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 generates cell- wall-based natural products known as muropeptides. Certain internalized muropeptides induce antibiotic resistance mechanisms. This process has led to obsolescence of many of the ?-lactam antibiotics against Gram-negative bacteria. These complex events are poorly understood, and are the subject of study in this grant proposal.
Two Specific Aims are proposed. I propose to study the periplasmic complexes involving lytic transglycosylases (LTs), which turn over the cell wall for the purpose of recycling, in Specific Aim 1. We have documented that there are 11 LTs in P. aeruginosa, whose individual reactions on the cell wall are described in this grant application. 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 would appear to be repair of cell wall upon exposure of bacteria to ?-lactam antibiotics. Bulgecin A, a natural product inhibitor of LTs, was shown to potentiate the activity of ?-lactam antibiotics. This takes place as bulgecin A impairs the function of the LTs.
Specific Aim 2 proposes to study bulgecin A and its derivatives by a detailed microbiological and enzymological evaluation of the compound. 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. The needs for novel strategies in treating Gram-negative bacterial infections have never been as accute within the past 50 years. The use of combinations of bulgecin A (or a variant thereof) and ?- lactam antibiotics holds a great promise, which deserves to be investigated.

Public Health Relevance

Bacteria become progressively resistant to all existing antibiotics, rendering them obsolete. Gram-negative bacteria are among the most notorious 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. The needs for novel strategies in treating Gram-negative bacterial infections perhaps have never been as urgent within the past 50 years. The study of these processes is the mission of this grant application. It is our expectation and hope that these studies will pave the way in devising stategies to overcoming these clinical difficulties.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM061629-18
Application #
9308348
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Fabian, Miles
Project Start
2001-04-01
Project End
2019-08-31
Budget Start
2017-09-15
Budget End
2018-08-31
Support Year
18
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Notre Dame
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
824910376
City
Notre Dame
State
IN
Country
United States
Zip Code
46556
Lee, Mijoon; Batuecas, María T; Tomoshige, Shusuke et al. (2018) Exolytic and endolytic turnover of peptidoglycan by lytic transglycosylase Slt of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 115:4393-4398
Tomoshige, Shusuke; Dik, David A; Akabane-Nakata, Masaaki et al. (2018) Total Syntheses of Bulgecins A, B, and C and Their Bactericidal Potentiation of the ?-Lactam Antibiotics. ACS Infect Dis 4:860-867
Byun, Byungjin; Mahasenan, Kiran V; Dik, David A et al. (2018) Mechanism of the Escherichia coli MltE lytic transglycosylase, the cell-wall-penetrating enzyme for Type VI secretion system assembly. Sci Rep 8:4110
Dik, David A; Fisher, Jed F; Mobashery, Shahriar (2018) Cell-Wall Recycling of the Gram-Negative Bacteria and the Nexus to Antibiotic Resistance. Chem Rev 118:5952-5984
Light, Samuel H; Cahoon, Laty A; Mahasenan, Kiran V et al. (2017) Transferase Versus Hydrolase: The Role of Conformational Flexibility in Reaction Specificity. Structure 25:295-304
Lee, Mijoon; Hesek, Dusan; Dik, David A et al. (2017) From Genome to Proteome to Elucidation of Reactions for All Eleven Known Lytic Transglycosylases from Pseudomonas aeruginosa. Angew Chem Int Ed Engl 56:2735-2739
Dik, David A; Marous, Daniel R; Fisher, Jed F et al. (2017) Lytic transglycosylases: concinnity in concision of the bacterial cell wall. Crit Rev Biochem Mol Biol 52:503-542
Acebrón, Iván; Mahasenan, Kiran V; De Benedetti, Stefania et al. (2017) Catalytic Cycle of the N-Acetylglucosaminidase NagZ from Pseudomonas aeruginosa. J Am Chem Soc 139:6795-6798
Lee, Mijoon; Hesek, Dusan; Lastochkin, Elena et al. (2017) Deciphering the Nature of Enzymatic Modifications of Bacterial Cell Walls. Chembiochem 18:1696-1702
Dik, David A; Domínguez-Gil, Teresa; Lee, Mijoon et al. (2017) Muropeptide Binding and the X-ray Structure of the Effector Domain of the Transcriptional Regulator AmpR of Pseudomonas aeruginosa. J Am Chem Soc 139:1448-1451

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