Phase-Variable Changes in the Position of O -Methyl Phosphoramidate Modifications on the Polysaccharide Capsule of Campylobacter jejuni Modulate Serum Resistance

Abstract

Campylobacter jejuni polysaccharide capsules (CPS) are characterized by the presence of nonstoichiometric O-methyl phosphoramidate (MeOPN) modifications. The lack of stoichiometry is due to phase variation at homopolymeric tracts within the MeOPN transferase genes. C. jejuni strain 81-176 contains two MeOPN transferase genes and has been shown previously to contain MeOPN modifications at the 2 and 6 positions of the galactose (Gal) moiety in the CPS. We demonstrate here that one of the two MeOPN transferases, encoded by CJJ81176_1435, is bifunctional and is responsible for the addition of MeOPN to both the 2 and the 6 positions of Gal. A new MeOPN at the 4 position of Gal was observed in a mutant lacking the CJJ81176_1435 transferase and this was encoded by the CJJ81176_1420 transferase. During routine growth of 81-176, the CJJ81176_1420 transferase was predominantly in an off configuration, while the CJJ81176_1435 transferase was primarily on. However, exposure to normal human serum selected for cells expressing the CJJ81176_1420 transferase. MeOPN modifications appear to block binding of naturally occurring antibodies to the 81-176 CPS. The absence of MeOPN-4-Gal resulted in enhanced sensitivity to serum killing, whereas the loss of MeOPN-2-Gal and MeOPN-6-Gal resulted in enhanced resistance to serum killing, perhaps by allowing more MeOPN to be put onto the 4 position of Gal.IMPORTANCE Campylobacter jejuni undergoes phase variation in genes encoding surface antigens, leading to the concept that a strain of this organism consists of multiple genotypes that are selected for fitness in various environments. Methyl phosphoramidate modifications on the capsule of C. jejuni block access of preexisting antibodies in normal human sera to the polysaccharide chain, thus preventing activation of the classical arm of the complement cascade. We show that the capsule of strain 81-176 contains more sites of MeOPN modifications than previously recognized and that one site, on the 4 position of galactose, is more critical to complement resistance than the others. Exposure to normal human serum selects for variants in the population expressing this MeOPN modification.

Enhanced Immunogenicity and Protective Efficacy of a Campylobacter jejuni Conjugate Vaccine Coadministered with Liposomes Containing Monophosphoryl Lipid A and QS-21

“…Anti-CPS ELISA. Anti-CPS total IgG or IgG subclass analysis was performed using oxidized CPS and Carbo-BIND plates as previously described (60). The following horseradish peroxidase-conjugated secondary antibodies were used for detection: goat anti-mouse IgG or goat anti-mouse IgA and A. nancymaae anti-CPS IgG responses were measured using goat anti-human IgG (SeraCare, Gaithersburg, MD).…”

Immunomodulatory Roles of Polysaccharide Capsules in the Intestine

“…The production of TNF-α and IL-8 production by human monocyte-derived macrophages was reduced in the presence of an EPS + Pediococcus parvulus strain compared to an EPS − strain (44). Campylobacter jejuni's CPS decreases activation of TLR4 and production of proinflammatory IL-1β, IFN-γ, and IL-6 (33). Expression of Salmonella's Vi capsular polysaccharide led to a decline in IL-6 and TNF-α secretion by BMDM in a TLR-dependent manner (47).…”

“…Vibrio cholera's CPS also protects against complement-mediated bacteriolysis and strains isolated from patients that contained less capsular material were more susceptible to the bactericidal activity of serum (53,54). The CPS of Campylobacter jejuni blocks antibody binding and activation of complement and non-stoichiometric O-methyl phosphoramidate (MeOPN) modifications at the 4 position of galactose has been shown to be the most critical to complement resistance (33). An acapsular strain of the Gram-negative symbiont Bacteroides thetaiotaomicron, which lacks expression of all B. thetaiotaomicron CPSs, also had a decrease in survival after treatment with normal human serum compared to WT, further suggesting that CPSs can enable bacteria to circumvent complement (25).…”

Update on Campylobacter vaccine development

“…Importantly, both the galactose 4-OH MeOPN transferase gene (cjj1420) and the galactose 2,6-OH MeOPN transferase gene (cjj1435) of 81-176 contain poly(C) tracts that can undergo slipped-strand mispairing, resulting in phase variable gene expression during infection. 48,53 This mechanism likely promotes optimal C. jejuni virulence and fitness during infection.…”

“…100,101 As described above, the CPS structure also contains non-stoichiometric modifications including methyl phosphoramidate (MeOPN) occurring on the Galactose residue at the 2-OH, 4-OH, or 6-OH group. 53 Those MeOPN are believed to be immunodominant and required for vaccine efficacy. To prevent potential autoimmune reactions resulting from trace contamination with the 81-176 sialylated LOS structures, the 81-176 strain was genetically manipulated to prevent expression of gangliosidelike mimics.…”