Studies on the molecular basis of penicillin-binding protein-mediated beta-lactam resistance in haemophilus influenza

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The molecular basis of clinical penicillin-binding protein-modulated beta-lactam resistance of the Haemophilus influenzae strain T-1,3 was studied. First, upon investigation of penicillin-binding proteins ( PBPs), H. influenzae strains were tested for whole-cell [3 eS] penicillin binding at either 20, 37 or Examination of the PBP profile from the beta-lactam resistant H. influenzae strain T-1,3 permitted identification of three PBPs (3a, 3b, and 4') with a diminished penicillin-binding activity compared to the isogenic sensitive strain Rd. These three PBPs were distinct from each other because they bound relatively different amounts of radiolabeled penicillin; they bound penicillin at different temperatures; they possess different penicillin saturability; and because only PBP 3a showed an altered electrophoretic mobility when eel ls were grown at 42°C. However, these three PBPs were related to each other because they were the preferred targets for three different beta-lactams in a competitive penicillin-binding assay. To understand how PEP-mediated beta-lactam resistance developed in H. influenzae, the cloning of the altered PBP expression gene f ram strain T-1, 3 was undertaken. Strain T-1, 3 DNA was used to construct a recombinant cosmid gene bank in Escherichia coli. Subcloning of individual fragments derived from one recombinant cosmid (pLB100) indicated that two adjacent fragments of DNA were both capable of transforming the susceptible strain Rd to betalactam resistance and altered PBP expression. Subsequently, by using a 3 -PJ-labeled probe made from the T-1,3 DNA region responsible for the altered PBP expression, larger homologous DNA fragments consisting in the "normal II and "altered" genes from H. inf luenzae Rd and T-1,3, respectively were cloned. Both insertions in pUC-18 vector coded for a 55-kDa product in E. coli minicells. The 55- kDa protein was unable to bind penicillin and was therefore not a PBP. A regulatory model in which the 55-kDa protein interacts with the penicillin-binding activity of PBPs was proposed. Finally, a 5-kb H. influenzae DNA fragment involved in the PEP-mediated beta-lactam resistance of strain T-1,3 was used as a probe for specific detection of H. influenzae strains in clinical specimens.
Bibliography: p. 277-311.
Malouin, F. (1988). Studies on the molecular basis of penicillin-binding protein-mediated beta-lactam resistance in haemophilus influenza (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from doi:10.11575/PRISM/15196