Outsmarting Beta-lactam resistance in Meningococci and Pneumococci – ORBiMP

Improving beta-lactams efficiency will address the escalating resistance problem of major bacterial pathogens expressing Penicillin-Binding Proteins (PBPs) with low affinity for the antibiotics. These variant PBPs are fully active in incorporating the peptidoglycan precursor in the cell wall despite the fact that beta-lactams are molecular mimics of the precursor. ORBiMP aims at solving this paradox. After 40 years of efforts, this is finally feasible owing to our recent ability to synthesize peptidoglycan in vitro using recombinant membrane enzymes and various forms of the precursor. We will compare the activities and structures of PBPs from resistant and susceptible strains from the Gram-positive pneumococcus and Gram-negative meningococcus, with various precursors, to reveal molecular features essential for functionality. These insights will guide the design of improved beta-lactams aimed at resistant pathogens.

More specifically, we will address the following questions using these approaches:

The growth of beta-lactam resistant bacteria is little affected suggesting that the altered PBPs can function appropriately. Is this truly the case?
We will compare the activities of PBPs from susceptible and beta-lactam resistant strains from N. meningitidis and S. pneumoniae.

Changes in peptidoglycan composition and cross-linking suggest that some compensatory mechanisms may operate. What are they?
We will examine if trimming of the peptides in N. meningitidis can be the result of a carboxypeptidases side activity of PBP2, and if it is decreased in “low affinity” variants from resistant strains.
We will compare the use of “branched” and linear peptide-containing precursors by PBPs from susceptible and resistant strains of S. pneumoniae.

Altered PBPs are better at catalysing transpeptidation than at reacting with drugs. What are the features of the substrate that contribute to maintaining the activity?
We will compare the use of variants of the precursor lipid II (“branched” and linear, and others) with PBPs from susceptible and resistant strains. We will measure if elements of the peptidoglycan modulate the reactivity of PBPs towards the drugs.

Is any defined organization imparted on the peptidoglycan by the PBPs alone?
We will attempt to observe peptidoglycan assembled in vitro using atomic force microscopy. We will compare the peptidoglycans produced by PBPs from the Gram-negative N. meningitidis and the Gram-positive S. pneumoniae, and from susceptible and resistant strains.