Abstract
Campylobacter jejuni is a leading cause of bacterial gastroenteritis in the developed world. Despite its prevalence, its mechanisms of pathogenesis are poorly understood. Peptidoglycan (PG) is important for helical shape, colonization, and host-pathogen interactions in C. jejuni. Therefore, changes in PG greatly impact the physiology of this organism. O-acetylation of peptidoglycan (OAP) is a bacterial phenomenon proposed to be important for proper cell growth, characterized by acetylation of the C6 hydroxyl group of N-acetylmuramic acid in the PG glycan backbone. The OAP gene cluster consists of a PG O-acetyltransferase A (patA) for translocation of acetate into the periplasm, a PG O-acetyltransferase B (patB) for O-acetylation, and an O-acetylpeptidoglycan esterase (ape1) for de-O-acetylation. In this study, reduced OAP in ΔpatA and ΔpatB had minimal impact on C. jejuni growth and fitness under the conditions tested. However, accumulation of OAP in Δape1 resulted in marked differences in PG biochemistry, including O-acetylation, anhydromuropeptide levels, and changes not expected to result directly from Ape1 activity. This suggests that OAP may be a form of substrate level regulation in PG biosynthesis. Ape1 acetylesterase activity was confirmed in vitro using p-nitrophenyl acetate and O-acetylated PG as substrates. In addition, Δape1 exhibited defects in pathogenesis-associated phenotypes, including cell shape, motility, biofilm formation, cell surface hydrophobicity, and sodium deoxycholate sensitivity. Δape1 was also impaired for chick colonization and adhesion, invasion, intracellular survival, and induction of IL-8 production in INT407 cells in vitro. The importance of Ape1 in C. jejuni biology makes it a good candidate as an antimicrobial target.
Highlights
Campylobacter jejuni is a leading bacterial cause of foodborne gastroenteritis in the developed world and the most common infectious antecedent to the autoimmune acute polyneuropathy Guillain-Barresyndrome [1, 2]
The OAP genes in C. jejuni were shown to contribute to PG O-acetylation/de-O-acetylation, consistent with their predicted functions
These genes were important for several key physiological and pathogenic properties. This was most notable for ape1, which was involved in PG de-O-acetylation and the only OAP gene significantly required for every phenotype examined
Summary
C. jejuni OAP Genes Were Identified by BLAST and Mutant and Complemented Strains Were Generated—The OAP gene cluster was identified in C. jejuni 81-176 wild type by BLAST analysis using the N. gonorrhoeae OAP gene sequences. O-Acetylation levels of C. jejuni 81-176, ⌬ape1, ⌬ape1C (complemented ⌬ape1), ⌬patB, ⌬patA, and ⌬oap (a mutant in which the entire cluster was deleted: ape, patB, and patA), as determined by basecatalyzed hydrolysis and release of acetate reported as a % O-acetylation relative to MurNAc content. Deletion of ape led to an increase in O-acetylation to 35.6 Ϯ 2.25% relative to total MurNAc content These results are in accordance with the functions described for homologs in N. gonorrhoeae and N. meningitidis [30, 42,43,44]. Analysis of the O-acetylation levels for ⌬patA and ⌬patB complements were not performed as, unlike the ⌬ape mutant, phenotypic differences between these mutants, ⌬oap and wild type, were in almost every case not statistically significant or were minimal (see below).
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