Non-conventional approaches for peptidoglycan cross-linking inhibition

Bacteria can become resistant against beta-lactam antibiotics (Penicillin) by replacing their 3-4 peptide crosslinks by 3-3 crosslinks in the peptidoglycan layer in their cell wall that gives the bacterium its shape and protects it against mechanical stress. Peptidolglycan consists of a network of sugars strands that are interconnected by peptide crossbridges. Hardly anything was known about the enzymes (LD-transpeptidases) that are responsible for crosslinking of the 3-3 bond.

Aim

The consortium set out to determine why and how bacteria could become resistant by using 3-3 crosslinks.

Research partners

The NAPLCI consortium consisted of scientist from England, The Netherlands, France and Canada.

Results

The bacterium Escherichia coli was made resistant by growing it in the presence of Ampicillin while overproducing one of the LD-TPases. Analysis of the resistant strain revealed that for the activity of the 3-3 crosslinking enzyme, also the transglycosylase activity of PBP1B and the carboxypeptidase activity of PBP5, which shortens the peptide side chain so that it can function as substrate for the LD-TPases. In addition, slow growth was needed. This fits with the growth behavior of pathogenic species that have a very high percentage of 3-3 crosslinks in their peptidoglycan layer. It appeared that the 3 LD-TPases has different activities at different pH and that one of them activated the others. This clearly will be of assistance when the bacteria have to survive in different environments. We showed that the normal function of the LD-TPases could be the repair of holes in the peptidoglycan layer. A series of substrates were chemically synthesized by our colleagues in France and they are presently testing which substrate works best for the various LD-TPases. Finally we developed an assay to measure the activity of LD-Tpases in living cells and also biochemically outside the cell. The former assay can be used to screen antibiotics.