Abnormally expressed ER stress response chaperone Gp96 in CD favours adherent-invasive Escherichia coli invasion

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Abstract

Background and aims

Crohn's disease (CD) ileal lesions are colonised by pathogenic adherent-invasive Escherichia coli (AIEC) producing outer membrane vesicles (OMVs) that contribute to the bacterial invasion process. In addition, increased expression of endoplasmic reticulum (ER)-localised stress response proteins, due to ER stress, is observed in patients with CD. The expression of the ER-localised stress response protein Gp96 in patients with CD and its biological role with regards to the ability of AIEC to invade intestinal epithelial cells were analysed.

Methods and results

Immunohistochemistry on tissue arrays showed that, together with CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6) or the ER stress protein Grp78, Gp96 is also strongly expressed at the apical plasma membrane of the ileal epithelial cells of 50% of patients with CD. Invasion experiments in the presence of antibodies raised against Gp96, or after transfection of Intestine-407 cells with gp96 small interfering RNA (siRNA), indicated that Gp96 is essential to promote AIEC LF82 invasion, allowing, via the recognition of the outer membrane protein OmpA, OMVs to fuse with intestinal epithelial cells.

Conclusions

Gp96 is overexpressed on the apical surface of ileal epithelial cells in patients with CD and acts as a host cell receptor for OMVs, promoting AIEC invasion. From the results shown here, it is speculated that AIEC could take advantage of the abnormal expression of Gp96 in patients with CD to invade the ileal mucosa.

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Most cited references 34

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Aberrant Mucin Assembly in Mice Causes Endoplasmic Reticulum Stress and Spontaneous Inflammation Resembling Ulcerative Colitis

Chad K Heazlewood, Matthew C. Cook, Rajaraman Eri … (2008)

Introduction Intestinal goblet cells produce the viscous mucus layer covering the intestinal epithelium. In the large intestine mucus completely fills the crypts and forms a thick coating over the mucosal surface. MUC2 mucin is the major macromolecular constituent of intestinal mucus, and is responsible for its high viscosity and forming a protease-resistant matrix that retains molecules vital to host defence. MUC2 contains a large central O-glycosylated domain and N- and C-terminal cysteine-rich domains that homo-oligomerise [1], forming a complex molecular lattice [2,3]. MUC2 is N-glycosylated in the ER, where initial homo-oligomerisation occurs [4], and the complex then moves into the Golgi apparatus where O-glycosylation, the final stages of oligomerisation, and packaging into granules occur [5]. Mucins stored intracytoplasmically in granules form the characteristic goblet cell theca from where they are secreted constitutively and in response to stimuli. Inflammatory bowel diseases (IBD), characterized by chronic or recurrent relapsing gastrointestinal inflammation, are broadly grouped by clinical and pathological features into two groups—Crohn's disease and ulcerative colitis (UC) [6–8]. Evidence from animal models indicates that failure to suppress immunity to the abundant intestinal foreign antigen load can cause inflammation. Maintaining the normal balance between competence to respond to intestinal pathogens while not generating an inflammatory response to commensals appears to depend on the integrity of the mucosal and epithelial barriers, proinflammatory signalling pathways (especially via NF-κB), and regulation of innate and adaptive immune responses in the intestine and draining lymphoid organs. Disruption of any of these components has been shown to result in intestinal inflammation in animal models [9,10]. Defects in these components have been implicated in human IBD, although fundamental knowledge of underlying pathogenesis remains very poorly understood, with even the most well-established defect, CARD15 mutations, elucidating only a subset of white patients with ileal Crohn's disease [9,10], and with a penetrance less than 4%. Furthermore, the pathogenic pathways that distinguish UC from Crohn's disease remain obscure. Goblet cell and secreted mucus phenotypes represent important differences between the two diseases: in Crohn's disease, there is typically an increase in goblet cells and a thicker mucus layer [11,12], whereas in UC there is a reduction in goblet cells, decreases in MUC2 production [13,14] and sulfation [14–16], accumulation of MUC2 precursor [16], and a reduction in secreted mucus. Although it remains unclear whether changes in mucus are causative or secondary to inflammation [17], the lack of these changes in Crohn's disease indicate they are not a universal consequence of intestinal inflammation. Furthermore, a reduction in a specific biochemical fraction of colonic mucin has been described in UC patients and in their unaffected monozygotic twins [18,19], and engineered changes in intestinal mucin glycosylation result in enhanced susceptibility to toxin-induced colitis [20], raising the possibility that genetic defects in mucin physiology or biochemistry predispose to UC. On the other hand, in Muc2 −/− mice the colitis phenotype appears to arise only on a permissive genetic background [21,22], and transgenic depletion of goblet cells not only does not trigger spontaneous inflammation, it unexpectedly reduces dextran sodium sulphate (DSS)-induced colonic injury [23]. Thus, a causal relationship between mucin abundance and colitis remains to be defined. In this study we used random mutagenesis to produce murine models of inflammatory bowel disease. Two models were produced and we sought to characterize the basis for the pathology and identify similarities between these models and human IBD, particularly UC. Materials and Methods Generation of Mice by N-Ethyl-N-Nitrosourea Mutagenesis All animal experimentation was approved by either the Australian National University or University of Queensland Animal Experimentation Ethics Committees. Mice were generated and housed in a PC2 specific pathogen-free facility and fed autoclaved food and water. For mutagenesis, we treated 8- to 15-wk-old male C57BL/6 mice three times at weekly intervals with 85 mg/kg N-ethyl-N-nitrosourea (ENU, Sigma-Aldrich) in 10% ethanol in citrate buffer (pH 5.0). After identifying heritable phenotypes, affected B6 mice were out-crossed with NOD H-2k congenic mice, and the F1 mice were intercrossed. DNA from affected F2 mice was scanned using simple sequence length polymorphism markers and agarose gel electrophoresis. For experiments mice were housed either under SPF or clean conventional conditions and showed the same colitis phenotype in both housing conditions. Assessment of Inflammation and Intestinal Permeability Scoring of aberrant crypt architecture (score range 0–5), increased crypt length (0–3), goblet cell depletion (0–3), general leukocyte infiltration (0–3), lamina propria neutrophil counts (0–3), crypt abscesses (0–3), and epithelial damage and ulceration (0–3) was performed on the proximal and distal colon at 6, 12, and 18 wk of age (full details in Table S1). The mesenteric lymph nodes (MLNs) were dissected free of fat, disaggregated between two microscope slides, and the number of recovered leukocytes determined. 2 × 106 MLN leukocytes were cultured in 1 ml of RPMI1640 containing 10% fetal calf serum and stimulated with 50 ng/ml PMA and 750 ng/ml ionomycin. The distal one-third of the colon was cleared of luminal material, weighed, washed four times in antibiotics, diced into 1 mm square pieces, and then cultured as explants for 24 h in 1 ml of RPMI1640 containing 10% fetal calf serum. MLN and explant culture supernatants were frozen at −70 °C until assayed for the inflammatory cytokines interleukin (IL)-1β, tumour necrosis factor (TNF)-α, interferon (IFN)-γ (BD Biosciences), and IL-13 (R&D Systems) according to the manufacturer's instructions. To assess intestinal permeability, mice were orally gavaged with 4 kDa FITC-dextran (Sigma, 400 mg/kg body weight in PBS), blood samples obtained at 2 and 5 h, and plasma FITC-dextran concentrations determined by measuring fluorescence at 520 nm emitted in a 96-well plate excited with a 474 nm laser using a FLA5100 scanner (Fuji) versus a FITC-dextran standard curve. Immunoglobulin coating of the faecal bacterial flora was determined using an adaptation of a flow cytometry technique used in human IBD [24]. Induction of Colitis with Dextran Sodium Sulfate Mice were treated with 0.5%, 2%, or 3% dextran sodium sulphate (DSS) in drinking water administered continuously for 3–63 d. Body weight, stool scores, and faecal occult blood were assessed daily. In 2% and 3% DSS experiments on day 7 blood was obtained for full blood counts and serum biochemistry, mice were humanely killed and dissected and the length of the large intestine was measured, and intestinal tissue was fixed for histological analysis. For assessment of DSS-induced inflammation, increased crypt length and goblet cell depletion were excluded from the colitis scores, because these parameters are fundamental components of the Winnie and Eeyore phenotypes. In the 0.5% DSS experiment, mice were weighed weekly, monitored daily, and humanely killed when too ill to continue based on a scoring system involving loss of body weight, diarrhoea, rectal prolapses and bleeding, behaviour, and appearance. Antibodies, Immunohistochemistry, Immunofluorescence, and Western Blotting Muc2 peptides (mM2.2, CPEDRPIYDEDLKK; mM2.3, NGLKPVRVPDADNC) were synthesized (Auspep), conjugated to BSA using glutaraldehyde, emulsified in Freund's adjuvant (Invitrogen), and used to immunize rabbits. Peptide-specific antibodies were purified from rabbit serum by affinity chromatography. The 4F1 antibody reactive with the MUC2 nonglycosylated VNTR peptide repeat [25] was purified from hybridoma supernatant. We purchased antibodies against FLAG (Sigma, clone M2), GRP78 (Santa Cruz, polyclonal N-20), and β-actin (Novus Biologicals, clone AC-15). Standard immunohistochemical procedures with HRP-polymer detection were used to detect MUC2 and GRP78. Standard immunofluorescence staining for Muc2 (detected with anti-rabbit Alexa 633, Invitrogen), the lectin Dolichos biflorus agglutinin (DBA, Sigma, detected with streptavidin Alexa 488, Invitrogen), and DAPI (Invitrogen) was analysed by multitracking on a LSM510 META confocal microscope (Zeiss). Standard PAGE (NuPAGE gels, Invitrogen) and Western blotting were performed with detection by chemiluminescence or dual-label infrared fluorescence on an Odyssey instrument (Li-Cor). Biochemical Characterization of Muc2 Faecal matter was gently removed from the small and large intestines before the mucosa was scraped from the submucosa using a coverslip and extracted in ten volumes of 6 M guanidine HCl extraction buffer, reduced, and alkylated as described [26], and subjected to agarose gel electrophoresis and Western blotting as described [27,28]. Cloning and Expression of Recombinant Truncated Muc2 N-Terminal Proteins The coding sequence for the murine Muc2 N-terminal D3 domain (AJ511872 mRNA nucleotides 2452–3696, hereafter called rMuc2-D3) was reverse transcribed and amplified from murine intestinal RNA extracted from both wild-type C57BL/6 and Winnie mice using Platinum Pfx Taq polymerase (Invitrogen) and the following primers: 5′-CCCAAGCTTCCTTGCATCCACAACAAAGA-3′, 5′-CTAGTCTAGAACCATCCTGGGTCATGTTAAG-3′. The amplicon was cloned into the HindIII and XbaI restriction sites of the pSecTag3xFLAG expression vector constructed by cloning three consecutive FLAG tags into the pSecTag vector (Invitrogen), establishing an N-terminal signal peptide followed by 3 FLAG tags, and a C-terminal myc tag. Correct cloning was confirmed by sequencing. MKN45 cells were transfected with wild-type and Winnie rMuc2-D3 with Lipofectamine 2000 (Invitrogen). After 48 h culture medium was replaced with serum-free medium for 24 h before collecting and concentrating it through a centrifugal membrane filter (Millipore). Cells were lysed in RIPA buffer and lysates subjected to Western blotting. Morphological Studies Intestinal tissue was fixed in 10% buffered formalin or frozen in OCT (Tissue Tek) and sections were stained with hematoxylin and eosin (H&E), or with Alcian blue/Periodic Acid Schiff (PAS). For electron microscopy (EM) tissue was fixed in 4% glutaraldehyde, then post-fixed in osmium tetroxide, embedded in resin, and semi-thin sections stained with toluidine blue and thin sections with uranyl acetate. Gene-Expression Analysis RNA was extracted separately from the distal and proximal large intestine of three C57BL/6, three Winnie, and three Eeyore 8-wk-old mice using Trizol (Invitrogen) and cleaned on RNeasy columns (Qiagen). RNA integrity (RNA integrity number >8) was verified using a Bio-analyser (Agilent) and equal quantities of RNA from the proximal and distal large intestine of each mouse pooled. Samples were labelled for GeneChip analysis using the One-Cycle Target Labelling and Control Reagents (Affymetrix). All steps of target labelling, hybridisation, and scanning were performed according to the manufacturer's protocol. The entire microarray dataset can be accessed at NCBI GEO Accession No. GSE9913 (http://www.ncbi.nlm.nih.gov/geo/) and a heat map of genes with altered expression is provided in Figure S1. For quantitative RT-PCR RNA was reverse-transcribed using Superscript III (Invitrogen) and amplified in a Rotor Gene RG-3000 (Corbett Life Science) using Platinum SYBR Green qPCR Supermix (Invitrogen) and the following cycling conditions and primers: Hspa5 (95 °C 10 s; 56 °C 20 s; and 72 °C 30 s: 5′-TGCAGCAGGACATCAAGTTC-3′ and 5′-GTTTGCCCACCTCCAATATC-3′), the unspliced isoform of XBP-1 (95 °C 10 s; 60 °C 45 s: 5′- CAGCACTCAGACTATGTGCACCTC-3′ and 5′- AAAGGATATCAGACTCAGAATCTGAAGA-3′), the spliced isoform of Xbp-1 (95 °C 10 s; 60 °C 45 s: 5′-GAGTCCGCAGCAGGTGC-3′ and 5′-CAAAAGGATATCAGACTCAGAATCTGAA-3′) and β-actin (94 °C 10 s; 53 °C 30 s; 72 °C 30 s: 5′-GAAATCGTGCGTGACATCAAA-3′ and 5′-CACAGGATTCCATACCCAAGA-3′). Assessment of Intestinal Hypertrophy and Apoptosis 20 crypts cut in longitudinal section at each region of the intestine were measured using a micrometer. To determine the percentage of cells undergoing apoptosis, all of the epithelial cell nuclei within ten consecutive longitudinal crypt sections were counted together with the number of apoptotic bodies. Additionally, TUNEL staining was conducted with an In Situ Death Detection Kit according to the manufacturer's instructions (Roche Diagnostics) and photographed with an Olympus BX60 fluorescence microscope. To assess proliferation mice were given 100 μg/g body weight 5-bromodeoxyuridine (BrdU) i.p. 2 h prior to killing for tissue removal. Sections from paraffin-embedded Swiss rolls of the small and large intestine were antigen-retrieved by heating for 20 min in 10 mM citric acid (pH 6) and cooled to room temperature, then placed in 2 N HCl for 60 min followed by boric acid-borate buffer (pH 7.6) for 1 min and then 0.01% trypsin (in 0.5 M Tris-HCl) for 3 min. Sections were then stained with the biotinylated anti-BrdU antibody and detected with streptavidin-HRP (BD Pharmingen). BrdU-positive and -negative nuclei were counted in ten crypts from proximal and distal colon and ten crypt–villus units from the small intestine. Human Tissue Samples For ultrastructural studies, intestinal tissue biopsies were obtained at colonoscopy from five female and three male patients; four of the total number had distal UC and four were unaffected individuals undergoing colorectal cancer screening. Paired biopsies from proximal and distal colon were taken for conventional H&E histology and for EM. Two of the UC patients were on no treatment, one was on maintenance thiopurine and 5-aminosalicylate treatments, and the other was on a tapering dose of prednisolone. For immunohistochemistry, archival colonic biopsy tissue was prepared from five male and five female UC patients aged 24–45 y with left-sided or extensive colitis. Three had colitis in complete endoscopic remission (one with no immunosuppressive or 5-aminosalicylate treatment), four had mild colitis (three no treatment), and three had moderately severe colitis (one no treatment). Immunohistochemical detection of the Muc2 and the ER chaperone protein GRP78 was conducted using paraffin sections from these ten UC patients and normal tissue obtained from resection margins of patients undergoing surgery for colorectal cancer. The collection of tissue and clinical data followed informed consent and was approved by the Mater Health Services Human Research Ethics Committee Approval No. 396A. Statistical Analyses Due to difficulties in verifying normality of distributions when the sample size is small, we have taken a conservative approach and used the nonparametric Mann-Whitney U-test or Kruskal-Wallis test with Dunn's multiple comparison test for multiple comparisons, and data are presented using box plots. For larger datasets (n > 10), data were assessed with probability plots to determine if they were normally distributed prior to parametric analysis by ANOVA followed by the Bonferroni post-hoc test. Survival analysis was conducted using Kaplan-Meier plots and the Mantel log-rank test. All statistical analyses were performed using Prism v4.03 (Graphpad Software) or Systat v10.2 (Systat Software). The statistical test used and the sample sizes for individual analyses are provided within the figure legends. Results Identification of Two Novel Goblet Cell Mutants We identified Winnie mice amongst the G3 progeny of an ENU-treated C57BL/6 founder by their visible phenotype of spontaneous watery diarrhoea and high incidence of rectal bleeding and prolapse. Compared with wild-type littermates, Winnie small and large intestines were characterized by fewer goblet cells with smaller thecae, the presence of PAS-positive Alcian blue-negative material in the cytoplasm, and a reduction in secreted mucus (Figure 1). This phenotype differs from Muc2 −/− mice, which completely lack Alcian blue-positive mucin stored in goblet cells and secreted mucus [21]. In order to map Winnie, we outcrossed an affected G4 to NODk mice, and intercrossed their F1 progeny; 46/172 (27%) F2 mice had the diarrheal phenotype, consistent with a fully penetrant recessive trait (Figure 2A). Genotyping of 19 affected mice for a panel of 115 microsatellite markers mapped the mutation to a 14.5 Mb interval on Chromosome 7 encoding 198 transcripts including the Muc2 and Muc6 mucin genes (Figure 2B). We sequenced exons for the two mucin genes and found a single missense mutation (G9492A, GenBank accession no. AJ511872, http://www.ncbi.nlm.nih.gov) resulting in substitution of cysteine with tyrosine in the D3 domain at the N terminus of Muc2 (Figure 2C). Figure 1 Histological Phenotype of Mice with Muc2 Mutations PAS/Alcian blue stained intestinal sections from Winnie and wild-type C57BL/6 mice. Note the reduced size of Alcian blue staining thecae (stored mucin) and the presence of PAS-positive/Alcian blue negative accumulations (arrows) in Winnie goblet cells. L, lumen. Figure 2 Generation and Characterization of Mice with Muc2 Mutations (A) Dendrogram showing genesis of the Winnie mutation; filled symbols indicate the diarrhoea phenotype. (B) Microsatellite genotype on Chromosome 7 of 17 affected Winnie F2 mice. (C) Domain organization of the Muc2 protein showing the N- and C-terminal vWF D-domains (D1–D4), the C-terminal B, C, and CK domains, and the two central glycosylated tandem repeat domains (VNTR). The sites of the Winnie and Eeyore mutations are shown, together with the region cloned to express the rMuc2-D3 recombinant protein. (D) Results of complementation cross of Win/Win and Eey/+. PAS-stained sections of representative distal large intestinal histological phenotypes demonstrate noncomplementation (preservation of the eey/eey histological phenotype in eey/win). The possibility existed that another, unidentified mutation might be present to explain Winnie. We identified a second strain, Eeyore, from a different G0 founder, with a similar phenotype also inherited as a fully penetrant recessive trait (Figure 2D). In a Win/Win × Eey/+ complementation test, eight of 22 offspring exhibited the clinical and histopathological phenotype (Figure 2D). Thus, the noncomplementation of Winnie with Eeyore provided strong evidence that both strains harbour a mutation in the same gene and that these mutations are solely responsible for the phenotype. Sequencing of Muc2Eey identified a unique missense mutation (T2996C, GenBank accession no. AJ511873) resulting in a serine to proline substitution in the C-terminal D4 domain (Figure 2C). Winnie and Eeyore remain the only characterized diarrheal/colitis phenotypes in the Australian Phenomics Facility and Phenomix Australia murine ENU mutagenesis programs. Muc2 Mutant Mice Develop Spontaneous Colitis The progressive incidence of rectal prolapse and colitis-associated mortality in Winnie and Eeyore mice is shown in Figure 3A. At 1 y approximately 40% of Eeyore and 25% of Winnie mice had died or were humanely killed due to the development of rectal prolapse or debility (based on a multifactorial scoring system, see Materials and Methods), whereas no wild-type littermates developed rectal prolapses. Proximal and distal colon were thickened and colon weight was greater in Winnie and Eeyore than in wild-type mice, and the thickening and weight increased progressively from 6 to 18 wk of age (Figure 3B). Despite its thickening, the colon was not shortened in mutant mice (Figure S2). Colitis was assessed histologically in Winnie mice at 6, 12, and 18 wk of age, revealing mild inflammation in the large intestine (Figure 3C). The inflammatory infiltrate was usually mild and did not become more severe with age. Classical signs of murine colitis, including crypt elongation, neutrophilic infiltrates, goblet cell loss, crypt abscesses, and focal epithelial erosions were present, particularly in the distal large intestine (examples shown in Figure 3F–3K). Figure 3 Spontaneous Colitis in Mice with Muc2 Mutations (A) Incidence of premature death or colitis-associated pathology requiring humane killing (chiefly rectal prolapse) in Winnie (n = 309) and Eeyore (n = 355) mice. Mice entering experiments or killed for other reasons were treated as censored observations (designated by upward ticks), and the number of uncensored mice remaining at 50 d intervals is included underneath the graph. Incidence rates in the two strains were compared using the Mantel Log-rank test. (B) Weight of the colon after removal of luminal faecal material in C57BL/6 (WT), Winnie (Win), and Eeyore (Eey) mice at 6 (Eey 6–9 wk), 12, and 18 (WT and Win only) wk of age, n = 4–9; box plots show median, quartiles, and range. (C) Histological colitis scores (see Materials and Methods) in WT and Win mice at 6, 12, and 18 wk of age, n = 4–6; scores from individual mice are shown. (D–K) Histology of normal distal colon from a C57BL/6 mouse (D and E) and examples of inflammation in the rectum (F–I) and distal large intestine (J and K) of untreated Winnie mice showing leukocytic infiltration (G and I), occasional branching crypts (F and H), crypt abscesses (J and K) and focal ulcerations (I); scale bars = 20 μm. Note the layer covering the mucosal surface in (F) is a granulocytic serous exudate. Statistics (B and C): p-values for Kruskal-Wallis nonparametric analysis are shown, Dunn's multiple comparison test versus wild type, ** p 15 kb) to express in vitro. Therefore, to ascertain whether the Winnie mutation affects biosynthesis of Muc2, we cloned partial cDNAs encoding the Winnie and wild-type Muc2 N-terminal D3 oligomerisation domain (rMuc2-D3, Figure 2C) and expressed these as recombinant proteins in MKN45 gastric cancer cells which produce the MUC5AC mucin (but not MUC2) and are therefore likely to express mucin-specific chaperones. Western blotting of cell lysates demonstrated increased oligomerisation of the D3 proteins carrying the Winnie mutation (Figure 7). Wild-type D3 proteins were present intracellularly mainly as monomers and dimers, with a small amount of tetramer (11%). In contrast, 61% of Winnie D3 proteins were in the form of a ladder of higher-order oligomers. Wild-type D3 proteins were secreted from MKN45 cells almost exclusively as dimers with a small tetramer band, whereas, despite production intracellularly, Winnie D3 proteins were not secreted, thus confirming the mutation leads to a defect in biosynthesis or secretion. Figure 7 Altered Muc2 N-Terminal Oligomerisation Caused by the Win Mutation (A) PAGE/Western blotting analysis of oligomerisation of the rMuc2-D3 wild-type proteins and proteins with the Win mutation in MKN45 cells and secretions following transfection (n = 4 separate transfections for each group). The position of the origin (o) and markers are shown. A reduced sample (D3 Red) on each gel shows the migration of the D3 monomer. Recombinant proteins were detected with the M2 anti-FLAG antibody, and no reactivity was seen with untransfected cells. Note the hyperoligomerisation of the Winnie D3 domain intracellularly and its failure to be secreted. (B) Densitometric analysis of oligomerisation of the rMuc2-D3 Winnie and wild-type proteins in MKN-45 cellular lysates following transfection. Relative expression of monomer, dimer, and higher-order oligomers was determined by densitometry and expressed as a percentage of the total densitometric value for each sample (lane). Statistics: individual data points and p-values from Mann-Whitney U-tests shown. Muc2 Mutations Lead to Accumulation in the ER Both Muc2 mutations result in a reduction of Muc2 secretion and altered oligomerisation, but the presence of cytoplasmic accumulations of Muc2 precursor raised the possibility of additional pathogenic effects. Since alterations resulting from point mutations can result in functional and sometimes toxic effects not observed when the protein is absent, such effects could contribute to the phenotype of Winnie and Eeyore and explain differences of these mutants from Muc2 −/− mice. To this end, we next examined both mutant strains for ultrastructural changes. Resin sections revealed that Winnie and Eeyore small and large intestines contained large numbers of cells distended with membranous vacuolar material, corresponding with the areas in which Muc2 precursor was detected by confocal microscopy. Vacuolisation was more frequent in the large intestine, and vacuolated cells usually contained stored mucin granules in thecae, demonstrating that they were goblet cells, although these thecae were smaller than in wild-type goblet cells (Figure 8A, 8E, 8I, 8M, 8P, and 8S). By transmission EM, goblet cells possessed dilated rough ER containing aggregates of variable electron density, which is the ultrastructural appearance of protein misfolding and ER stress (Figures 8F–8H, 8J–8L, 8Q, 8R, 8T, and 8U). The size and extent of these ER accumulations varied; cells with more were substantially distended, usually lacked stored mucin granules and often contained swollen mitochondria with disrupted cristae (Figure 8V). The two distinct lineages of mucus-producing cells in the proximal colon both showed vacuolisation, although vacuoles were more extensive in the longer-lived lineage that resides in the base of the crypts [32]. Some vacuoles were surrounded by membranes lacking ribosomes, possibly representing accumulations in the Golgi apparatus. Vacuole accumulation was restricted to Muc2-producing cells, with most nongoblet epithelial cells showing normal ultrastructure. However, some enterocytes and Paneth cells also contained ER vacuoles, although these vacuoles were fewer and smaller than in goblet cells, consistent with the comparatively low levels of Muc2 production that we observed in these cells by immunohistochemistry (Figure 8X). Figure 8 Ultrastructural Evidence of Endoplasmic Reticulum Stress in the Intestinal Epithelium of Mice with Muc2 Mutations Semi-thin sections from resin embedded large intestine of wild-type (A), Winnie (E), and Eeyore (I) mice and small intestine of wild-type (M), Winnie (P), and Eeyore (S) mice stained with toluidine blue. Transmission electron micrographs from the large intestine (B–D, F–H, J–L, and V) and small intestine (N, O, Q, R, T, U, W, and X) of wild-type (B–D, N, and O), Winnie (F–H, Q, R, and V), and Eeyore (J–L, T, and U) mice. Note the reduced size of goblet cell thecae (indicated by a T) containing stored mucin granules and the presence of vacuoles (indicated by a V) surrounded by rough endoplasmic reticulum (RER) in Winnie and Eeyore. Other abbreviations: G, Paneth cell granule; GA, Golgi apparatus; L, lumen. Scale bars 40 μm (A, E, and I), 20 μm (L, O, and R), 5 μm (B, F, J, and U), 2 μm (C, G, K, M, P, S, and V), 1 μm (W), 50 nm (D, H, L, N, Q, and T). Given the increased susceptibility of heterozygous mice to DSS, to assess whether one mutant allele could initiate ER stress we examined heterozygous mutants. These mice lacked the diarrhoea phenotype, and their intestinal morphology appeared normal by standard light microscopy. However, glutaraldehyde fixation and resin embedding revealed variable, though often extensive, vacuolisation in goblet cells deep in the crypts of the proximal colon (Figure 9), demonstrating subclinical pathology and establishing that these mutations are not simple mendelian recessive traits. In heterozygotes the surface goblet cell lineage in the proximal colon and the morphologically similar goblet cells in the distal colon and small intestine were mostly free of vacuolisation (Figure 9). However, vacuoles were present in some Paneth cells in the small intestine despite their lower level of Muc2 production, which may be due to the extended lifespan of these cells (unpublished data). Figure 9 Evidence of Mucin Misfolding in Heterozygous Mice Intestinal tissue from mice heterozygous for the Win mutation (Win/+) was fixed with glutaraldehyde, embedded in resin, and semi-thin sections stained with toluidine blue. Note the variable degree of vacuolisation of the goblet cells in the base of the crypts in the proximal large intestine (solid white arrows) and the lack of/minimal vacuolisation in surface goblet cells in the proximal large intestine (hollow arrows), distal large intestine and small intestinal villi. Tissue from wild-type C57BL6 mice is shown for comparison. Scale bars = 20 μm. Accumulation of nonglycosylated Muc2 precursor (Figure 6C), altered electrophoretic migration (Figure 6A), ER vacuolisation (Figure 8), and the hyperoligomerisation of the Winnie D3 domain (Figure 7) together demonstrate that a proportion (but not all) of Muc2 produced in Winnie and Eeyore goblet cells is inappropriately assembled, accumulates in the ER, and leads to ER vacuolisation, smaller goblet cell thecae, and reduced secretion of mature Muc2. It appears that at some stage of their life cycle these goblet cells cease producing mature mucin, whilst still retaining Muc2 precursor in ER vacuoles (see middle right image of large intestine in Figure 6C). Aberrant Mucin Assembly Triggers an ER Stress Response GRP78 is a heat shock protein that chaperones proteins in the ER during folding and is up-regulated in ER stress when it accumulates with misfolded proteins, ensuring they do not exit the ER normally but are removed for degradation [33]. Quantitative PCR showed a 2- to 3-fold increase in Hspa5 (which encodes GRP78) mRNA in Winnie and Eeyore proximal and distal colon (Figure 10A). Protein misfolding engages a series of molecular events that result in reduced protein translation and transcription of genes involved in the unfolded protein response (UPR) [34]. Splicing of the Xbp-1 mRNA by the enzyme IRE-1 is one key component of the UPR and we observed significantly decreased levels of the unspliced form of Xbp-1 in the proximal colon of Winnie and increased levels of the spliced form of Xbp-1 in the proximal colon of Eeyore mice (Figure 10A). The ratio of spliced to unspliced Xbp-1, which is used as a measure of UPR activation [35], increased 2.5- and 2.7-fold in Winnie and Eeyore proximal colon, respectively. Western blotting demonstrated increased GRP78 protein in both Winnie and Eeyore intestinal tissue (Figure 10B). In goblet cells from mutant mice GRP78 was detected immunohistochemically within the membranous accumulations, consistent with an association with misfolded Muc2, but notably not within thecae containing mucin packaged in granules for secretion (Figure 10C). Analysis of the Winnie and Eeyore intestinal transcriptome demonstrated increased transcription of the genes encoding the GRP78 and GRP94 chaperones, the γ-subunit of the SEC61 pore through which misfolded proteins exit the ER, ubiquitin peptidases, and key elements of Ca2+ metabolism and signalling pathways consistent with disrupted Ca2+ sequestration ensuing from ER stress (see Table S2). Figure 10 Evidence of ER Stress and UPR Activation in Mice with Muc2 Mutations (A) mRNA expression of hspa5 (GRP78) and the unspliced and spliced forms of Xbp-1 in the proximal and distal colon of wild-type C57BL/6 (WT), Winnie (Win), and Eeyore (Eey) mice determined by quantitative PCR and expressed relative to β-actin with the mean ratio for the WT group in each tissue corrected to 1. Statistics: n = 3 individual data points shown, Kruskal-Wallis nonparametric analysis (p-values shown) with Dunn's multiple comparison test (*p 2 Winnie:wild type, green for 0.5 and <2.0. The entire microarray dataset can be accessed at NCBI GEO Accession No. GSE9913 (http://www.ncbi.nlm.nih.gov/geo/). (57 KB PDF) Click here for additional data file. Figure S7 Evidence of MUC2 Precursor Accumulation in Ulcerative Colitis Individual confocal images of the composite confocal images shown in Figure 12B. Staining with the MUC2 precursor antibody 4F1, DBA lectin, and DAPI as indicated. Scale bars = 10 μm. (560 KB PDF) Click here for additional data file. Figure S8 Morphological Evidence of ER Stress in Ulcerative Colitis Intestinal biopsies from two healthy individuals, the unaffected proximal colon of three UC patients, and the affected distal colon of one of those UC patients, were examined by light (left) and electron (right) microscopy. Arrows indicate the presence of vacuoles in goblet cells. Abbreviations: NG, nongranular material; RER, rough endoplasmic reticulum; T, theca/mucin granules; V, vacuole. Scale bars are individually annotated. (2.9 MB PDF) Click here for additional data file. Table S1 Histological Scoring of Murine Colitis (67 KB DOC) Click here for additional data file. Table S2 Comparison of the Intestinal Transcriptome of C57BL/6, Winnie and Eeyore Mice—Genes Involved in ER Stress, Antimicrobial Defence, Wound Repair, Epithelial Growth, Cell Cycle, and Apoptosis (61 KB DOC) Click here for additional data file. Table S3 Comparison of the Intestinal Transcriptome of C57BL/6, Winnie and Eeyore Mice—Genes Involved in Inflammation, Metabolism, Detoxification, and the Mucus Barrier (60 KB DOC) Click here for additional data file.

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Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn's disease involving the ileum.

Martin Baumgart, Belgin Dogan, Mark Rishniw … (2007)

Intestinal bacteria are implicated increasingly as a pivotal factor in the development of Crohn's disease, but the specific components of the complex polymicrobial enteric environment driving the inflammatory response are unresolved. This study addresses the role of the ileal mucosa-associated microflora in Crohn's disease. A combination of culture-independent analysis of bacterial diversity (16S rDNA library analysis, quantitative PCR and fluorescence in situ hybridization) and molecular characterization of cultured bacteria was used to examine the ileal mucosa-associated flora of patients with Crohn's disease involving the ileum (13), Crohn's disease restricted to the colon (CCD) (8) and healthy individuals (7). Analysis of 16S rDNA libraries constructed from ileal mucosa yielded nine clades that segregated according to their origin (P<0.0001). 16S rDNA libraries of ileitis mucosa were enriched in sequences for Escherichia coli (P<0.001), but relatively depleted in a subset of Clostridiales (P<0.05). PCR of mucosal DNA was negative for Mycobacterium avium subspecies paratuberculosis, Shigella and Listeria. The number of E. coli in situ correlated with the severity of ileal disease (rho 0.621, P<0.001) and invasive E. coli was restricted to inflamed mucosa. E. coli strains isolated from the ileum were predominantly novel in phylogeny, displayed pathogen-like behavior in vitro and harbored chromosomal and episomal elements similar to those described in extraintestinal pathogenic E. coli and pathogenic Enterobacteriaceae. These data establish that dysbiosis of the ileal mucosa-associated flora correlates with an ileal Crohn's disease (ICD) phenotype, and raise the possibility that a selective increase in a novel group of invasive E. coli is involved in the etiopathogenesis to Crohn's disease involving the ileum.

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Long-Distance Delivery of Bacterial Virulence Factors by Pseudomonas aeruginosa Outer Membrane Vesicles

Jennifer M. Bomberger, Daniel MacEachran, Bonita Coutermarsh … (2009)

Introduction Nosocomial infections contribute $4.5 billion to annual healthcare costs in this country alone, with an estimated 2 million nosocomial infections occurring in the US annually, resulting in 99,000 deaths [1]. Many of these nosocomial infections are caused by Gram-negative pathogens, and interaction of these pathogens with the host is often mediated by secreted virulence factors. Bacteria have evolved mechanisms for the secretion of virulence factors into the host cell to alter host cell biology and enable bacterial colonization, and these mechanisms typically require that bacteria be in intimate contact with the host. For example, the Type III secretion system (T3SS) and Type IV secretion system (T4SS) deliver proteins directly into the host cytoplasm from an extracellular bacterial pathogen's cytoplasm [2] utilizing transport machines that act as macromolecular syringes [3]. Delivery of extracellular bacteria or bacterial products can also occur via endocytosis initially into the lumen of the host endocytic compartment, then movement to the host cytoplasm via lysis of the endocytic compartment or delivery of the proteins across the endocytic membrane via the Type III Secretion System (T3SS) [3]. For several decades, work by Beveridge's group has characterized bacterial-derived outer membrane vesicles (OMV) to be a novel secretion mechanism employed by bacteria to deliver various bacterial proteins and lipids into host cells, eliminating the need for bacterial contact with the host cell [4]–[7]. OMV are 50–200 nm proteoliposomes constitutively released from pathogenic and non-pathogenic species of Gram-negative bacteria [8],[9]. Biochemical and proteomic analyses have revealed that OMV are comprised of lipopolysaccharide, phospholipids, outer membrane proteins, and soluble periplasmic proteins [8],[9]. Many virulence factors that are periplasmic proteins are enriched in OMV, for example, Escherichia coli cytolysin A (ClyA), enterotoxigenic E. coli heat labile enterotoxin (LT), and Actinobacillus actinomycetemcomitans leukotoxin [10]–[12]. Beveridge's group and others have reported that some secreted virulence factors from P. aeruginosa, including β-lactamase, hemolytic phospholipase C, alkaline phosphatase, pro-elastase, hemolysin, and quorum sensing molecules, like N-(3-oxo-dodecanoyl) homoserine lactone and 2-heptyl-3-hydroxy-4-quinolone (PQS) [6],[7],[13],[14], are also associated with P. aeruginosa OMV [8],[9]. Whether these secreted virulence factors packaged in OMV are eventually delivered to the host and the mechanism by which this occurs is currently unknown. A recent study suggested that E. coli OMV fuse with lipid rafts in the host colonic epithelial cell, but the delivery and intracellular trafficking of the OMV cargo was not characterized [15]. Thus, we investigated the possibility that OMV deliver multiple secreted virulence factors into the host cell through a lipid raft-mediated pathway, eliminating the need for intimate contact of the pathogen with the host. Results Outer membrane vesicles deliver toxins to airway epithelial cells Based on reports that multiple virulence factors are packaged in OMV, we hypothesized that these virulence factors could be simultaneously delivered in a coordinated manner in OMV to the host cell by the microbe. We tracked four P. aeruginosa secreted factors, including alkaline phosphatase, β-lactamase, hemolytic phospholipase C, and Cif, previously reported to be packaged in OMV [6],[7],[13],[14],[16]. We chose these secreted virulence factors because they play important roles in host colonization, for example alkaline phosphatase promotes biofilm formation [17],[18], β-lactamase degrades host antimicrobial peptides, hemolytic phospholipase C is cytotoxic and promotes P. aeruginosa virulence [19], and Cif is a recently characterized toxin that inhibits CFTR-mediated chloride secretion in the airways [16] and thereby likely reduces mucociliary clearance. To purify OMV from bacterial products not packaged in OMV, like pilus, that may also elicit a host response, we modified a published protocol [14] utilizing high-speed differential centrifugation and density gradient fractionation to isolate OMV from an overnight P. aeruginosa culture supernatant. The Cif protein, as well as a protein present in the membrane of OMV, Omp85 [20], were identified in purified bacterial-derived OMV (Figure S1). When airway cells were treated with isolated and purified OMV for ten minutes all four OMV proteins examined were detected in host airway epithelial cell lysate (Figure 1A). By contrast, these virulence factors were not detected in lysates of control cells treated with vehicle (Figure 1A). Therefore, OMV deliver multiple virulence factors to host airway epithelial cells in the absence of bacteria, thus providing a mechanism for bacteria to alter host cell physiology without the need for intimate contact with the host. 10.1371/journal.ppat.1000382.g001 Figure 1 OMV deliver multiple toxins to induce cytotoxicity in airway epithelial cells. (A) Purified P. aeruginosa OMV deliver multiple virulence factors (Cif, alkaline phosphatase, β-lactamase, and PlcH) into the cytoplasm of airway epithelial cells. Western blot analysis of cell lysates of airway epithelial cells treated with purified OMV. CTRL, no OMV added. Δcif OMV applied to airway epithelial cells, cells lysed, and probed by Western blot analysis for Cif did not detect Cif in airway cell lysates (data not shown). (B) OMV elicit time-dependent cytotoxicity in host airway cells as determined by the CellTiter 96 AQueous One cytotoxicity assay. (C) OMV elicit time-dependent cytotoxicity in host airway cells that is not dependent on the presence of Cif, as determined by the CellTiter 96 AQueous One cytotoxicity assay. Δcif OMV, OMV purified from a strain of P. aeruginosa lacking the cif gene. Black bars, wild-type OMV (WT OMV); white bars, Δcif OMV. (D) Airway epithelial cells treated with lysed OMV components demonstrated a reduction in cellular cytotoxicity as determined by the CellTiter 96 AQueous One cytotoxicity assay. Data are presented as mean+/−SEM. n = 3, * p<0.05, OMV versus Control, intact OMV versus lysed OMV. To explore the significance of OMV in the delivery of virulence factors into the host cytoplasm, we examined the cytotoxic effect of P. aeruginosa OMV on host airway cells using the CellTiter 96 AQueous One cytotoxicity assay. OMV were cytotoxic after a delay of 8 hours (Figure 1B), although virulence factors could be detected in the cytoplasm of host cells after 10 minutes (Figure 1A). The time-dependent increase in cytotoxicity induced by OMV was not dependent on Cif expression in OMV, given that Δcif OMV did not produce a statistically significant difference in cytotoxicity compared to wild-type OMV (Figure 1C). To determine if intact OMV are required for cytotoxicity, purified OMV were lysed with 0.1 M EDTA and the lysate was applied to airway epithelial cells for 8 h (Figure S2, Figure 1D). This method was previously employed by Horstman et al. to effectively lyse E. coli OMV [21]. The lysed OMV did not have a cytotoxic effect on airway epithelial cells, demonstrating that cytotoxicity is mediated by virulence factors delivered into the host cell cytoplasm by bacterial-derived OMV (Figure 1D). In the next series of experiments we began to examine the mechanism whereby OMV deliver virulence factors into the cytoplasm of the host airway epithelial cell. Previously we reported that purified, recombinant Cif, a virulence factor secreted in OMV by P. aeruginosa, is necessary and sufficient to reduce apical membrane expression of CFTR and P-glycoprotein (Pgp) in human airway epithelial cells [16],[22], thus reducing mucociliary clearance and xenobiotic resistance of the host cells, respectively. In the current study we use Cif as a model protein to investigate how OMV deliver virulence factors into the cytoplasm of human airway epithelial cells. First, experiments were conducted to confirm our previous observation that Cif is secreted in purified OMV and second, to determine if Cif is an intravesicular component of OMV. Cif was detected in OMV derived from P. aeruginosa expressing the cif gene, but not in OMV derived from P. aeruginosa in which the cif gene was deleted (Figure S3). The inability of Proteinase K (0.1 µg/ml), which does not enter the lumen of OMV, to degrade OMV-associated Cif indicates that this virulence factor is an intravesicular component of OMV (Figure S3). Thus, these studies demonstrate that Cif maintains an intravesicular localization in purified OMV. Next, studies were conducted to determine if the Cif virulence factor packaged in OMV was functional when delivered to airway epithelial cells. Cif function was measured by examining the ability of Cif to reduce apical plasma membrane CFTR abundance in airway epithelial cells. Purified OMV containing Cif reduced apical plasma membrane CFTR in a time-dependent manner (Figure 2A), whereas purified OMV from P. aeruginosa deleted for the cif gene had no effect on CFTR membrane expression (Figure 2B). Taken together these studies confirm and extend our previous observations that OMV-packaged Cif reduces plasma membrane CFTR. 10.1371/journal.ppat.1000382.g002 Figure 2 OMV deliver functional Cif virulence factor to airway epithelial cells, across a mucus layer. (A) Purified OMV applied to the apical surface of airway epithelial cells reduce CFTR in the plasma membrane in a time-dependent manner, compared to the buffer control (Control), as measured by Western blot analysis. Overnight supernatant (PA14 O/N Sup), previously reported to decrease apical membrane CFTR, serves as a positive control [16]. (B) OMV purified from a P. aeruginosa Δcif mutant strain did not reduce apical membrane expression of CFTR, as measured by Western blot analysis. Black bar, wild-type OMV; striped bar, Δcif OMV. (C) Cif-containing OMV decrease apical membrane CFTR in airway epithelial cells (CFBE) and mucous-producing airway epithelial cells (Calu-3), as measured by Western blot analysis. Black bar, CFBE cells; striped bar, Calu-3 cells. Data are presented as mean+/−SEM. n = 3, * p<0.05 versus Control. If OMV modulate host physiology of lung cells without direct bacteria-host contact, we would predict that OMV secreted by bacteria should overcome barriers such as the mucus overlying human airway cells [23]. OMV containing Cif reduced CFTR in the apical plasma membrane of airway epithelial cells that have a thick layer of mucus on the apical surface (Calu-3 cells, Figure 2C). A delay in the Cif-mediated reduction of apical membrane CFTR abundance was observed in Calu-3 cells, compared to airway cells that lack a overlying mucus layer, suggesting the mucus only delays OMV from diffusing to host airway epithelial cells. Thus, OMV allow the long distance delivery of secreted bacterial factors to the host cell in the absence of direct bacteria-host contact. Host cell detergent-resistant membranes (aka, lipid rafts) are required for OMV fusion and toxin delivery We next explored the mechanism whereby OMV deliver bacterial proteins into the host cell using the Cif virulence factor as a model. Based on a recent study showing that Filipin III disrupts E. coli OMV association with host cells [15], we hypothesized that OMV deliver secreted bacterial proteins to host cells by fusing with lipid raft microdomains. Five minutes after addition of OMV to epithelial cells, Cif, as well as a protein documented to be associated with OMV, Omp85 [20], were detected in membrane lipid raft fractions (Figure 3A). The lipid raft (i.e. detergent insoluble membranes) fractions were separated with density gradient fractionation and characterized by labeling with the flotillin-1 antibody. The fusion of OMV with membrane rafts was observed visually by confocal microscopy using cholera toxin B subunit (labeled with FITC), a documented lipid raft marker [24], which co-localized with rhodamine-R18 labeled OMV five minutes after OMV were added to the apical side of airway cells (Figure 3B). The rhodamine-R18 dye is quenched when loaded in bilayer membranes at a high concentration and is subsequently dequenched, fluorescing in the red channel upon membrane fusion, which allows dilution of the probe and fluorescence detection. Pearson's correlation and Mander's overlap coefficients demonstrated a high degree of co-localization of cholera toxin B subunit and OMV (0.771+/−0.018 and 0.952+/−0.012 versus control levels of 0.153+/−0.026 and 0.259+/−0.026, respectively, p<0.0001). In further support that Cif-containing OMV fuse with lipid rafts, Cif co-immunoprecipitated with the glycosylphosphatidylinisotol-anchored protein p137 [25], a documented lipid raft-associated protein, from the lipid raft fraction of airway cells that had been treated with OMV for five minutes (Figure 3C). 10.1371/journal.ppat.1000382.g003 Figure 3 Cif-containing OMV fuse with the epithelial cell lipid raft microdomains. (A) Cif virulence factor and Omp85 localize to the lipid raft fraction. Omp85, a documented OMV protein, and Cif virulence factor co-fractionate with the lipid raft marker, flotillin-1, when raft and non-raft fractions are isolated from airway epithelial cells treated with OMV for 5 min, as measured by Western blot analysis. Right blot represents raft and non-raft fractions from the left blot, which are combined and the proteins precipitated to concentrate Cif signal to allow antibody detection. (B) Rhodamine R18-labeled OMV, which only fluoresce red upon fusion with the host plasma membrane, co-localize with the FITC-conjugated lipid raft marker cholera toxin B subunit (CtxB). All OMV co-localize with lipid rafts with the exception of five OMV that are indicated with small white arrows (see lower right panel). Scale bar equals 10 µm. (C) Cif virulence factor interacts with GPIp137 in the lipid raft fraction. Immunoprecipitation (IP) experiments demonstrate that the GPI-anchored protein p137 (GPIp137), a lipid raft marker, interacts with Cif in the lipid raft fractions of airway epithelial cells treated with wild-type OMV for 5 min. Serving as a negative control, cif mutant OMV (Δcif OMV)–treated airway epithelial cells cannot immunoprecipitate Cif and therefore do not demonstrate immunoprecipitation of GPIp137. 5% of total lysate run in Lysate lane. IB, immunoblot. Experiments repeated three times; representative blots/images are shown. To determine if host cell lipid raft microdomains are required for OMV fusion, the cholesterol-sequestering agent Filipin III complex was used to disrupt lipid raft domains and OMV fusion was assessed. The rhodamine-R18 dye was utilized to allow visualization and quantitation of OMV fusion with host cells. Rhodamine-R18 only fluoresces upon OMV fusion to host cells, thus an increase in fluorescence is interpreted as an increase in OMV fusion. Rhodamine-R18 labeled OMV applied to the apical membrane of airway epithelial cells produced a time-dependent increase in fluorescence (Figure 4A). In contrast, the fluorescence did not increase above background levels in samples containing only airway epithelial cells or only rhodamine-R18 labeled-OMV (Figure 4A). Filipin III eliminated the fusion of OMV with epithelial cells, indicated by a lack of fluorescence detected when compared to control epithelial cells (Figure 4B). Microscopy studies were confirmed by the quantitative, fluorescence-based assay, described in Figure 4A, which also demonstrated that OMV fusion to the host cell was blocked with Filipin III pretreatment of the host cells (Figure 4C). 10.1371/journal.ppat.1000382.g004 Figure 4 Disruption of lipid rafts blocks virulence factor delivery and function in airway epithelial cells. (A) Rhodamine-R18 labeled OMV were applied to the apical side of polarized airway epithelial cells, and fluorescence was measured over time as readout for OMV fusion. Rhodamine-R18 signal is quenched at high concentration in OMV, but fluorescence increases as the rhodamine probe is diluted by fusion of the OMV with the host plasma membrane. OMV and cells alone serve as negative controls where the fluorescence does not change over time (background fluorescence). Black square, OMV+cells; black triangle, OMV alone; black inverted triangle, cells alone. Data are presented as mean fluorescence intensity. (B,C) Visualization and quantitative assay demonstrating that disruption of lipid rafts with the cholesterol-sequestering reagent, filipin III complex (10 µg/ml), inhibits OMV fusion with airway epithelial cells. Rhodamine-R18 labeled OMV were applied to the apical side of polarized airway epithelial cells in the presence or absence of filipin III, and fluorescence was measured over time. Wheat germ agglutin (WGA-blue) is a marker of host epithelial cell membranes. Scale bars equal 10 µm. Black square, control; black triangle, filipin III. (D) Filipin III prevents delivery of Cif to host cell endosomes, as measured by cytoplasmic and endosomal fractionation and Western blot analysis. Rab5 GTPase and actin are endosomal and cytoplasmic markers, respectively. Cyto, cytoplasm; Endo, endosome. (E) Disruption of lipid rafts blocks Cif-mediated reduction in apical membrane CFTR expression, compared to buffer control (Control), as measured by Western blot analysis. “Centrifuge Sup” refers to the overnight culture supernatant of P. aeruginosa PA14 depleted of OMV, which serves as a negative control in this experiment. (F) Filipin III complex disruption of host cell lipid raft microdomains reduces the cytotoxic effect of OMV on host airway cells with 8 h OMV treatment, as measured with the CellTiter 96 AQueous One cytotoxicity assay. Data are presented as mean+/−SEM. n = 3, * p<0.05, OMV versus Control; # p<0.05, OMV versus OMV+Filipin III. We next tracked the Cif virulence factor biochemically to determine if lipid raft microdomains are required for virulence factor delivery and function in host cells. Five minutes after OMV are exposed to host airway epithelial cells, the Cif virulence factor is detected by Western blot analysis in the endosomal sub-fraction of the host cell lysate, but not in the cytoplasmic fraction (Figure 4D). The Filipin III complex prevented the appearance of Cif in the endosomal fraction of host cells (Figure 4D) and blocked the ability of Cif to reduce apical membrane CFTR (Figure 4E), demonstrating a requirement for the host lipid raft machinery for Cif delivery and function in host cells. Furthermore, disruption of lipid raft microdomains with Filipin III, and thus blocking OMV fusion with airway epithelial cells, reduced the cytotoxicity induced in the airway epithelial cells with 8 h of OMV treatment (Figure 4F). Thus, lipid raft microdomains are required for OMV-mediated delivery and function of secreted virulence factors in host cells. Actin cytoskeleton is required for OMV fusion, toxin entry, and function The actin cytoskeleton, in particular the neuronal WASP (N-WASP)–initiated actin assembly, is critical to the internalization of select lipid raft-associated cargo [26],[27]. Based on these previous studies, we investigated the role of the actin cytoskeleton, in general, and N-WASP-mediated cytoskeletal rearrangements specifically, in OMV fusion to the plasma membrane of the airway epithelial cell. Both cytochalasin D (an actin monomer-sequestering agent) and wiskostatin (an inhibitor of neuronal WASP (N-WASP) induced actin polymerization) disrupted the actin cytoskeleton in airway epithelial cells (Figure 5A), resulting in a loss of OMV fusion (Figure 5B,C), as measured by a reduction in OMV-dependent fluorescence in airway cells pretreated with cytochalasin D or wiskostatin. Thus, wiskostatin and cytochalasin blocked OMV fusion to human airway epithelial cells, demonstrating a need for N-WASP induced actin polymerization for OMV fusion to host cells. 10.1371/journal.ppat.1000382.g005 Figure 5 Intact N-WASP–induced actin cytoskeleton is required for OMV fusion and virulence factor delivery. (A) Wiskostatin and cytochalasin D disrupt actin cytoskeleton in airway cells. Alexa 647–conjugated phalloidin was used to label actin to monitor disruption of the actin cytoskeleton by two actin-disrupting agents, wiskostatin (Wisko, 10 µM for 30 min) and cytochalasin D (Cyto D, 2 µM for 45 min), by immunofluorescence microscopy. Scale bar equal to 20 µm. (B,C) Visualization and quantitative assay demonstrating that disruption of the actin cytoskeleton with either of the two actin-disrupting agents, wiskostatin (Wisko, 10 µM) or cytochalasin D (Cyto D, 2 µM), inhibits OMV fusion with airway epithelial cells. Rhodamine-R18 labeled OMV were applied to the apical side of polarized airway epithelial cells in the presence or absence of wiskostatin or cytochalasin D, and fluorescence was measured over time. Scale bar equals 10 µm (B). Representative images of three experiments revealing that OMV do not fuse with cells in which actin has been disrupted. WGA, wheat germ agglutin (blue) to label the cell surface. Black square, control; black triangle, Cytochalasin D; open circle, Wiskostatin. * p<0.05 (Control versus Cytochalasin D and Wiskostatin). (D) Wiskostatin or cytochalasin D treatment of airway epithelial cells prevents delivery of Cif virulence factor to endosomal fraction, as measured by cytoplasm and endosome fractionation and Western blot analysis. Rab5 GTPase and actin are endosomal and cytoplasmic markers, respectively. Cyto, cytoplasm; Endo, endosome. (E) Disruption of the actin cytoskeleton with wiskostatin blocks the Cif virulence factor–mediated reduction in CFTR apical membrane expression in airway epithelial cells, as measured by Western blot analysis. Data are presented as mean+/−SEM. n = 3, * p<0.05 versus Control. Wiskostatin and cytochalasin D were also utilized to determine if the actin cytoskeleton is required for OMV delivery of Cif to airway epithelial cells. Cytoplasmic and endosomal fractions were purified from airway epithelial cells pretreated with vehicle, cytochalasin D or wiskostatin in the presence or absence of Cif-containing OMV. In control cells Cif localized to the endosomal fraction, as described above, whereas cytochalasin D and wiskostatin (Figure 5D) blocked the entry of Cif into the endosomal and cytoplasmic fractions. Furthermore, wiskostatin pretreatment blocked the Cif toxin-mediated reduction of CFTR from the apical membrane of airway epithelial cells (Figure 5E). Because cytochalasin D changes the rate of CFTR endocytosis, we cannot assess the effects of this inhibitor on Cif virulence factor-mediated reduction of CFTR. In addition, the purified Cif virulence factor alone did not induce morphological changes to the actin cytoskeleton (data not shown). These results reveal that Cif does not alter the cytoskeleton and establishes the requirement for an intact actin cytoskeleton, specifically N-WASP-mediated actin polymerization, for OMV fusion and virulence factor delivery to the host airway epithelial cells. Cif toxin is localized to the cytoplasmic face of early endosomes after entry into host cell The data above strongly suggest that OMV deliver the Cif virulence factor into the interior of the host cell and allow this virulence factor to associate with an endosomal compartment (Figures 1A, 4D and 5D). To more precisely identify which endosomal compartment was the target of Cif, OMV were applied apically to airway cells for ten minutes, the airway cells were lysed and endosomes were purified by differential centrifugation. From the purified endosomal fraction, Cif co-immunoprecipitated with Rab5 GTPase, a marker of early endosomes, and the early endosomal antigen (EEA)-1 (Figure 6A). In contrast, Cif did not co-immunoprecipitate with Rab4 (a marker of sorting endosomes), Rab7 (a marker of late endosomes) or Rab11 (a marker of recycling endosomes) (Figure S4). Proteinase K, which does not degrade luminal endosomal proteins but can degrade proteins on the cytoplasmic face of endosomes, eliminated Cif from the endosomal fraction (Figure 6B). As expected, proteinase K did not affect the endosomal association of the transferrin receptor, a luminal endosomal protein that is resistant to proteinase K treatment [28]. However, the transferrin receptor was not resistant to proteinase K degradation in the presence of 0.1% Triton X-100, which disrupts the endosomal membrane and allows proteinase K access to luminal endosomal proteins (Figure 6B). These data reveal that OMV-delivered Cif is localized to the cytoplasmic face of the early endosomes after entry into the epithelial cell. 10.1371/journal.ppat.1000382.g006 Figure 6 Cif virulence factor is delivered to the cytoplasm and localizes to the cytoplasmic face of early endosomes. (A) Cif localizes to the early endosomal (Rab5 GTPase, early endosomal antigen (EEA)-1 labeled) compartment after entry into airway epithelial cells. Airway epithelial cells were treated with OMV for 10 min, cells lysed, and endosomes were purified. Cif was immunoprecipitated from the endosomal fraction, and Western blot analysis was performed for Rab5 GTPase and EEA-1, early endosomal markers. IgG IP is a non-immune control immunoprecipitation experiment. (B) The ability of proteinase K (PK) to degrade Cif from the early endosomal fraction reveals that the Cif virulence factor localizes to the cytoplasmic face of early endosomes, as measured by Western blot analysis. The transferrin receptor serves as a control for a luminal endosomal protein marker, which is only exposed to proteinase K after treatment with Triton X-100 (TX). (C) Cif entry into airway epithelial cells is not altered by disruption of endosomal acidification by NH4Cl (5 mM). Rab5 GTPase and actin are endosomal and cytoplasmic markers, respectively. (D) Entry of Cif virulence factor into airway cells is unaffected by inhibition of retrograde transport with Brefeldin A (1 µM), as measured by Western blot analysis. Rab5 GTPase and actin are endosomal and cytoplasmic markers, respectively. (E) Retrograde transport to the endoplasmic reticulum is not required for Cif to reduce plasma membrane CFTR in airway cells. Airway epithelial cells pretreated with Brefeldin A (1 µM), which inhibits retrograde transport, were treated with OMV. Brefeldin A had no effect on the ability of Cif to reduce plasma membrane CFTR, as measured by Western blot analysis. Data are presented as mean+/−SEM. n = 3, * p<0.05 versus Control. To determine if OMV-delivered Cif enters the host cell cytoplasm by penetrating the membrane of endosomal vesicles, cells were treated with ammonium chloride, a lysosomotropic drug that inhibits vesicle acidification, and thereby inhibits the movement of virulence factors from endosomal vesicles into the cytoplasm [3]. Ammonium chloride had no effect on the ability of Proteinase K to decrease the amount of Cif in the endosomal fractions (Figure 6C), indicating that the Cif virulence factor does not reach the cytoplasm via penetrating intracellular vesicular membranes. Some intracellular bacteria and virulence factors move through the retrograde pathway from endosomes, to the Golgi apparatus and endoplasmic reticulum, from which they enter the host cytoplasm. However, Brefeldin A, a pharmacologic inhibitor of retrograde transport, had no effect on the entry of the Cif into the airway cell and the appearance of Cif in the endosomal fraction (Figure 6D), or the Cif-mediated reduction in apical membrane CFTR abundance (Figure 6E). Thus, our data demonstrate that OMV deliver Cif directly to the host cytoplasm rather than requiring passage across an endosomal membrane or through the retrograde transport pathway. Interestingly, PlcH and alkaline phosphatase also localized to the endosomes after entry into the airway epithelial cells, whereas β-lactamase was detected in the cytoplasmic fraction, as determined by subcellular fractionation and Western blot analysis (data not shown). Thus, virulence factors with differing functions are distributed to different subcellular locations after entry into the host cytoplasm. OMVs are a physiological delivery mechanism for secreted virulence factors We propose that rather than secretion of virulence factors into the surrounding medium, OMV are a physiologically- and clinically-relevant mechanism utilized by Gram-negative bacterium, in particular P. aeruginosa, to deliver secreted products into the host cell. In support of this hypothesis, Cif packaged in OMV was 17,000-fold more effective than purified, recombinant Cif in reducing plasma membrane CFTR, with 3 ng of Cif in OMV- reducing plasma membrane CFTR expression as effectively as 50 µg of purified, recombinant Cif protein (Figure 7A). Cif was detected in lysates of airway epithelial cells exposed to OMV (3 ng Cif) and 50 µg of recombinant Cif (Figure 7B), but Cif was not detected in cells exposed to up to 10 ng of recombinant Cif, correlating the presence of the virulence factor inside the host cell with virulence factor function. Moreover, airway epithelial cells treated with lysed OMV (Figure S2) showed a dramatic reduction in the ability of the Cif toxin to reduce apical membrane CFTR, as compared to cells treated with intact OMV (Figure 7C). Therefore, OMV-mediated delivery of virulence factors to airway epithelial cells increases the efficacy of these virulence factors in altering host cell physiology. 10.1371/journal.ppat.1000382.g007 Figure 7 OMV required for virulence factor delivery to host airway cells. (A) OMV delivery enhances the ability of Cif to reduce plasma membrane CFTR in airway cells by 17,000-fold. Cif was applied to airway epithelial cells as 1 ng, 10 ng, 50 µg recombinant protein or 3 ng in OMV, and its effect on CFTR expression at the apical plasma membrane was measured by Western blot analysis. (B) Cif was detected in airway cells treated with 50 µg Cif or 3 ng Cif packaged in OMV. Airway epithelial cells were treated with vehicle (Control-CTRL), 1 ng, 10 ng, or 50 µg of recombinant Cif protein, or with 3 ng of Cif packaged in OMV (OMV). Cif was detected in the airway cell cytoplasm by Western blot analysis. The presence of Cif protein in the airway cell cytoplasm correlated with the ability of Cif to reduce plasma membrane CFTR (A). (C) Intact OMV required for reduction of apical membrane CFTR expression (i.e., Cif virulence factor function). Airway epithelial cells were treated with OMV or lysed OMV components, and airway epithelial cells were assayed for the Cif-mediated reduction in apical membrane CFTR, as measured by Western blot analysis. Data are presented as mean+/−SEM. n = 3, * p<0.05 versus Control. Discussion We have demonstrated that P. aeruginosa OMV deliver multiple virulence factors, simultaneously, into host airway epithelial cells via a mechanism of OMV fusion with host cell lipid raft machinery and trafficking via an N-WASP induced actin pathway to deliver OMV cargo directly to the host cytoplasm. The OMV-delivered Cif virulence factor is then localized to the cytoplasmic face of the early endosomal compartment (Figure 8). E. coli OMV association with host cells had previously been shown to be sensitive to Filipin III treatment, and thus was proposed to be lipid raft-dependent, but whether the OMV actually delivered cargo into the host cells and the mechanism by which this occurred was not characterized [15]. Fiocca et al. demonstrated that VacA packaged in OMV from H. pylori was internalized into a cytoplasmic vacuole in gastric epithelial cells, but did not investigate a mechanism [29]. We propose the first mechanism for the entry and intracellular fate of OMV-delivered bacterial virulence factors. 10.1371/journal.ppat.1000382.g008 Figure 8 Proposed model for P. aeruginosa OMV fusion with airway epithelial cells. OMV are released from P. aeruginosa, diffuse to the host cell plasma membrane, and subsequently fuse with host cell lipid raft microdomains. Virulence factors are subsequently released into the cytoplasm of the airway epithelial cell, through an actin-dependent pathway. Considering the pioneering work of Beveridge to characterize OMV and our current mechanistic studies, we propose that OMV-mediated virulence factor delivery should be considered for designation as a secretion system [4]–[7]. Like the T3SS, OMV can deliver bacterial proteins directly to the host cell cytoplasm without releasing the naked bacterial proteins into the extracellular environment where they could be degraded by secreted proteases [30]–[32]. OMV deliver fully-folded, enzymatically-active secreted virulence factors into host cells, ready for immediate action upon delivery. By delivering multiple, active OMV-packaged virulence factors, the pathogen may be able to impact the host on multiple levels. For example, simultaneously altering epithelial cell function by perturbing surfactant abundance or tight junction integrity, and the innate immune response to bacteria by stimulating pro-inflammatory cytokine production [14], [33]–[36]. Based on our studies in P. aeruginosa and published reports of OMV production by E. coli, H. pylori, A. actinomycetemcomitans, V. cholerae and N. meningitidis, it is likely that other bacteria package multiple secreted virulence factors in OMV for efficient transfer to host cells and thus, the studies proposed here likely represent a general strategy utilized by Gram-negative bacteria in their interactions with the host [10]–[12],[37],[38]. In contrast to known secretion systems, OMV-mediated direct delivery of bacterial proteins to the host can occur at a distance, and in the absence of bacteria, thus obviating the need for the pathogen to interact directly with the host cell to cause cellular cytotoxicity and alter host cell biology to promote colonization. Furthermore, OMV can deliver bacterial factors across host barriers, such as mucus layers. We believe that our work should prompt those studying bacterial pathogens to reconsider how secreted virulence factors impact host cells. That is, our data suggest that secreted virulence factors are not released individually into the surrounding milieu where they may randomly contact the surface of the host cell, but are released in a strategic manner, packaged with multiple virulence factors in OMV for coordinated delivery directly into the host cell cytoplasm. It is also possible that OMV provide a mechanism for delivering a concentrated bolus of virulence factors to the host, instead of individual toxins being delivered one at a time to the host cell. Moreover, OMV-mediated, long distance delivery of virulence factors might help explain observations such as, bacterial colonization of catheters causing systemic symptoms in kidney dialysis patients, ocular keratitis occurring in patients who do not have cultivatable pathogens, and the significant lung damage in cystic fibrosis, bronchiectasis, and chronic obstructrive pulmonary disease patients resulting from chronic infections with P. aeruginosa suspended in mucus above the airway epithelium. This mechanism of OMV-mediated protein secretion is reminiscent of the long distance delivery of signaling proteins between and among eukaryotic cells via exosomes [39], and may represent a general protein secretion strategy used by both pathogen and host. Materials and Methods Antibodies and reagents The antibodies used were: rabbit anti-Cif antibody (Covance Research Products, Denver, Pa [16]); rabbit anti-OprF antibody (a generous gift from Nobuhiko Nomura, Graduate School of Life and Environmental Sciences, University of Tsukuba); rabbit anti-pilus antibody (a generous gift from Michael Zegans, Dartmouth Medical School); goat anti-phospholipase C-H antibody (a generous gift from Michael Vasil); mouse anti-human CFTR C-terminus antibody (clone 24-1; R&D systems, Minneapolis, MN); mouse anti-CFTR antibody (clone M3A7; Upstate Biotechnology, Lake Placid, NY); mouse anti-EEA1 antibody, mouse anti-ezrin antibody, mouse anti-flotillin-1 antibody, mouse anti-Rab5 antibody, mouse anti-actin antibody (BD Biosciences, San Jose, CA); cholera toxin B subunit-FITC (Sigma-Aldrich, St. Louis, MO); rabbit anti-GPIp137 antibody (Abgent, San Diego, CA); Alexa 647-conjugated phalloidin (Molecular Probes, Carlsbad, CA); rabbit anti-Rab4 antibody, rabbit anti-Rab7 antibody (Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-Rab11 antibody, mouse anti-transferrin receptor antibody (Zymed, San Francisco, CA); mouse anti-β lactamase antibody (Novus Biologicals, Littleton, CO); rabbit anti-alkaline phosphatase antibody (GeneTex, Inc., San Antonio, TX) and horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies (Bio-Rad, Hercules, CA). Other reagents include: Filipin III complex, ammonium chloride, Optiprep, proteinase K, and cytochalasin D (Sigma-Aldrich), wiskostatin (Calbiochem, San Diego, CA), Triton X-100 (Bio-Rad, Hercules, CA). All antibodies and reagents were used at the concentrations recommended by the manufacturers or as indicated in the figure legends. Cell culture Two airway epithelial cell lines were studied to examine outer membrane vesicle fusion and toxin delivery to host epithelial cells. First, human bronchial epithelial CFBE cells (ΔF508/ΔF508) were stably transduced with WT-CFTR (generous gift from Dr. J. P. Clancy, University of Alabama at Birmingham, Birmingham, AL; hereafter referred to as airway epithelial cells) [40]. CFBE WT-CFTR cells were polarized on 24-mm transwell permeable supports (0.4-µm-pore size; Corning, Corning, NY) coated with vitrogen plating medium containing human fibronectin, as described previously [41]. Second, human airway epithelial cells (Calu-3) were obtained from the American Type Culture Collection (Manassas, VA) and polarized on 24-mm transwell permeable supports, as described previously [42]. Pseudomonas aeruginosa cultures Lysogeny broth (LB) was inoculated with P. aeruginosa strain UCBPP-PA14 (PA14) [43] and cultures were prepared as previously reported [16]. Outer membrane vesicle purification OMV were purified using a differential centrifugation and discontinuous Optiprep gradient protocol adapted from Bauman et al. [14] OMV were lysed, when noted, with 100 mM EDTA at 37°C for 60 minutes. Cell compartment fractionation To study the localization of the Cif toxin after OMV fusion with the airway epithelial cell, differential centrifugation and fractionation techniques were used to isolate cytosolic and early endosomal compartments. Early endosomes were isolated using a protocol adapted from Butterworth et al. [44]. Immunoprecipitation To characterize proteins interacting with the Cif toxin in lipid raft microdomains, Cif was immunoprecipitated from airway epithelial cell lipid raft fractions by methods described previously [45]. Detergent-resistant membrane fractionation To determine if OMV fuse with lipid raft microdomains of the host, detergent-resistant membranes were purified from airway epithelial cells that had been exposed to OMV. These studies were performed using a discontinuous Optiprep gradient in a protocol adapted from Pike et al. [46]. OMV fusion assay To monitor the fusion of OMV with airway epithelial cells, OMV were fluorescently labeled with a probe that fluoresces upon membrane fusion. OMV purified with the method described above were resuspended in labeling buffer (50 mM Na2CO3, 100 mM NaCl, pH 9.2). Rhodamine isothiocyanate B-R18 (Molecular Probes), which integrates in the membrane of the OMV, was added at a concentration of 1 mg/ml for 1 hour at 25°C, followed by ultracentrifugation at 52,000×g for 30 min at 4°C. Rhodamine isothiocyanate B-R18 fluorescence is quenched at high concentrations in bilayer membranes, and fluorescence is dequenched when the probe is diluted upon vesicle fusion. Subsequently, rhodamine labeled-OMV were resuspended in PBS (0.2 M NaCl) and pelleted at 52,000×g for 30 min a 4°C. After a final centrifugation step, the labeled-OMV were resuspended in 1 ml PBS (0.2 M NaCl) containing a protease inhibitor cocktail tablet (Complete Protease Inhibitor Tablet, Roche). Labeled-OMV were applied to the apical side of airway epithelial cells at 1∶4 dilution of labeled-OMV to Earle's Minimal Medium (MEM, Invitrogen) and fluorescence was detected over time as indicated on a fluorescent plate reader (Ex 570 nm; Em 595 nm). Fluorescence intensity was normalized for fluorescence detected by labeled-OMV in the absence of airway epithelial cells at the indicated time points. Confocal microscopy To visualize the fusion and localization of OMV with airway epithelial cells, rhodamine R18-labeled OMV (see OMV Fusion Assay method) were applied to the apical membrane of cells and confocal sections were captured over time. Airway epithelial cells were seeded at 0.1×106 on collagen-coated, glass-bottom MatTek dishes (MatTek, Ashland, MA) and grown for 6–7 days in culture at 37°C. For wheat germ agglutin (WGA, which labels the plasma membrane) studies, nonpermeabilized cells were incubated for 5 minutes with Alexa-647 WGA (1 µg/ml, 37°C; Molecular Probes) following 15-minute vesicle incubation. Z-stacks of all labeled cells were acquired with a Nikon Sweptfield confocal microscope (Apo TIRF 60× oil immersion 1.49 NA objective) fitted with a QuantEM:512sc camera (Photometrics, Tuscon, AZ) and Elements 2.2 software (Nikon, Inc.). For OMV fusion experiments, a single confocal section (0.4 µm) at the apical membrane of the airway epithelial cells is presented. Experiments were repeated three times, with five fields imaged for each experiment. Cell-surface biotinylations and Western blot analysis To examine the effect of OMV on the apical membrane expression of CFTR, cell surface biotinylation was performed as described in detail previously by our laboratory [41]. Protein band intensity was analyzed as described previously using NIH image software, version 1.63 (Wayne Rasband, NIH, USA; http://rsb.info.nih.gov). Cytotoxicity assay To determine if P. aeruginosa OMV are cytotoxic to airway cells, cells were incubated with OMV in serum-free media for the indicated time points. Cytotoxicity was measured using the CellTiter 96 AQueous One Solution Reagent (Promega, Madison, WI), according to the manufacturer's protocol. Data analysis and statistics Statistical analysis of the data was performed using Graphpad Prism version 4.0 for Macintosh (Graphpad, San Diego, CA). Means were compared using a Students t-test or one-way ANOVA, followed by a Tukey-Kramer post hoc test using a 95% confidence interval. Data are expressed as means+/−SEM. Accession numbers Cif (PA2934, NP 251624.1); PlcH (PA0844, YP 792433.1); alkaline phosphatase (PA3296, YP 789857.1); β-lactamase (PA1797, YP 791446.1); N-WASP (NP 003932); Omp85 (PA3648, YP 789516); GPIp137 (NP 005889). Supporting Information Figure S1 The Cif virulence factor is packaged in P. aeruginosa OMV. From an overnight P. aeruginosa PA14 culture, Optiprep density gradient centrifugation was utilized to purify OMV from the bacteria and possible contaminants, including pilus (PilA). Purified OMVs retrieved from fractions 2 and 3 were pooled for use in all experiments described. Experiment repeated three times; representative blot shown. (0.35 MB TIF) Click here for additional data file. Figure S2 EDTA effectively lyses OMV. EDTA (0.1 M) disrupted OMV membranes to allow proteinase K (PK)-mediated degradation of Cif, an intravesicular OMV component, as measured by Western blot analysis. Experiment repeated three times; representative blot shown. (0.18 MB TIF) Click here for additional data file. Figure S3 Cif virulence factor is an intravesicular OMV component. Isolated OMV treated with Proteinase K (PK: 100 µg/ml) for 1 h at 37°C to degrade proteins on the exterior of OMV. Δcif: OMV purified from a P. aeruginosa Δcif mutant strain. Experiment repeated three times; representative blot shown. (0.35 MB TIF) Click here for additional data file. Figure S4 Cif does not localize to Rab4, Rab7, or Rab11-labeled endosomes. Cif does not localize to the sorting endosomal (Rab4 GTPase-labeled), late endosomal (Rab7 GTPase-labeled), or recycling endosomal (Rab11 GTPase-labeled) compartments after entry into airway epithelial cells. Airway epithelial cells were treated with OMV for 10 min, cells lysed, and endosomes were purified. Cif was immunoprecipitated from the endosomal fraction and Western blot analysis was performed for Rab4, 7, and 11 GTPases. IgG IP is a non-immune control immunoprecipitation experiment. (0.60 MB TIF) Click here for additional data file.

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Journal ID (nlm-ta): Gut

Journal ID (publisher-id): gut

Journal ID (hwp): gutjnl

Title: Gut

Publisher: BMJ Group (BMA House, Tavistock Square, London, WC1H 9JR )

ISSN (Print): 0017-5749

ISSN (Electronic): 1468-3288

Publication date (Electronic): 29 June 2010

Publication date (Print): October 2010

Publication date PMC-release: 29 June 2010

Volume: 59

Issue: 10

Pages: 1355-1362

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[1 ]Clermont Université, Université d'Auvergne, Clermont-Ferrand, France

[2 ]INRA, Clermont-Ferrand, France

[3 ]INSERM ERI-21/EA 4319, Laboratoire de Pathologie Clinique et Expérimentale et CRB INSERM, Hôpital Pasteur, et Faculté de Médecine, Université de Nice Sophia Antipolis, Nice, France

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Correspondence to Arlette Darfeuille-Michaud, Pathogénie Bactérienne Intestinale, Université d'Auvergne, CBRV, 28 place Henri Dunant, 63000 Clermont-Ferrand, France; arlette.darfeuille-michaud@ 123456u-clermont1.fr

This manuscript is dedicated to our friend and co-author Dr Anne-Lise Glasser who sadly passed away on 7 September 2009.

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Publisher ID: gutjnl207456

DOI: 10.1136/gut.2010.207456

PMC ID: 2976078

PubMed ID: 20587550

SO-VID: 1c4b501a-2127-4521-a390-fee305ebf00a

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