Many members of Enterobacteriaceae and Pseudomonas aeruginosa (subject of this grant application) have the ability to sense damage inflicted to their cell wall by ?-lactam antibiotics. A primary mechanism for this sensing involves the events of cell-wall recycling, and results in the induction of resistance mechanisms. These events have led to the obsolescence of many of the ?-lactam antibiotics against these Gram-negative bacteria. These multiple complex steps are poorly elucidated, and are the subject of this grant proposal.
Three Specific Aims are proposed.
In Specific Aim 1 I propose to elucidate the reactions of all lytic transglycosylases, enzymes that initiate the cell-wall recycling events. This undertaking utilizes methodologies developed in my lab for highly sensitive high-resolution identification of reaction products of these enzymes by proteomics approaches. Furthermore, a link between the function of penicillin-binding protein 4 (PBP4) of Pseudomonas aeruginosa and the sensing of the presence of the ?-lactam antibiotic has been made. I disclose my views on how this process could take place and I propose the means to the identification of the signal molecule, which triggers the antibiotic resistance processes.
Specific Aim 2 proposes to study and identify a key enzyme in the resistance induction process, the AmpD protease. Three homologous enzymes have been annotated in P. aeruginosa for this activity. We have outlined studies that will delineate which of the three, or a subset thereof, are bona fide enzymes involved in cell-wall recycling and antibiotic resistance induction.
Specific Aim 3 will delineate the transcriptional events that lead to ?-lactam antibiotic resistance in P. aeruginosa. This includes identification of the key transcriptional activator molecule and the kinetics of the processes. I anticipate that the successful completion of this proposed science will not only lead to the elucidation of these events, but also will identify opportunities for their interruption as means to circumvent the elaborate mechanisms of resistance.

Public Health Relevance

Bacteria become progressively resistant to all existing antibiotics, rendering therapeutic options in treatment of infections rather limited. There exit organisms that are extremely difficult to treat with the currently available antibiotics, which mad investigations of the mechanism of antibiotic resistance important in understanding how to redress the clinical problem. A group of Gram-negative bacterial pathogens has devised 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 study of these processes is the mission of this grant application.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM061629-14
Application #
8575183
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Fabian, Miles
Project Start
2000-07-01
Project End
2017-06-30
Budget Start
2013-09-01
Budget End
2014-06-30
Support Year
14
Fiscal Year
2013
Total Cost
$315,400
Indirect Cost
$107,900
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; Dhar, Supurna; De Benedetti, Stefania et al. (2016) Muropeptides in Pseudomonas aeruginosa and their Role as Elicitors of β-Lactam-Antibiotic Resistance. Angew Chem Int Ed Engl 55:6882-6
Sandalova, Tatyana; Lee, Mijoon; Henriques-Normark, Birgitta et al. (2016) The crystal structure of the major pneumococcal autolysin LytA in complex with a large peptidoglycan fragment reveals the pivotal role of glycans for lytic activity. Mol Microbiol 101:954-67
Lenz, Jonathan D; Stohl, Elizabeth A; Robertson, Rosanna M et al. (2016) Amidase Activity of AmiC Controls Cell Separation and Stem Peptide Release and Is Enhanced by NlpD in Neisseria gonorrhoeae. J Biol Chem 291:10916-33
Lee, Mijoon; Hesek, Dusan; Blázquez, Blas et al. (2015) Catalytic spectrum of the penicillin-binding protein 4 of Pseudomonas aeruginosa, a nexus for the induction of β-lactam antibiotic resistance. J Am Chem Soc 137:190-200
Rico-Lastres, Palma; Díez-Martínez, Roberto; Iglesias-Bexiga, Manuel et al. (2015) Substrate recognition and catalysis by LytB, a pneumococcal peptidoglycan hydrolase involved in virulence. Sci Rep 5:16198
Fisher, Jed F; Mobashery, Shahriar (2014) The sentinel role of peptidoglycan recycling in the β-lactam resistance of the Gram-negative Enterobacteriaceae and Pseudomonas aeruginosa. Bioorg Chem 56:41-8
Artola-Recolons, Cecilia; Lee, Mijoon; Bernardo-García, Noelia et al. (2014) Structure and cell wall cleavage by modular lytic transglycosylase MltC of Escherichia coli. ACS Chem Biol 9:2058-66
Martínez-Caballero, Siseth; Lee, Mijoon; Artola-Recolons, Cecilia et al. (2013) Reaction products and the X-ray structure of AmpDh2, a virulence determinant of Pseudomonas aeruginosa. J Am Chem Soc 135:10318-21
Zhang, Weilie; Lee, Mijoon; Hesek, Dusan et al. (2013) Reactions of the three AmpD enzymes of Pseudomonas aeruginosa. J Am Chem Soc 135:4950-3
Johnson, Jarrod W; Fisher, Jed F; Mobashery, Shahriar (2013) Bacterial cell-wall recycling. Ann N Y Acad Sci 1277:54-75

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