Effects of sequential Campylobacter jejuni 81-176 lipooligosaccharide core truncations on biofilm formation, stress survival, and pathogenesis

Abstract

Campylobacter jejuni is a highly prevalent human pathogen for which pathogenic and stress survival strategies remain relatively poorly understood. We previously found that a C. jejuni strain 81-176 mutant defective for key virulence and stress survival attributes was also hyper-biofilm and hyperreactive to the UV fluorescent dye calcofluor white (CFW). We hypothesized that screening for CFW hyperreactive mutants would identify additional genes required for C. jejuni pathogenesis properties. Surprisingly, two such mutants harbored lesions in lipooligosaccharide (LOS) genes (waaF and lgtF), indicating a complete loss of the LOS outer core region. We utilized this as an opportunity to explore the role of each LOS core-specific moiety in the pathogenesis and stress survival of this strain and thus also constructed DeltagalT and DeltacstII mutants with more minor LOS truncations. Interestingly, we found that mutants lacking the LOS outer core (DeltawaaF and DeltalgtF but not DeltagalT or DeltacstII mutants) exhibited enhanced biofilm formation. The presence of the complete outer core was also necessary for resistance to complement-mediated killing. In contrast, any LOS truncation, even that of the terminal sialic acid (DeltacstII), resulted in diminished resistance to polymyxin B. The cathelicidin LL-37 was found to be active against C. jejuni, with the LOS mutants exhibiting modest but tiled alterations in LL-37 sensitivity. The DeltawaaF mutant but not the other LOS mutant strains also exhibited a defect in intraepithelial cell survival, an aspect of C. jejuni pathogenesis that has only recently begun to be clarified. Finally, using a mouse competition model, we now provide the first direct evidence for the importance of the C. jejuni LOS in host colonization. Collectively, this study has uncovered novel roles for the C. jejuni LOS, highlights the dynamic nature of the C. jejuni cell envelope, and provides insight into the contribution of specific LOS core moieties to stress survival and pathogenesis.

FIG. 1.

C. jejuni 81-176 lipooligosaccharide (LOS) structure and mutant strains used in this study. (A) Structure of the C. jejuni strain 81-176 LOS. Inner and outer core regions are delineated. Transferase genes responsible for the addition of each sugar residue are shown in bold. Abbreviations: GalNAc, N-acetylgalactosamine; NeuAc, N-acetylneuraminic acid (sialic acid); Gal, galactose; Glc, glucose; Hep, heptose; PEtn, phosphoethanolamine; Kdo, 2-keto-3-deoxymannooctulosonic acid. cgtA is phased off in our 81-176 strain; thus, CgtA and the terminal GalNAc are shown in gray. (B) LOS mutants isolated in the CFW screen. The ΔwaaF* mutant harbors the solo transposon, encoding kanamycin resistance, in the intergenic region between waaF and waaV. The ΔlgtF mutant harbors the picard transposon, encoding chloramphenicol resistance, upstream of the first glucosyltransferase domain, at the approximate site shown. Gene numbers are those annotated for strain 81-176 and are abbreviated versions of the “cjj81176_XXXX” nomenclature in the NCBI database. (C) Targeted LOS mutants. An aphA-3 cassette encoding kanamycin resistance but no downstream transcriptional terminator was used to disrupt waaF, galT, and cstII. Specifically, aphA-3 was used to remove 556 bp of waaF, including ∼75% of the single heptosyltransferase gene (ΔwaaF), and 446 bp of galT, including ∼75% of the single glycosyltransferase domain (ΔgalT). The ΔcstII mutant was constructed by inserting aphA-3 into cstII. Gene numbers are those annotated for strain 81-176 and are abbreviated versions of the “cjj81176_XXXX” nomenclature in the NCBI database.

FIG. 2.

LOS profile and structures of the 81-176 mutant and complemented strains. (A) The LOS of C. jejuni strains was resolved by SDS-PAGE and visualized by silver staining. Complemented strains are denoted by “-c”. (B) Using mass spectrometry analyses, the core LOS structure of each mutant strain was deduced by comparing the observed mass species of the mutant strains to those of the wild type, each other, and the wild-type 81-176 LOS core structure reported by Guerry et al. and Kanipes et al. (25, 43). Abbreviations: GalNAc, N-acetylgalactosamine; NeuAc, N-acetylneuraminic acid (sialic acid); Gal, galactose; Glc, glucose; Hep, heptose; PEtn, phosphoethanolamine; Kdo, 2-keto-3-deoxymannooctulosonic acid.

FIG. 3.

CFW reactivity, biofilm formation, and broth growth properties of LOS mutant strains. (A) CFW reactivity after 48 h of growth on BHI plates containing 0.002% CFW was visualized under long-wave UV light. All strains were assessed on the same plate, with spot rearrangement necessary for presentation purposes. (B) Biofilm formation was assessed after 1, 2, and 3 days of incubation in MH broth in polypropylene plates. Biofilms were stained with crystal violet, dissolved, and quantified by measuring the absorbance at 550 nm. Complemented strains are denoted by “-c”. Error bars were calculated from triplicate readings and are representative of three independent assays. The asterisk (*) indicates statistically significant differences from wild-type 81-176 (P < 0.01). (C, D) Growth in shaking MH broth cultures was assessed by absorbance at 600 nm (C) and plating of serial dilutions to determine CFU/ml of culture (D). (D) Points represent means derived from triplicate readings and are representative of three independent assays.

FIG. 4.

Complement-mediated killing of LOS mutant strains. Strains were incubated in PBS containing 10% pooled human serum or 10% heat-killed human serum (HK) for 0, 40, and 80 min, and survival was assessed by plating serial dilutions to determine numbers of CFU/ml. Complemented strains are denoted by the “-c”. Bracketed group “1” contains data points for all strains incubated with heat-killed serum. Bracketed group “2” contains data points for, from the top down, wild-type 81-176 and the ΔcstII, ΔgalT, ΔlgtF-c, ΔgalT-c, and ΔwaaF-c strains. The ΔlgtF and ΔwaaF mutants in 10% normal pooled human serum are noted on the graph as well as in the symbol key. Error bars are present but in most cases are too small to see. The asterisks represent statistically significant differences from wild-type 81-176, with the double asterisk (**) representing a P of <0.005 and the single asterisk (*) representing a P of <0.02.

FIG. 5.

Invasion and intracellular survival. A gentamicin protection assay was used to assess invasion and intracellular (IC) survival in Caco-2 (intestinal epithelial) cells for all LOS mutant strains constructed. Only the ΔwaaF mutant differed from wild-type 81-176 and is the only strain shown. The “invasion” time point represents 3 h of infection followed by 2 h of gentamicin treatment to kill extracellular bacteria. For “intracellular survival,” the cells were incubated in fresh medium for an additional 19 h following the gentamicin treatment prior to bacterial enumeration. (A) Numbers of CFU/well recovered at each time point. The asterisk (*) denotes a statistically significant difference for the ΔwaaF mutant compared to both wild-type 81-176 and the ΔwaaF-c strain at the “intracellular survival” time point (P < 0.05), as assessed by both total numbers of bacteria recovered and the amounts of inocula recovered as percentages of the wild-type inoculum recovered. (B) The percentage of the inoculum recovered was calculated for each strain at each time point. Numbers shown represent the percent recovered relative to the wild-type (WT).

FIG. 6.

In vivo colonization of the ΔlgtF and ΔwaaF mutants. (A, B) BALB/c ByJ mice were fed a 1:1 mixture of wild-type 81-176 and either the ΔlgtF (A) or ΔwaaF (B) mutant, and colonization levels were determined at 7, 14, and 21 days postinfection. Mean colonization levels are denoted by horizontal bars. Both the ΔwaaF and ΔlgtF mutants colonized at levels that were statistically significantly different from the wild-type 81-176 level (P < 0.01 for both strains at all time points). Symbols located on the x axis represent colonization levels below the limit of detection. (C, D) Shaking MH broth cultures were inoculated with a 1:1 mixture of wild-type 81-176 and either the ΔlgtF or ΔwaaF mutant. Growth and recovery were assessed by measuring the absorbance at 600 nm (C) and plating for CFU/ml (D) on plates identical to those used to assess colonization.