Key Role of Capsular Polysaccharide in the Induction of Systemic Infection and Abortion by Hypervirulent Campylobacter jejuni

Abstract

Campylobacter jejuni is a zoonotic pathogen, and a hypervirulent clone, named clone SA, has recently emerged as the predominant cause of ovine abortion in the United States. To induce abortion, orally ingested Campylobacter must translocate across the intestinal epithelium, spread systemically in the circulation, and reach the fetoplacental tissue. Bacterial factors involved in these steps are not well understood. C. jejuni is known to produce capsular polysaccharide (CPS), but the specific role that CPS plays in systemic infection and particularly abortion in animals remains to be determined. In this study, we evaluated the role of CPS in bacteremia using a mouse model and in abortion using a pregnant guinea pig model following oral challenge. Compared with C. jejuni NCTC 11168 and 81-176, a clone SA isolate (IA3902) resulted in significantly higher bacterial counts and a significantly longer duration of bacteremia in mice. The loss of capsule production via gene-specific mutagenesis in IA3902 led to the complete abolishment of bacteremia in mice and abortion in pregnant guinea pigs, while complementation of capsule expression almost fully restored these phenotypes. The capsule mutant strain was also impaired for survival in guinea pig sera and sheep blood. Sequence-based analyses revealed that clone SA possesses a unique CPS locus with a mosaic structure, which has been stably maintained in all clone SA isolates derived from various hosts and times. These findings establish CPS as a key virulence factor for the induction of systemic infection and abortion in pregnant animals and provide a viable candidate for the development of vaccines against hypervirulent C. jejuni.

Keywords: Campylobacter; abortion; bacteremia; capsule; sheep; systemic infection.

FIG 1

Quantitation of systemic infection by IA3902, 81-176, and NCTC 11168 in CD-1 mice. Mice were orally challenged with 10⁸ CFU of each strain. At each time point, 5 mice were sacrificed, and the numbers of C. jejuni bacteria in cardiac blood (A) and liver tissue (B) were determined. Each bar represents the log₁₀ CFU per milliliter of blood or gram of liver (means ± SEM) for 5 mice. *, P < 0.05 (statistically significant). Data were collected in three different trials, which were performed under similar conditions.

FIG 2

Analysis of capsule expression of C. jejuni IA3902 with alcian blue staining. Lanes: 1, wild-type strain IA3902; 2, capsule mutant strain (ΔkpsS); 3, complemented mutant strain (ΔkpsS-C). Positions of capsule (CPS) and lipooligosaccharide (LOS) are shown.

FIG 3

Effect of CPS on systemic infection of mice by C. jejuni. CD-1 mice (n = 8/group) were challenged via gastric gavage with 10⁸ CFU of either wild-type strain IA3902 or the ΔkpsS or ΔkpsS-C mutant, and CFU were determined at different time points. Each bar represents the log₁₀ CFU per milliliter of blood (A) or gram of liver (B) (means ± SEM) for 5 mice. *, P < 0.05 (statistically significant). Data were collected in two different trials, which were performed under similar conditions.

FIG 4

(A) Abortion rates following oral inoculation with the C. jejuni IA3902 wild-type, capsule mutant (ΔkpsS), and complemented capsule mutant (ΔkpsS-C) strains in pregnant guinea pigs. (B) Percentages of culture-positive pregnant guinea pigs (n = 10, 8, and 9 in the wild-type, mutant, and complemented groups, respectively) by each sample type processed at necropsy following abortion or at termination of the experiment 21 days after oral inoculation with the C. jejuni IA3902 wild-type, capsule mutant (ΔkpsS), or complemented capsule mutant (ΔkpsS-C) strain.

FIG 5

Survival of the C. jejuni IA3902 wild-type, capsule mutant (ΔkpsS), and complemented capsule mutant (ΔkpsS-C) strains in 20% fresh guinea pig serum (A) or in whole sheep blood (B). Data from a single experiment are shown (triplicates); similar results were obtained from an independent experiment. Significant differences (*, P < 0.05) are indicated.

FIG 6

Capsule of C. jejuni as detected by Tricine SDS-PAGE with alcian blue staining. Clone SA isolates of sheep abortions from Iowa (lanes 3 and 4), South Dakota (lanes 5 and 6), and Idaho (lanes 7 and 8) are depicted. As controls for non-clone SA isolates, NCTC 11168 (lane 1), 81-176 (lane 2), and OF48 (lane 9) (from feces of healthy sheep) are included. M, prestained protein size markers (Bio-Rad). Positions of CPS and lipooligosaccharide (LOS) are shown on the right.