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.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM061629-15
Application #
8725674
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Fabian, Miles
Project Start
Project End
Budget Start
Budget End
Support Year
15
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Notre Dame
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Notre Dame
State
IN
Country
United States
Zip Code
46556
Fisher, Jed F; Mobashery, Shahriar (2014) The sentinel role of peptidoglycan recycling in the ?-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
Johnson, Jarrod W; Fisher, Jed F; Mobashery, Shahriar (2013) Bacterial cell-wall recycling. Ann N Y Acad Sci 1277:54-75
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
Lee, Mijoon; Hesek, Dusan; Llarrull, Leticia I et al. (2013) Reactions of all Escherichia coli lytic transglycosylases with bacterial cell wall. J Am Chem Soc 135:3311-4
Lee, Mijoon; Artola-Recolons, Cecilia; Carrasco-Lopez, Cesar et al. (2013) Cell-wall remodeling by the zinc-protease AmpDh3 from Pseudomonas aeruginosa. J Am Chem Soc 135:12604-7
Martinez-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
Boudreau, Marc A; Fisher, Jed F; Mobashery, Shahriar (2012) Messenger functions of the bacterial cell wall-derived muropeptides. Biochemistry 51:2974-90
Artola-Recolons, Cecilia; Carrasco-Lopez, Cesar; Llarrull, Leticia I et al. (2011) High-resolution crystal structure of MltE, an outer membrane-anchored endolytic peptidoglycan lytic transglycosylase from Escherichia coli. Biochemistry 50:2384-6
Maestro, Beatriz; Novakova, Linda; Hesek, Dusan et al. (2011) Recognition of peptidoglycan and ýý-lactam antibiotics by the extracellular domain of the Ser/Thr protein kinase StkP from Streptococcus pneumoniae. FEBS Lett 585:357-63

Showing the most recent 10 out of 28 publications