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United States Department of Agriculture

Agricultural Research Service

Research Project: Molecular Biology of Human Pathogens Associated with Food

Location: Produce Safety and Microbiology Research

Title: O-Methyl Phosphoramidate Modifications on the Capsular Polysaccharide of Campylobacter Jejuni Are Involved in Serum Resistance, Infection, and Insecticidal Activity

Authors
item Van Alphen, Lieke -
item Fodor, Chistopher -
item Wenzel, Cory -
item Ashmus, Roger -
item Miller, William
item Stinzi, Alain -
item Lowary, Todd -
item Szymanski, Christine -

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: August 10, 2011
Publication Date: N/A

Interpretive Summary: Campylobacter jejuni are the most commonly reported agent of bacterial foodborne illness in North America. C. jejuni decorates its outer surface with a series of sugar structures and phosphorylated groups. One such groups is O-methyl phosphoramidate. These decorations are common among C. jejuni strains and in related organisms. The genes necessary for synthesizing these decorations were recently confirmed through mutational analysis. Mutants lacking these decorations were more sensitive to blood serum proteins, however they showed a greater invasion potential into human cells. A wax moth larval model was used to study these decorated cells – cells so decorated kill wax moth larvae. Mutational analysis of the O-methyl phosphoramidate genes indicated that disruption of some genes still lead to a reduced killing response. The experiments suggested that the decorated sugar structures still killed moth larvae even though they were never displayed on the outer surface.

Technical Abstract: Campylobacter jejuni is the most commonly reported cause of bacterial foodborne illness in North America. C. jejuni decorates its surface polysaccharides with a variety of variable phosphorylated structures, including O-methyl phosphoramidate (MeOPN) modifications on the capsular polysaccharide. Although MeOPN moieties are present in ~70% of C. jejuni isolates as well as several other members of the epsilonproteobacteria, little is known about their biological role. The C. jejuni genes involved in MeOPN biosynthesis and transfer to capsule have previously been identified in strain 11168, and our group has recently confirmed via site-directed mutagenesis that homologous genes in the highly virulent strain 81-176 are required for the biosynthesis and transfer of MeOPN. To determine the biological significance of MeOPN expression in C. jejuni, a variety of in vitro and in vivo assays were performed comparing the wild-type 81-176 strain to knockout mutants deficient in biosynthesis (cj1416) or transfer (orf7/orf20) of MeOPN. Mutants lacking MeOPN were more sensitive to serum and exhibited 10-fold greater invasion of Caco-2 epithelial cells. To investigate the potential insecticidal effects of the MeOPN structure in vivo, we used the Galleria mellonella (wax moth larvae) model system in which live C. jejuni bacterial cells are injected into the hemolymph of the larvae. In this model, wild type C. jejuni 81-176 efficiently killed G. mellonella in a dose-dependent manner and the cj1416 mutant displayed reduced killing, while complementation of this mutant resulted in wild-type killing. Interestingly, the orf7/orf20 mutant killed G. mellonella as efficiently as wild-type, suggesting that accumulation of MeOPN within the bacterial cells might be killing the insects. Infection experiments with piglets demonstrated that the cj1416 mutant was outcompeted by the wild-type strain. In contrast, the orf7/orf20 mutant showed similar levels of colonization as wild-type.

Last Modified: 10/24/2014
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