Biological Roles of the O-Methyl Phosphoramidate Capsule Modification in Campylobacter jejuni

Abstract

Campylobacter jejuni is a major cause of bacterial gastroenteritis worldwide, and the capsular polysaccharide (CPS) of this organism is required for persistence and disease. C. jejuni produces over 47 different capsular structures, including a unique O-methyl phosphoramidate (MeOPN) modification present on most C. jejuni isolates. Although the MeOPN structure is rare in nature it has structural similarity to some synthetic pesticides. In this study, we have demonstrated, by whole genome comparisons and high resolution magic angle spinning NMR, that MeOPN modifications are common to several Campylobacter species. Using MeOPN biosynthesis and transferase mutants generated in C. jejuni strain 81–176, we observed that loss of MeOPN from the cell surface correlated with increased invasion of Caco-2 epithelial cells and reduced resistance to killing by human serum. In C. jejuni, the observed serum mediated killing was determined to result primarily from activation of the classical complement pathway. The C. jejuni MeOPN transferase mutant showed similar levels of colonization relative to the wild-type in chickens, but showed a five-fold drop in colonization when co-infected with the wild-type in piglets. In Galleria mellonella waxmoth larvae, the MeOPN transferase mutant was able to kill the insects at wild-type levels. Furthermore, injection of the larvae with MeOPN-linked monosaccharides or CPS purified from the wild-type strain did not result in larval killing, indicating that MeOPN does not have inherent insecticidal activity.

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

“…When both mutants were complemented with their respective, repaired alleles (called CJJ1420 Ϫ /C or CJJ1435 Ϫ /C), the serum resistance returned to levels comparable to that of the wild type. However, mutation of both MeOPN transferases (strain 3479) resulted in enhanced sensitivity over the CJJ1420 Ϫ mutant and showed levels of sensitivity similar to those reported previously for another double transferase mutant (24) and for the mpnC mutant (23).…”

“…Although van Alphen et al (24) reported that the population of their strain of 81-176 had the CJJ1420 gene in an off configuration, they constructed a double mutant mutated in both putative transferase genes and showed that this mutant was sensitive to complement killing, an observation consistent with earlier work with the mpnC mutant (23). When the variable regions of both MeOPN transferases were sequenced from the population of our version of strain 81-176, CJJ1420 was also in an off configuration, while CJJ1435 was on.…”

“…Complement-mediated killing of C. jejuni has been reported to occur primarily by the classical pathway (24,34), and it is thought that the CPS likely functions to shield the cell from naturally occurring antibodies in NHS that cross-react with surface proteins. Data presented here, however, suggest that MeOPN moieties also serve to protect the polysaccharide chain from preexisting antiglycan antibodies in NHS.…”

“…Capsules are a major factor in resistance to complement-mediated killing and, in the case of C. jejuni 81-176 (HS23/36), decoration of the CPS with MeOPN is essential for complement resistance. Thus, mutants of 81-176 expressing a CPS lacking MeOPN were as sensitive to complement-mediated killing as a mutant lacking CPS (23,24,28). CPS of two serotypes, HS2 and HS23/36, have also been shown to have immunomodulatory effects in vitro (23,27), and the immunomodulatory effects for the 81-176 CPS have been confirmed in vivo in a mouse model (29).…”

“…jejuni CPS have been shown to be important for pathogenesis. Nonencapsulated mutants were attenuated in a ferret model of diarrheal disease and were reduced in their ability to colonize chickens, mice, and piglets (21)(22)(23)(24)(25), although there are conflicting reports on the role of MeOPN in pathogenesis of a Galleria mellonella model of disease (26,27) and in the invasion of intestinal epithelial cells in vitro (24,28). Capsules are a major factor in resistance to complement-mediated killing and, in the case of C. jejuni 81-176 (HS23/36), decoration of the CPS with MeOPN is essential for complement resistance.…”