Grant: Discovery Grants Program - Individual

This proposal concerns the continuation of my research program that investigates the structure and function relationship of carbohydrate-active enzymes. Over the past thirty plus years, my lab has developed expertise in the study of enzymes involved in the metabolism of the bacterial cell wall heteropolymer peptidoglycan (PG) and the biodegradation of the plant cell wall polymers cellulose and xylan. The research program builds on our findings to date on the specificity, structure and function relationship, and enzymatic control of the lytic transglyosylases (LTs), endogenous bacterial autolysins that play essential roles in the biosynthesis, maturation, and turnover of PG. Given their potentially catastrophic autolytic activity (i.e., lysis of PG leading to cell rupture and death), these enzymes have to be controlled at the enzyme and/or substrate level. Our research has shown that many bacteria accomplish this control by the O-acetylation of their substrate PG. We discovered and continue to characterize the enzymes that both perform this modification and remove the controlling O-acetylation to permit continued PG metabolism. However, a large number of Gram-negative bacteria  (including Escherichia coli and Pseudomonas aeruginosa and related species, respectively) lack this ability to modify their PGs. Instead, we found that an LT from P. aeruginosa is efficiently inhibited by "inhibitors of vertebrate lysozyme" (Ivy), proteins thought to provide protection to Gram-negative bacteria from the defensive action of the lysozymes of host innate immune systems. Moreover, we found a direct and strict correlation between those bacteria that produce Ivy like proteins with those that do not O acetylate their PG. Hence, we have postulated that the true physiological function of Ivy and its orthologs and paralogs is to control the endogenous autolysins of the producing bacteria. Thus, the overarching aims of this research program are to fully characterize the role and activity of the LTs by testing two specific hypotheses. The first is that LT activity is controlled by the various proteinaceous inhibitors of lysozyme that have been discovered. We also propose to address the fundamental question of why the LTs perform a transglycosylation reaction generating an intramolecular 1,6-anhydro bond in muramoyl products rather than a simple hydrolysis as catalyzed by the muramidases (lysozymes) found in eukaryotic organisms as components of innate immune systems? In this regard, we will test our hypothesis that the LTs retain the cleaved ß-(1,4) glycosyl linkage as the 1,6 anhydro bond for subsequent re-ligation to appropriate acceptors (eg., GlcNAc residues on neighboring PG strands or proteins of wall-spanning structures) during the process of PG re-modelling. Insight to these fundamental questions will greatly further our understanding of the role and function of the LTs in the metabolism of the essential component of bacterial cell walls, PG.