Cell-Wall Recycling and Nexus to Antibiotic Resistance

Project Summary/AbstractCell-wall recycling is a fundamental process in bacteria, whereby it allows for remodeling of the cell wall in thecourse of the normal growth and in response to antibiotics that inflict damage to the cell wall for theirmechanisms of action. Enterobacteriaceae and Pseudomonas aeruginosa (subject of this grant application)sense damage inflicted to their cell wall by ?-lactam antibiotics. The sensing event is linked to cell-wallrecycling, which leads to the formation of cell-wall-based natural products known as muropeptides. Certainmuropeptides are internalized to the cytoplasm, where they induce the bacterial response to the antibiotic(antibiotic-resistance mechanisms). This process has led to obsolescence of many of the ?-lactam antibioticsagainst Gram-negative bacteria. My lab has studied this system for the past several years and what I disclosein this MIRA application is the path that the lab will chart in the immediate future. I propose to study theperiplasmic complexes involving lytic transglycosylases (LTs), which turn over the cell wall for the purpose ofrecycling or in response to damage by antibiotics. My lab has documented that there are 11 known LTs in P.aeruginosa, whose individual reactions with the cell wall have been described by us. These enzymes areproposed to be involved in complexes with other proteins within the periplasm, whose identities are not knownand represent a major gap in our knowledge of cell-wall processes. Whereas all the functions of LTs are notunderstood, one is repair of cell wall upon exposure of bacteria to ?-lactam antibiotics. Muropeptides are thedegradation products of cell-wall recycling, which are internalized to the cytoplasm for this purpose. As anoffshoot of the recycling events, certain muropetides activate the AmpR transcriptional regulator in expressionof the AmpC ?-lactamase, the resistance determinant for ?-lactam antibiotics. We will study the interactions ofthe key mutropeptides with the AmpR protein in elucidating the system. Furthermore, four additionalcytoplasmic enzymes that have been identified in P. aeruginosa for the key events of the muropeptiderecycling will be investigated for their chemical reactions, the details of the catalytic cycles and for theirstructures. I anticipate that the successful completion of this proposed science will not only lead to theelucidation of these important events regulating the cell wall, but also will identify opportunities for theirinterruption as a means to circumventing the elaborate mechanism of ?-lactam resistance that Gram-negativebacteria have evolved. These complex events are poorly understood, and they will be studied in detail in mylab in the course of the proposed reseach.