Transpeptidase-Mediated Incorporation of <scp>d</scp>-Amino Acids into Bacterial Peptidoglycan

Lupoli, Tania J.; Tsukamoto, Hirokazu; Doud, Emma H.; Wang, Tsung-Shing Andrew; Walker, Suzanne; Kahne, Daniel

doi:10.1021/ja2040656

Cited by 129 publications

(135 citation statements)

References 48 publications

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“…FDAA probes are thought to indicate regions of active transpeptidase activity by acting as a mimic of D-alanine that reverses a covalent acyl-intermediate formed between PBPs and PG tetrapeptides (33,58). This side reaction of PBP transpeptidases restores PG pentapeptides that contain fluorescent D-Ala at their ends.…”

Section: Resultsmentioning

confidence: 99%

Minimal Peptidoglycan (PG) Turnover in Wild-Type and PG Hydrolase and Cell Division Mutants of Streptococcus pneumoniae D39 Growing Planktonically and in Host-Relevant Biofilms

et al. 2015

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We determined whether there is turnover of the peptidoglycan (PG) cell wall of the ovococcus bacterial pathogen Streptococcus pneumoniae (pneumococcus). Pulse-chase experiments on serotype 2 strain D39 radiolabeled with N-acetylglucosamine revealed little turnover and release of PG breakdown products during growth compared to published reports of PG turnover in Bacillus subtilis. PG dynamics were visualized directly by long-pulse-chase-new-labeling experiments using two colors of fluorescent D-amino acid (FDAA) probes to microscopically detect regions of new PG synthesis. Consistent with minimal PG turnover, hemispherical regions of stable "old" PG persisted in D39 and TIGR4 (serotype 4) cells grown in rich brain heart infusion broth, in D39 cells grown in chemically defined medium containing glucose or galactose as the carbon source, and in D39 cells grown as biofilms on a layer of fixed human epithelial cells. In contrast, B. subtilis exhibited rapid sidewall PG turnover in similar FDAA-labeling experiments. High-performance liquid chromatography (HPLC) analysis of biochemically released peptides from S. pneumoniae PG validated that FDAAs incorporated at low levels into pentamer PG peptides and did not change the overall composition of PG peptides. PG dynamics were also visualized in mutants lacking PG hydrolases that mediate PG remodeling, cell separation, or autolysis and in cells lacking the MapZ and DivIVA division regulators. In all cases, hemispheres of stable old PG were maintained. In PG hydrolase mutants exhibiting aberrant division plane placement, FDAA labeling revealed patches of inert PG at turns and bulge points. We conclude that growing S. pneumoniae cells exhibit minimal PG turnover compared to the PG turnover in rod-shaped cells. IMPORTANCEPG cell walls are unique to eubacteria, and many bacterial species turn over and recycle their PG during growth, stress, colonization, and virulence. Consequently, PG breakdown products serve as signals for bacteria to induce antibiotic resistance and as activators of innate immune responses. S. pneumoniae is a commensal bacterium that colonizes the human nasopharynx and opportunistically causes serious respiratory and invasive diseases. The results presented here demonstrate a distinct demarcation between regions of old PG and regions of new PG synthesis and minimal turnover of PG in S. pneumoniae cells growing in culture or in host-relevant biofilms. These findings suggest that S. pneumoniae minimizes the release of PG breakdown products by turnover, which may contribute to evasion of the innate immune system. P eptidoglycan (PG) biosynthesis and placement are dynamic processes that determine the shapes, sizes, chaining, and resistance to turgor of bacterial cells (1-6). In Gram-positive bacteria, PG also serves as the scaffolding for covalent attachment of surface wall teichoic acid, capsule, and sortase-attached proteins (7-9). The seminal work of Park and Uehara demonstrated that PG is rapidly turned over and the breakdown components recycled in...

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Section: Resultsmentioning

confidence: 99%

Minimal Peptidoglycan (PG) Turnover in Wild-Type and PG Hydrolase and Cell Division Mutants of Streptococcus pneumoniae D39 Growing Planktonically and in Host-Relevant Biofilms

et al. 2015

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“…Such competition cannot occur in the peptidoglycan, as the site of noncanonical Damino acid incorporation into the peptidoglycan peptide side chain excludes molecules with an L center (11). Rather, the specific rescue of biofilm formation by L-enantiomers suggests that Damino acids inhibit biofilm formation by interfering with protein synthesis.…”

Section: Methodsmentioning

confidence: 99%

D-Amino Acids Indirectly Inhibit Biofilm Formation in Bacillus subtilis by Interfering with Protein Synthesis

Leiman¹,

May²,

Lebar³

et al. 2013

Journal of Bacteriology

189

139

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The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of D-leucine, D-methionine, D-tryptophan, and D-tyrosine and was reported to inhibit biofilm formation via the incorporation of these D-amino acids into the cell wall. Here, we show that L-amino acids were able to specifically reverse the inhibitory effects of their cognate D-amino acids. We also show that D-amino acids inhibited growth and the expression of biofilm matrix genes at concentrations that inhibit biofilm formation. Finally, we report that the strain routinely used to study biofilm formation has a mutation in the gene (dtd) encoding D-tyrosyl-tRNA deacylase, an enzyme that prevents the misincorporation of D-amino acids into protein in B. subtilis. When we repaired the dtd gene, B. subtilis became resistant to the biofilm-inhibitory effects of D-amino acids without losing the ability to incorporate at least one noncanonical D-amino acid, D-tryptophan, into the peptidoglycan peptide side chain. We conclude that the susceptibility of B. subtilis to the biofilm-inhibitory effects of D-amino acids is largely, if not entirely, due to their toxic effects on protein synthesis.

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“…The same conclusion applies to HMM PBPs, which, as solubilized forms in vitro, also do not in general turn over small peptidoglycan-mimetic substrates at rates sufficient to support bacterial growth; 34,35,66 larger model substrates may be needed. 67,68 There is some evidence of alternative conformations of particular HMM PBPs from crystal structures 69−71 and solution studies. 72−74 It is thus clear that an understanding of the molecular details of catalysis in vivo by the most important of these enzymes has not yet been achieved.…”

Section: ■ Conclusionmentioning

confidence: 99%

Substrate Specificity of Low-Molecular Mass Bacterial dd-Peptidases

et al. 2011

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The bacterial DD-peptidases or penicillin-binding proteins (PBPs) catalyze the formation and regulation of cross-links in peptidoglycan biosynthesis. They are classified into two groups, the high-molecular mass (HMM) and lowmolecular mass (LMM) enzymes. The latter group, which is subdivided into classes A−C (LMMA, -B, and -C, respectively), is believed to catalyze DD-carboxypeptidase and endopeptidase reactions in vivo. To date, the specificity of their reactions with particular elements of peptidoglycan structure has not, in general, been defined. This paper describes the steady-state kinetics of hydrolysis of a series of specific peptidoglycan-mimetic peptides, representing various elements of stem peptide structure, catalyzed by a range of LMM PBPs (the LMMA enzymes, Escherichia coli PBP5, Neisseria gonorrhoeae PBP4, and Streptococcus pneumoniae PBP3, and the LMMC enzymes, the Actinomadura R39 DD-peptidase, Bacillus subtilis PBP4a, and N. gonorrhoeae PBP3). The R39 enzyme (LMMC), like the previously studied Streptomyces R61 DD-peptidase (LMMB), specifically and rapidly hydrolyzes stem peptide fragments with a free N-terminus. In accord with this result, the crystal structures of the R61 and R39 enzymes display a binding site specific to the stem peptide N-terminus. These are water-soluble enzymes, however, with no known specific function in vivo. On the other hand, soluble versions of the remaining enzymes of those noted above, all of which are likely to be membrane-bound and/or associated in vivo and have been assigned particular roles in cell wall biosynthesis and maintenance, show little or no specificity for peptides containing elements of peptidoglycan structure. Peptidoglycan-mimetic boronate transition-state analogues do inhibit these enzymes but display notable specificity only for the LMMC enzymes, where, unlike peptide substrates, they may be able to effectively induce a specific active site structure. The manner in which LMMA (and HMM) DD-peptidases achieve substrate specificity, both in vitro and in vivo, remains unknown. T he bacterial DD-peptidases catalyze the final steps of peptidoglycan (cell wall) biosynthesis 1,2 and are efficiently inhibited by β-lactam antibiotics.3 Resistance to these antibiotics continues to emerge, particularly from evolution of new β-lactamases, enzymes that catalyze hydrolytic destruction of β-lactams, but also by the evolution and dispersal of β-lactam-resistant DD-peptidases. and Neisseria gonorrhoeae. 10Many attempts have been made to develop new β-lactams effective against mutant 11,12 but, to date, these have not been completely successful. The ideal of a broadspectrum β-lactam not susceptible to loss of effectiveness through DD-peptidase mutation has been difficult to achieve. To a considerable extent, this is likely due to the largely trial and error approach to new β-lactams. The alternative approach of targeting the reaction center and the common and essential structural features of the DD-peptidase active site has not been pursued as vigorously. C...

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Transpeptidase-Mediated Incorporation of d-Amino Acids into Bacterial Peptidoglycan

Cited by 129 publications

References 48 publications

Minimal Peptidoglycan (PG) Turnover in Wild-Type and PG Hydrolase and Cell Division Mutants of Streptococcus pneumoniae D39 Growing Planktonically and in Host-Relevant Biofilms

Minimal Peptidoglycan (PG) Turnover in Wild-Type and PG Hydrolase and Cell Division Mutants of Streptococcus pneumoniae D39 Growing Planktonically and in Host-Relevant Biofilms

D-Amino Acids Indirectly Inhibit Biofilm Formation in Bacillus subtilis by Interfering with Protein Synthesis

Substrate Specificity of Low-Molecular Mass Bacterial dd-Peptidases

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