A healthy cell wall is indispensable for survival of bacteria. The building blocks of the bacterial peptidoglycan, the major constituent of cell wall, are assembled within the cytoplasm, then they are transported to the surface of the cytoplasmic membrane. The final steps of cell wall assembly take place on the cytoplasmic membrane and they involve the interplay among the activities of a set of enzymes referred to as penicillin-binding proteins (PBPs) and lytic transglcosylases. PBPs catalyze the final steps of cell wall maturation. The roles of lytic transglycosylases are less well understood. They are involved in recycling of cell wall, as well as in restructuring of the cell wall for incorporation of macromolecular systems such as various secretion systems, uptake systems and pilus that requires localized degradation/alteration of the cell wall. Investigations of the activities of lytic transglycosylases are disclosed in this grant application.
Three Specific Aims are offered.
Specific Aim 1 is to clone the genes, to express and to purify the proteins and to perform studies on the reactions of the six Escherichia coli lytic transglycosylases. Synthetic substrates that closely mimic the polymeric substrates for these enzymes are to be developed for detailed study of the microscopic steps of the reactions of these enzymes.
Specific Aim 2 describes the detailed studies of inhibition of lytic transglycosylases. These studies will prepare molecules as both mechanistic probes and as potential antibiotics.
Specific Aim 3 will deal with a key step in cell wall maturation catalyzed by Peptidoglycan Undecaprenyl Releasing Enzyme. It is expected that the outcome of these studies will shed definitive light on the biochemical mechanisms of lytic transglycosylases and pave the way for discoveries of novel inhibitors as potential future antibiotics.

Public Health Relevance

Bacteria require a healthy cell wall for survival. Processes that lead to the formation of cell wall are complex and are targets for antibiotics. This grant application proposes to investigate cell wall and its formation and degradation. The knowledge from the proposed study will provide greater understanding of how bacterial cell wall exists and functions and how potential antibiotic molecules can be generated that target the cell wall.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061629-13
Application #
8240113
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Fabian, Miles
Project Start
2000-07-01
Project End
2013-08-31
Budget Start
2012-04-01
Budget End
2013-08-31
Support Year
13
Fiscal Year
2012
Total Cost
$301,381
Indirect Cost
$100,460
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
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

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