Peptidoglycan-Modifying Enzyme Pgp1 Is Required for Helical Cell Shape and Pathogenicity Traits in Campylobacter jejuni

The impact of bacterial morphology on virulence and transmission attributes of pathogens is poorly understood. The prevalent enteric pathogen Campylobacter jejuni displays a helical shape postulated as important for colonization and host interactions. However, this had not previously been demonstrated experimentally. C. jejuni is thus a good organism for exploring the role of factors modulating helical morphology on pathogenesis. We identified an uncharacterized gene, designated pgp1 (peptidoglycan peptidase 1), in a calcofluor white-based screen to explore cell envelope properties important for C. jejuni virulence and stress survival. Bioinformatics showed that Pgp1 is conserved primarily in curved and helical bacteria. Deletion of pgp1 resulted in a striking, rod-shaped morphology, making pgp1 the first C. jejuni gene shown to be involved in maintenance of C. jejuni cell shape. Pgp1 contributes to key pathogenic and cell envelope phenotypes. In comparison to wild type, the rod-shaped pgp1 mutant was deficient in chick colonization by over three orders of magnitude and elicited enhanced secretion of the chemokine IL-8 in epithelial cell infections. Both the pgp1 mutant and a pgp1 overexpressing strain – which similarly produced straight or kinked cells – exhibited biofilm and motility defects. Detailed peptidoglycan analyses via HPLC and mass spectrometry, as well as Pgp1 enzyme assays, confirmed Pgp1 as a novel peptidoglycan DL-carboxypeptidase cleaving monomeric tripeptides to dipeptides. Peptidoglycan from the pgp1 mutant activated the host cell receptor Nod1 to a greater extent than did that of wild type. This work provides the first link between a C. jejuni gene and morphology, peptidoglycan biosynthesis, and key host- and transmission-related characteristics

HPLC elution profile of <i>C. jejuni</i> muropeptides and proposed muropeptide structures.

<p>Purified PG was digested with cellosyl and the resulting muropeptides were reduced with sodium borohydride and separated on a Prontosil 120-3-C18 AQ reverse-phase column. HPLC profiles are shown for <b>A, </b><i>C. jejuni</i> wild-type 81-176; <b>B,</b> Δ<i>pgp1</i>; <b>C,</b> the complement Δ<i>pgp1</i>c; <b>D,</b> the <i>pgp1</i> overexpressing strain, 81-176+<i>pgp1</i>. Peak numbers correspond to the main muropeptide peak fractions of <i>C. jejuni</i> 81-176 analyzed by LTQ-FT-MS (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002602#ppat.1002602.s005" target="_blank">Table S3</a>) to determine the structures shown in <b>E</b>. G, N-acetylglucosamine; M, reduced N-acetylmuramic acid; L-Ala, L-alanine; D-iGlu, D-isoglutamic acid; D-Glu, D-glutamic acid; <i>meso-</i>DAP, <i>meso-</i>diaminopimelic acid; Gly. Glycine; Ac, O-acetyl groups at the C-6 hydroxyl group of MurNAc; Anh, 1,6-anhydro group at MurNAc. The asterisk (*) indicates that it is not known on which MurNAc residue the modification occurs.</p

Summary of muropeptide composition of <i>C. jejuni</i> wild-type 81-176, Δ<i>pgp1</i> mutant, Δ<i>pgp1</i> complement (Δ<i>pgp1</i>c), and <i>pgp1</i> overexpression (81-176+<i>pgp1</i>) strains, and the resultant Δ<i>pgp1</i> PG profiles of Pgp1 activity assays consisting of Δ<i>pgp1</i> PG incubated without enzyme, with Pgp1 in the presence of ZnCl₂, and with Pgp1 without ZnCl₂ but with EDTA.

1<p>Numbers represent the percent area of each muropeptide from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002602#ppat.1002602.s006" target="_blank">Table S4</a> calculated to give a total of 100%. Values indicated with an asterisk (*) represent an equal to or greater than 20% difference in comparison to wild-type 81-176 or Δ<i>pgp1</i> PG to which no enzyme was added; bolded asterisked values (<b>*</b>) indicate a greater than 30% change.</p>2<p>Values for the percentage of O-acetylated species do not represent the true level of PG O-acetylation in these strains, as most O-acetyl groups are lost in the standard alkaline muropeptide reduction procedure used in this study.</p

Pgp1 has metal-dependent DL-carboxypeptidase activity on Δ<i>pgp1</i> PG, cleaving monomeric tripeptide disaccharides to dipeptides.

<p><b>A,</b> SDS-PAGE analysis of affinity purified Pgp1 with a predicted molecular weight of 67.9 kDa, indicated by an arrow. HPLC chromatograms of <b>B,</b> purified Δ<i>pgp1</i> PG; <b>C,</b> Δ<i>pgp1</i> PG incubated with purified Pgp1 and ZnCl₂; and <b>D,</b> Δ<i>pgp1</i> PG with purified Pgp1 and EDTA. Peaks corresponding to monomeric disaccharide dipeptides and tripeptides are indicated. <b>E,</b> a schematic diagram of the Pgp1 cleavage site indicated with an arrow. G, N-acetylglucosamine; M, reduced N-acetylmuramic acid; L-Ala, L-alanine; D-iGlu, D-isoglutamic acid; <i>meso-</i>DAP, <i>meso-</i>diaminopimelic acid.</p

<i>C. jejuni</i> 81-176 <i>pgp1</i> gene locus.

<p>Δ<i>pgp1</i> was constructed by deleting 880 bp of <i>pgp1</i>and inserting the non-polar <i>aphA-3</i> Km^R cassette; the approximate location of this insertion is shown above the gene cluster and is denoted by the Δ<i>pgp1</i> strain designation. The regions cloned into the integrative vectors pRRC (pEF35R; Cm^R) or pRRK (pEF20; Km^R) used for complementation and overexpression, respectively, are shown below the gene cluster. An R after the plasmid name indicates that the region is cloned in the opposite direction as the antibiotic resistance cassette promoter.</p

The role of cell shape and PG composition on host related phenotypes.

<p><b>A,</b> Δ<i>pgp1</i> is defective for chick colonization. Each point represents the log CFU/g cecal contents of an individual chick 6 days following infection with 10⁴ CFUs of the indicated <i>C. jejuni</i> strains. The geometric mean is denoted by a black bar. <b>B–D,</b> to assay the ability of <i>C. jejuni</i> wild type and Δ<i>pgp1</i> PG to activate Nod proteins, human embryonic kidney cells (HEK293T) were co-transfected with either <i>C. jejuni</i> 81-176 or Δ<i>pgp1</i> PG at 0.1 µg/mL, vectors for a NF-κB luciferase reporter, and either human Nod1 (<b>B,</b> hNod1), mouse Nod1 (<b>C,</b> mNod1) or human Nod2 (<b>D,</b> hNod2). Nod activation was determined by measuring the activity of the NF-κB luciferase reporter in comparison to the non-stimulated (NS) negative control. Positive controls used were TriDAP, FK565, and MDP. Data represent the mean ± SEM of three independent experiments. <b>E,</b> Deletion of <i>pgp1</i> increases IL-8 secretion in the INT407 epithelial cell line. Quantification of IL-8 levels was performed by ELISA. Data represent the mean ± SEM of three independent experiments. The asterisk (*) indicates a statistically significant difference using the unpaired Student's t-test, with * or *** indicating <i>p</i><0.05 or <i>p</i><0.001, respectively.</p

A <i>pgp1</i> mutant has a straight morphology and defects in CFW reactivity, motility, and biofilms.

<p>Negatively stained TEM images of <b>A,</b> the helical <i>C. jejuni</i> 81-176 strain; <b>B,</b> the straight Δ<i>pgp1</i> strain with intact flagella; and <b>C,</b> the complemented strain Δ<i>pgp1</i>c with restored helical morphology in 95% of the population. <b>D,</b> Δ<i>pgp1</i> is hypofluorescent after 48 h of growth on plates containing 0.002% CFW which is restored by complementation. <b>E,</b> Δ<i>pgp1</i>exhibits a slight motility defect, as assayed by measuring halo diameters in soft agar plates. Standard error of the mean was calculated from 12 measurements. <b>F,</b> Δ<i>pgp1</i> is defective for biofilm formation, partially complemented in Δ<i>pgp1</i>c. Biofilm formation was assessed by crystal violet staining of standing cultures in borosilicate tubes and quantification of dissolved crystal violet at 570 nm. Standard errors of the mean were calculated from triplicate cultures and are representative of three independent experiments. The asterisk (*) indicates a statistically significant difference using the unpaired Student's t-test, with * or ** indicating <i>p</i><0.05 or <i>p</i><0.01, respectively. Numerous other phenotypes showed no difference from wild type (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002602#ppat.1002602.s004" target="_blank">Table S2</a>).</p