Highly infectious pathogenic strains of bacteria are becoming increasingly resistant to the current clinical antibiotics which have created a dire need for the development of novel antibiotics. O-Acetylpeptidoglycan esterase 1 (Ape1) is a periplasmic esterase present in several peptidoglycan (PG) O-acetylating pathogenic species of Gram-positive and all Gram-negative bacteria that perform this modification to this essential cell wall polymer. Inhibition of this growth-limiting enzyme may prove the principle that Ape1 has the potential to be the target for the development of a novel class of antibiotics. Ape1 plays a crucial role in bacterial growth by regulating PG turnover through catalytic removal of the C-6 acetyl group from O-acetylPG. This activity is required for the continued metabolism of PG because the major autolytic enzymes involved, the lytic transglycosylases, require a free C-6 hydroxyl group to produce their reaction product, 1,6-anhydromuramic acid. Several of the compounds that have been identified to effectively inhibit Ape1, were re-evaluated by determining their kinetic parameters. Work presented in this thesis explored the inhibitory potential of these compounds, belonging to the anthraquinone (alizarin, quinizarin, quinalizarin, emodin, sennoside A) or tannin (ellagic acid) families of compounds, both in vitro and in vivo, among species of bacteria that are known to O-acetylate their PG. Of the inhibitory compounds tested, ellagic acid was found to be most effective in vitro, with an IC50 value of 0.91 µM ± 0.06, Ki 1.18 ± 0.04 and in vivo it was shown to reduce bacterial growth.

The O-acetylation of peptidoglycan (PG) is currently known to occur in greater than 50 eubacterial species, including numerous pathogens. This modification, which occurs at the C-6 hydroxyl of the N-acetylmuramoyl residues within the heteropolymer’s glycan backbone, serves as a cell wall autolytic regulatory mechanism, and contributes to pathogenesis and persistence within a host. Despite these significant physiological and pathobiological roles however, the identity of the pathway(s) responsible for the modification was only recently elucidated, for which two unrelated systems were identified, viz., the O-acetylpeptidoglycan (OAP) cluster-encoded multi-component system typical of Gram-negative species and the singular OatA of Gram-positives. As part of the OAP PG O-acetylation system, our group previously identified O-acetylpeptidoglycan esterase (Ape) as an enzyme responsible for the removal of the modification, permitting the continued metabolism of the PG sacculus. Herein, studies were performed to assess the postulated viability of this class of enzyme as a novel antibacterial target. Specifically, recombinant Ape1 from Neisseria gonorrhoeae was purified to homogeneity and the inhibitory effect of purpurin, a natural product identified as such, evaluated in detail. Kinetic analysis demonstrated that the compound elicited a competitive mode of inhibition (Kic ~3.7 μM), while the in vivo treatment of an array of environmental and pathogenic species was found to result in growth arrest for those cells containing both O-acetylPG and Ape. Evaluation of modification levels, cell wall morphology, and viability indicated a bacteriostatic effect. Taken together these data provide proof of principle that this class of enzyme presents a worthy therapeutic target. In addition to the presence of an Ape, the OAP system further differs from that of OatA through the use of two PG O-acetyltransferases. While purported to be mutually exclusive and evolutionarily divergent, in silico genomic analyses indicated their potential copresence in Bacillus anthracis and other closely related organisms. Indeed, purpurin-mediated differential growth inhibition between several such isolates and other bacilli indicated Ape activity therein. To investigate this possibility, the hypothetical Ape3 protein from Bacillus cereus ATCC 10987 was overproduced, purified, and its function assessed. Data from activity assays involving natural and synthetic substrates indicated that the protein possesses basal esterase activity in vitro. Phenotypic analysis of B. anthracis mutants deficient in each of the organism’s putative integral membrane PG O-acetyltranslocases subsequently indicated that Ape3 preferentially functions as a PG O-acetyltransferase (Pat) in vivo and that the OAP-mediated system is required for the separation of daughter cells following division. In addition, the presence of an Oat homologue was also confirmed. Thus, this is the first report of a bacterium known to possess both types of PG O-acetylation systems. / Natural Sciences and Engineering Research Council of Canada (NSERC)