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Structural insights into the modulation of bacterial peptidoglycan synthase activity

University of British Columbia

The bacterial cell wall plays a crucial role in cellular viability and is an important drug target. The bacterial cell wall is constructed using a complex biosynthetic pathway, which ultimately terminates in the activity of peptidoglycan synthases. These synthases act to polymerize the lipid-linked cell wall precursors and crosslink these strands into the existing sacculus. The activity of these synthases can be modulated in two key ways. First, there are proteins which modulate the type of synthase activity. In most bacteria, the peptidoglycan synthase crosslinking reaction uses D,D-transpeptidase activity. However, an alternate crosslinking mechanism involving the formation of a complex between peptidoglycan synthases and the L,D-transpeptidase YcbB can lead to bypass of D,D-transpeptidation, β-lactam resistance, typhoid toxin release, and stress linked cell wall crosslinking. Second, there are proteins which associate with peptidoglycan synthases to stimulate or inhibit the canonical synthase activity. In E. coli, there are the positive regulators LpoA and LpoB, and the negative regulator CpoB. These proteins seen in E. coli are conserved across a large number of Gram-negative organisms, though the human pathogen P. aeruginosa is seen to have an alternative to LpoB, known as LpoP. Here, we provide insight into the structure and function of YcbB and LpoP. We show that the crystallographic structure of YcbB from E. coli, S. Typhi, and C. rodentium consists of a conserved L,D-transpeptidase catalytic domain, substrate capping loop subdomain, peptidoglycan-binding domain and scaffolding domain. Meropenem and ertapenem acylation of YcbB gives insight into inhibition by carbapenems, the singular antibiotic class with significant activity against L,D- transpeptidases. Additionally, we probe the interaction network of this pathway, assay β-lactam resistance in vivo, and provide insight into the role of YcbB in acute bacterial infection. Second, we show that the crystallographic structure of LpoP consists of tandem tetratricopeptide repeats, distinct from the structure of the canonical LpoB. Using in vitro glycosyltransferase and transpeptidase assays, we compare and contrast the structure of LpoP- and LpoB-PBP1b systems. As a whole, this thesis provides insight into both forms of modulation of peptidoglycan synthase activity and lays the groundwork for further understanding of this complex biosynthetic pathway.

Text

2021-07-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/75160

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/