in cell wall turnover.

Project description:BackgroundListeria monocytogenes is a common cause of bacterial meningitis. We developed an animal model of listerial meningitis.MethodsIn survival studies, C57BL/6 mice received intracisternal injections with different L. monocytogenes sequence type 1 (ST1) colony forming units per milliliter (CFU; n = 48, 105, 106, 107, 108, and 109 CFU/ml). Second, mice were inoculated with 108 CFU/ml ST1 and sacrificed at 6 h and 24 h (n = 12/group). Outcome parameters were clinical score, CFUs, cyto- and chemokine levels, and brain histopathology. Third, 84 mice were inoculated (109 CFU/ml ST1) to determine optimal antibiotic treatment with different doses of amoxicillin and gentamicin. Fourth, mice were inoculated with 109 CFU/ml ST1, treated with amoxicillin, and sacrificed at 16 h and 24 h (n = 12/group) for outcome assessment. Finally, time point experiments were repeated with ST6 (n = 24/group).ResultsMedian survival time for inoculation with 108 and 109 CFU/ml ST1 was 46 h and 40 h; lower doses of bacteria led to minimal clinical signs of disease. Brain levels of IL-6, IL-17A, and IFN-γ were elevated at 24 h, and IL-1β, IL-6, IL-10, IFN-γ, and TNF-α were elevated in blood at 6 h and 24 h. Histopathology showed increased meningeal infiltration, vascular inflammation of meningeal vessels, hemorrhages, and ventriculitis. In the treatment model, brain levels of IL-6 and IL-17A and blood levels of IL-6 and IFN-γ were elevated. Compared to ST6, infection with ST1 led initially to higher levels of IL-1β and TNF-α in blood and more profound neuropathological damage. At 16 h post inoculation, IL-1β, IL-10, and TNF-α in blood and IL-6, IL17A, TNF-α, and IFN-γ levels in brain were higher in ST1 compared to ST6 without differences in CFUs between STs. At 24 h, neuropathology score was higher in ST1 compared to ST6 (p = 0.002) infected mice.ConclusionsWe developed and validated a murine model of listerial meningitis. ST1-infected mice had a more severe inflammatory response and brain damage as compared to ST6-infected mice.

Dataset Information

Characterization of the bifunctional glycosyltransferase/acyltransferase penicillin-binding protein 4 of Listeria monocytogenes.

Publications

Characterization of the bifunctional glycosyltransferase/acyltransferase penicillin-binding protein 4 of Listeria monocytogenes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets