Influence of MeOPN on colonization.

<p>(<b>A</b>) Colonization levels of <i>C. jejuni</i> 81-176 wild-type and <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> (MeOPN transferase mutant) in a chicken colonization model, 6 days post-infection. (<b>B</b>) Relative colonization levels of <i>C. jejuni</i> 81-176 wild-type and the MeOPN transferase mutant in a competitive piglet infection model. Note that no significant difference was observed between strains in the chicken colonization model. In the competitive piglet infection model, the MeOPN transferase mutant displays a 5-fold reduction in colonization relative to wild-type comparing the measured wild-type-to-mutant ratio of the inoculum to the ratio recovered from the piglet intestine following infection (p = 0.0005). Statistical significance was determined using a Mann−Whitney test. Horizontal bars represent median values.</p

1D ¹H–³¹P HSQC spectra of select <i>Campylobacter</i> species containing orthologues of the <i>cj1416</i>-<i>cj1418</i> genes.

<p>Spectra contain the following resonances corresponding to MeOPN: <i>C. insulaenigrae</i> RM5435, peak at 3.74 ppm; <i>C. lari</i> UPTC NCTC 11845, peak at 3.70 ppm; <i>C. lari</i> subsp. <i>concheus</i> LMG 11760, peak at 3.70 ppm; <i>C. subantarcticus</i> RM8523, peak at 3.75 ppm; <i>C. cuniculorum</i> LMG 24588, peak at 3.76 ppm; <i>C. upsaliensis</i> RM3195, peak at 3.70 ppm; <i>C. upsaliensis</i> RM3940, peaks at 3.75 and 3.71 ppm; <i>C. helveticus</i> CCUG 30566, peak at 3.68 and 3.61 ppm; <i>C. jejuni</i> NCTC 11168, peaks at 3.71 and 3.68 ppm.</p

1D ¹H–³¹P HSQC analyses of <i>C. jejuni</i> 81-176 wild-type and the MeOPN biosynthesis and transferase mutants.

<p>Intact whole cells of <i>C. jejuni</i> were analysed by HR-MAS NMR after 48 h growth on MH agar. Depicted are the 1D ¹H–³¹P HSQC spectra, which specifically show the MeOPN resonance at 3.8 ppm in 81-176 wt (A), but not in the <i>cjj81176_1415</i>::<i>kan^r</i> MeOPN biosynthesis mutant (B), and <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> MeOPN transferase mutant (C).</p

Loss of MeOPN results in a decrease in serum resistance.

<p>Survival of <i>C. jejuni</i> 81-176 wild-type, <i>cjj81176_1415</i>::<i>kan^r</i> (MeOPN biosynthesis mutant), <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> (MeOPN transferase mutant), and <i>kpsM</i>::<i>kan^r</i> (capsule mutant) in 10% natural human complement serum relative to survival in heat inactivated serum. Serum resistance was significantly different between <i>C. jejuni</i> 81-176 wild-type, the <i>cjj81176_1415</i>::<i>kan^r</i> (MeOPN biosynthesis mutant), and the <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> mutant at both 30 and 60 min (p<0.0001). Serum resistance was significantly different between <i>C. jejuni</i> 81-176 wild-type and the <i>kpsM</i>::Km^R mutant at both 30 and 60 min (p = 0.005 and p = 0.014, respectively) using an unpaired t-test. Results represent the mean (± SEM) of three independent experiments.</p

MeOPN modifications provide protection against serum mediated killing by interfering with the classical complement activation pathway.

<p>Survival of <i>C. jejuni</i> 81-176 wild-type and <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> (MeOPN transferase mutant) in 10% natural human complement serum. The samples were tested in the presence or absence of 50 mM EGTA after 60 min incubation, relative to survival in heat inactivated serum. Serum resistance was significantly different between <i>C. jejuni</i> 81-176 wild-type in the presence versus absence of EGTA (p = 0.0030), the <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> mutant in the presence versus absence of EGTA (p = 0.0014), and the 81-176 wild-type versus the <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> mutant in the absence of EGTA (p = 0.0047) using an unpaired t-test. Results represent the mean (± SEM) of three independent experiments. Note that both the wild-type and MeOPN transferase mutant approach 100% survival in the presence of EGTA.</p

MeOPN does not have insecticidal activity against <i>Galleria mellonella</i>.

<p>(A) Insecticidal activity of <i>C. jejuni</i> 81-176 wild-type, <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> (MeOPN transferase mutant), <i>C. jejuni</i> 81-176 <i>kpsM</i>::<i>kan^r</i> (capsule mutant), <i>C. jejuni</i> 11168H wild-type, and <i>C. jejuni</i> 11168H <i>cj1421-1422</i>::<i>kan^r</i> (MeOPN transferase mutant). (B) Insecticidal activity of <i>C. jejuni</i> 81-176 wild-type, <i>cjj81176_1415</i>::<i>kan^r</i> (MeOPN biosynthesis mutant), <i>C. jejuni</i> 11168H wild-type, and <i>C. jejuni</i> 11168H <i>cj</i>1416::<i>kan^r</i> (MeOPN biosynthesis mutant). (C) Insectidal activity of <i>C. jejuni</i> 81-176 wild-type, <i>C. jejuni</i> 81-176 purified CPS, or MeOPN-containing compounds in a <i>G. mellonella</i> larval model. (D) Chemical structures of MeOPN-containing compounds. Glc – methyl α-D-glucopyranoside; 2-MeOPN-Glc - methyl 2-<i>O</i>-(methylphosphoramidyl)-α-D-glucopyranoside; 6-MeOPN-Glc - methyl 6-<i>O</i>-(methylphosphoramidyl)-α-D-glucopyranoside; Gal – methyl α-D-galactopyranoside; 2-MeOPN-Gal - methyl 2-<i>O</i>-(methylphosphoramidyl)-α-D-galactopyranoside; 6-MeOPN-Glc - methyl 6-<i>O</i>-(methylphosphoramidyl)-α-D-galactopyranoside; CPS – purified capsular polysaccharide from 81-176 wild-type. Survival percentages were significantly different between wild-type 81-176 and the <i>kpsM</i>::<i>kan^r</i> mutant (p = 0.0031), the <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> and <i>kpsM</i>::<i>kan^r</i> mutants (p = 0.0026), and between wild-type 81-176 and all tested MeOPN-containing compounds (p<0.005 in all cases). Note that no statistically significant difference was observed between the wild-type and MeOPN transferase mutant in either the 81-176 (p = 0.264) or 11168H (p = 0.063) strains. Results represent the mean (±SEM) of five independent experiments in (A) and four independent experiments in (C) except for 81-176 CPS, which was the result of a single experiment.</p

Prevalence of MeOPN biosynthesis genes within the <i>Campylobacter</i> genus.

<p>Dendrogram of AtpA amino acid sequences of representative <i>Campylobacter</i> species, with strains containing orthologues of the MeOPN biosynthesis genes (<i>cj1416-1418</i>) indicated with an asterisk. The scale bar represents substitutions per site. Bootstrap values of >75%, generated from 1000 replicates using the Neighbor-Joining algorithm, are shown in the nodes. The Cj1416-Cj1418 orthologues for <i>C. jejuni, C. lari</i> and <i>C. fetus</i> are present in the NCBI database: the accession number for <i>C. jejuni subsp. jejuni</i> strain NCTC 11168 is AL111168; the accession number for <i>C. coli</i> strain 76339 (genes BN865_14280-14300) is HG326877; the accession number for <i>C. lari subsp. lari</i> strain RM2100 (genes Cla_0314-316) is CP000932; the accession number for <i>C. fetus subsp. fetus</i> strain 82–40 (genes CFF8240_1630-1632) is CP000487; and the accession number for <i>C. fetus subsp. venerealis</i> strain NCTC 10354 (genes CFV354_1762-1764) is CM001228. The accession numbers for the <i>atp</i> sequences and new Cj1416-Cj1418 orthologues from the other <i>Campylobacter</i> species are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087051#pone.0087051.s002" target="_blank">Table S1</a>.</p