Citation: van Alphen LB, Wenzel CQ, Richards MR, Fodor C, Ashmus RA, Stahl M, et al. (2014)
Biological Roles of the O-Methyl Phosphoramidate Capsule Modification in Campylobacter jejuni. PLoS ONE 9(1): e87051. doi:10.1371/journal.pone.0087051
Editor: Mikael Skurnik, University of Helsinki, Finland
Received: September 7, 2013;
Accepted: December 18, 2013;
Published: January 30, 2014
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: LVA held an Alberta Innovates Health Solutions Postdoctoral Fellowship. RAA is an Alberta Innovates Technology Futures Scholarship awardee. CMS is an Alberta Innovates Scholar. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
[h=2]Introduction[/h]
Campylobacter jejuni is a Gram-negative bacterium that is a leading cause of bacterial gastroenteritis worldwide
[1]. The handling and consumption of contaminated poultry are the most common routes of infection in developed countries
[2], although contaminated water and dairy products can also be significant sources
[3]. In developing countries, campylobacteriosis is endemic and represents a major cause of diarrheal disease and infant mortality. Symptoms of infection typically range from mild to severe inflammatory diarrhea,
but in rare cases infection leads to the life-threatening autoimmune disorder known as the Guillain-Barr? syndrome [4].
Capsular polysaccharide (CPS) forms the outermost structure on most bacteria and plays key roles in the interaction between the organism, host, and environment. In the case of
C. jejuni, CPS is the major determinant of the Penner serotyping scheme
[5] and has been demonstrated to be important for both serum resistance and invasion of epithelial cells
[6]. The CPS produced by
C. jejuni strains are structurally complex and highly variable, due in part to the addition of phase-variable modifications such as
O-methyl, ethanolamine, aminoglycerol, and
O-methyl phosphoramidate (MeOPN) groups
[7],
[8],
[9].
Although phosphoramidates are rare in nature, our previous studies identified the presence of MeOPN modifications in ∼70% of C. jejuni isolates [10]. In
C. jejuni strain 11168H, we identified four genes required for the biosynthesis of MeOPN,
cj1415−cj1418, and two phase variable genes,
cj1421 and
cj1422, that encode transferases responsible for the addition of MeOPN to C-3 of β-D-Gal
fNAc or to C-4 of D-
glycero-α-L-
gluco-Hep, respectively
[10].
While it is unknown whether MeOPN modifications are present in other
Campylobacteraceae, their commonality among
C. jejuni strains suggests an important biological role for these surface-expressed modifications within the species, and we have recently demonstrated that MeOPN is a receptor for several
C. jejuni lytic bacteriophages
[11],
[12].
MeOPN has structural similarity to some synthetic pesticides [13], and infection studies with
C. jejuni 11168H in the
Galleria mellonella model showed a substantial decrease in insecticidal activity for a MeOPN biosynthesis mutant relative to the wild-type strain
[14]. More recently, Maue
et al.
[15] demonstrated that disruption of a MeOPN biosynthetic gene in the virulent
C. jejuni 81?176 strain correlated to reductions in both serum resistance and colonization in a mouse intestinal model, relative to the wild-type strain. In combination, these studies indicate that, when present, MeOPN biosynthesis is important for
C. jejuni cellular interactions and infection.
In this study, we have determined that MeOPN modifications are prevalent among several
Campylobacter species, and not limited to
C. jejuni. We have mutated the MeOPN transferase gene homologues in
C. jejuni strains 81?176 and 11168H. Both mutants exhibited similar levels of insecticidal activity compared to wild-type in the
G. mellonella infection model, and no insecticidal activity was observed when
G. mellonella was injected with either purified CPS or synthesized compounds containing MeOPN. However, the MeOPN mutant exhibits enhanced invasion of Caco-2 cells and reduced resistance to serum, primarily due to activation of the classical complement pathway. The mutant shows no difference in colonization of chickens compared to the wild-type, but the mutant shows a drop in colonization in piglets in co-infection studies with the wild-type.
Our data suggests that, when present, MeOPN has a contributory role in pathogenesis.
