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

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

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

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

<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

Pulmonary function tests of mutant mice with reduced bioavailable VEGF indicate a restrictive lung disease.

<p>Plethysmography was performed at 12 weeks on anesthetized mutant mice and littermate controls. (A) Pressure volume curves of mutant mice were shifted downward compared to control mice. Data are expressed as mean ± SEM. (B, C) Mutant mice also demonstrated a 30% decrease in hysteresis and compliance. (D, E) They also showed a 1.5-fold increase in static elastance and a 2-fold increase in resistance, all indicative of a restrictive lung disease. All differences were statistically significant. Green bars represent mean values, **P<0.01, ***P<0.001, ****P<0.0001 compared to controls.</p

Mesenchymal VEGF sequestration does not affect pulmonary angiogenesis but affects acinar air space morphogenesis.

<p>Western blot analyses of (A) total VEGFR-2 and phosphorylated VEGFR-2, and (B) endothelial markers PECAM-1 and VE-cad. No significant difference in expression between control and mutant mice was noted on either analysis. (C-D) Representative H&E images of peripheral lung sections from control and mutant mice demonstrate larger acinar air spaces in mutants. This was confirmed by mean linear intercept analysis. Images at 20x magnification, scale bars represent 100 μm.</p

Reduced VEGF disrupts the parenchyma around airways and vasculature.

<p>(A, B) Representative images of immunofluorescent staining for α smooth muscle actin (αSMA) reveals fewer putative myofibroblasts surrounding the airways in mutant mice (B) compared to controls (A). (C) Quantification by Western blot confirms a statistically significant decrease in the relative abundance of αSMA in mutants. (D, E) Representative images of Sirius red stain for Type 1 collagen demonstrates decreased peri-vascular staining in mutants (E) compared to controls (D). (F) Mutant mice express significantly less collagen around pulmonary veins based on optical density quantification. <i>White and black arrows</i> point to magnified areas shown in insets. Scale bars represent 50 μm. Data are expressed as mean ± SD, *P< 0.05, **P< 0.01 versus control.</p

VEGF sequestration decreases PI3K-Akt and MAPK-ERK pathway signaling.

<p>Western blot data for (A) PI3K, (B) phosphorylated and total Akt, and (C) phosphorylated and total ERK1/2 demonstrate decreased activation of these signaling pathways in mutants. (D) Western blot data also show decreased relative production of HIF-1α in mutant mice. (E) Mutant mice also express decreased relative levels of activated EGFR, which could further decrease surfactant production through its synergistic effect on VEGF expression. Data are expressed as mean ± SD, *P<0.05 vs control.</p