Cell-wall recycling is a fundamental process in bacteria, whereby it allows for remodeling of the cell wall in the course of the normal growth and in response to antibiotics that inflict damage to the cell wall for their mechanisms 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-wall recycling, which leads to the formation of cell-wall-based natural products known as muropeptides. Certain muropeptides 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 antibiotics against Gram-negative bacteria. My lab has studied this system for the past several years and what I disclose in this MIRA application is the path that the lab will chart in the immediate future. I propose to study the periplasmic complexes involving lytic transglycosylases (LTs), which turn over the cell wall for the purpose of recycling 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 are proposed to be involved in complexes with other proteins within the periplasm, whose identities are not known and represent a major gap in our knowledge of cell-wall processes. Whereas all the functions of LTs are not understood, one is repair of cell wall upon exposure of bacteria to ?-lactam antibiotics. Muropeptides are the degradation products of cell-wall recycling, which are internalized to the cytoplasm for this purpose. As an offshoot of the recycling events, certain muropetides activate the AmpR transcriptional regulator in expression of the AmpC ?-lactamase, the resistance determinant for ?-lactam antibiotics. We will study the interactions of the key mutropeptides with the AmpR protein in elucidating the system. Furthermore, four additional cytoplasmic enzymes that have been identified in P. aeruginosa for the key events of the muropeptide recycling will be investigated for their chemical reactions, the details of the catalytic cycles and for their structures. I anticipate that the successful completion of this proposed science will not only lead to the elucidation of these important events regulating the cell wall, but also will identify opportunities for their interruption as a means to circumventing the elaborate mechanism of ?-lactam resistance that Gram-negative bacteria have evolved. These complex events are poorly understood, and they will be studied in detail in my lab in the course of the proposed reseach.
Bacteria become progressively resistant to all existing antibiotics, rendering them obsolete. Gram-negative bacteria are among the most nefarious human pathogens that have evolved a host of resistance mechanisms. These are among the most difficult pathogens to treat clinically. These bacteria have evolved an inducible mechanism for resistance to ?-lactam antibiotics (such as penicillins, cephalosporins, carbapenems, etc.) as an offshoot of the physiological processes of cell-wall recycling. The mechanism is complex and the outcome of resistance to a broad range of antibiotics is disconcerting. Furthermore, cell-wall recycling, the subject of study in the proposed reaserch, is a fundamental biochemical process in bacteria, itself a target for antibiotics. The study of these processes is the mission of this grant application. The needs for novel strategies in treating Gram-negative bacterial infections perhaps have never been as urgent within the past 50 years. It is our expectation that these studies will pave the way in elucidating a very important bacterial system and in devising stategies to overcoming these clinical difficulties.
