Gram-negative bacteria, members of Enterobacteriaceae and Pseudomonas aeruginosa, which are among the most serious human pathogens, have evolved an inducible resistance mechanism against �-lactam antibiotics. It has been shown that mutational inactivation of penicillin-binding protein 4 (PBP4) leads to high-level expression of a class C �-lactamase, an enzyme of resistance to �-lactam antibiotics. Mutational alteration of PBPs in Gram-negative bacteria in manifestation of resistance to �-lactam antibiotics is not common. We investigated the reactions of PBP4 of P. aeruginosa in processing of the bacterial cell wall in shedding light on its physiological role. We also investigated the consequence of the alteration of cell wall when P. aeruginosa was grown in the presence of a high inducer of �-lactamase production. The dacB gene was cloned from the strain P. aeruginosa PA01 and the PBP4 protein was purified to homogeneity. The function of PBP4 was assessed by the use of two types of substrates�bacterial peptidoglycan and synthetic samples as surrogates of cell-wall peptidoglycan. PBP4 turns over bacterial peptidoglycan and turnover products were characterized by LC/MS. Moreover its biochemical functions were further characterized by use of synthetic samples with more defined structures, which contain pentapeptide (as substrate of DD-carboxypeptidase) and crosslinked heptapeptide (as substrate of endopeptidase). The results show PBP4 of P. aeruginosa is a bifunctional DD-carboxypeptidase/endopeptidase. Peptidoglycan analysis of sacculus grown in the presence of a high inducer (which results in the inhibition of PBP4) shows the increase of muropeptides containing pentapeptide and 4,3-crosslinked peptide-substrates of PBP4. Together with biochemical analysis with sacculus and synthetic samples, this suggests that the accumulation of PBP4 substrates or the imbalance between its substrates and products could be the trigger for b-lactamase induction in P. aeruginosa. Detailed biochemical analysis of PBP4 and chemical syntheses of synthetic substrates will be discussed.

Reactions of penicillin-binding protein 4 of <i>Pseudomonas aeruginosa</i> and its implication to �-lactam antibiotics resistance

248th National Meeting (2014)

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Reactions of penicillin-binding protein 4 of Pseudomonas aeruginosa and its implication to �-lactam antibiotics resistance