Proteomic Analysis of the Intestinal Epithelial Cell Response to Enteropathogenic Escherichia coli

We present the first large scale proteomic analysis of a human cellular response to a pathogen. Enteropathogenic Escherichia coli (EPEC) is an enteric human pathogen responsible for much childhood morbidity and mortality worldwide. EPEC uses a type III secretion system (TTSS) to inject bacterial proteins into the cytosol of intestinal epithelial cells, resulting in diarrhea. We analyzed the host response to TTSS-delivered EPEC effector proteins by infecting polarized intestinal epithelial monolayers with either wild-type or TTSS-deficient EPEC. Host proteins were isolated and subjected to quantitative profiling using isotope-coded affinity tagging (ICAT) combined with electrospray ionization tandem mass spectrometry. We identified over 2000 unique proteins from infected Caco-2 monolayers, of which ∼13% are expressed differentially in the presence of TTSS-delivered EPEC effector proteins. We validated these data in silico and through immunoblotting and immunofluorescence microscopy. The identified changes extend cytoskeletal observations made in less relevant cell types and generate testable hypotheses with regard to host proteins potentially involved in EPEC-induced diarrhea. These data provide a framework for future biochemical analyses of host-pathogen interactions. We present the first large scale proteomic analysis of a human cellular response to a pathogen. Enteropathogenic Escherichia coli (EPEC) is an enteric human pathogen responsible for much childhood morbidity and mortality worldwide. EPEC uses a type III secretion system (TTSS) to inject bacterial proteins into the cytosol of intestinal epithelial cells, resulting in diarrhea. We analyzed the host response to TTSS-delivered EPEC effector proteins by infecting polarized intestinal epithelial monolayers with either wild-type or TTSS-deficient EPEC. Host proteins were isolated and subjected to quantitative profiling using isotope-coded affinity tagging (ICAT) combined with electrospray ionization tandem mass spectrometry. We identified over 2000 unique proteins from infected Caco-2 monolayers, of which ∼13% are expressed differentially in the presence of TTSS-delivered EPEC effector proteins. We validated these data in silico and through immunoblotting and immunofluorescence microscopy. The identified changes extend cytoskeletal observations made in less relevant cell types and generate testable hypotheses with regard to host proteins potentially involved in EPEC-induced diarrhea. These data provide a framework for future biochemical analyses of host-pathogen interactions. Enteropathogenic Escherichia coli (EPEC) 1The abbreviations used are: EPEC, enteropathogenic Escherichia coli; TTSS, type III secretion system; wt, EPEC E2348/69; N-, EPEC E2348/69ΔescN; ICAT, isotope-coded affinity tag; PBS, phosphate-buffered saline; ILK, integrin-linked kinase. is the leading cause of bacterial-mediated diarrhea in children and is a major endemic health threat in the developing world. EPEC binds to intestinal epithelial cells, forming a characteristic lesion (attaching/effacing (A/E)) resulting from localized microvilli destruction and the formation of an underlying pedestal-like projection composed of epithelial-derived cytoskeletal components (1Donnenberg M.S. Kaper J.B. Finlay B.B. Trends Microbiol. 1997; 5: 109-114Abstract Full Text PDF PubMed Scopus (211) Google Scholar). Bacteria remain adherent on these cup-like projections, rarely penetrating the intestinal barrier. The bacterial factors responsible for the formation of attaching/effacing lesions and diarrheal disease are produced and regulated by a pathogenicity island described as the locus of enterocyte effacement (2Elliott S.J. Wainwright L.A. McDaniel T.K. Jarvis K.G. Deng Y.K. Lai L.-C. McNamara B.P. Donnenberg M.S. Kaper J.B. Mol. Microbiol. 1998; 28: 1-4Crossref PubMed Scopus (551) Google Scholar). The locus of enterocyte effacement (LEE) encodes a Type III secretion system (TTSS), a cellular receptor, numerous secreted/translocated proteins, and over 20 open reading frames of unknown function. The TTSS is a molecular syringe that directs the active transport of proteins from the bacterial cytoplasm across the inner and outer bacterial membranes, directly into the cytoplasm of an associated eukaryotic cell (3Hueck C.J. Microbiol. Mol. Biol. Rev. 1998; 62: 379-433Crossref PubMed Google Scholar). TTSSs are widely conserved among a diverse array of animal and plant pathogens and critical for their virulence. Of major interest is the TTSS-specific response of human intestinal epithelial cells to EPEC infection. Previous research has focused primarily on morphological changes to the host as inferred from immunostaining. Particular interest has developed in the elucidation of host components contributing to pedestal formation. Transcriptional profiling of the host has been attempted, although not yet in a relevant cell type (4de Grado M. Rosenberger C.M. Gauthier A. Vallance B.A. Finlay B.B. Infect. Immun. 2001; 69: 6217-6224Crossref PubMed Scopus (51) Google Scholar). A fundamental understanding of how the intestinal epithelial proteome is altered by this pathogen and its TTSS effector proteins is needed, as few host pathways have been examined. We therefore undertook a quantitative analysis of the proteome of polarized Caco-2 intestinal epithelial cells infected with either wild-type or TTSS-deficient EPEC. Microcapillary liquid chromatography combined with electrospray ionization tandem mass spectrometry (ESI μLC-MS/MS) identifies proteins from mixtures without prior electrophoretic separation. Labeling of protein lysates prior to μLC-MS/MS with isotope-coded affinity tags (ICAT) specific to sulfhydryl groups greatly reduces sample complexity, allows detection of low abundance proteins, and aids in quantification of relative protein abundance between samples (5Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Nat. Biotechnol. 1999; 17: 994-999Crossref PubMed Scopus (4350) Google Scholar). We employed ICAT technology to both identify and quantify TTSS-dependent alterations to host protein expression, performed in silico validation, and further confirmed our results through immunoblotting and immunofluorescence microscopy. We discuss the predominant functional categories of differentially regulated proteins and their implications to the future study of EPEC-host interactions and present novel testable hypotheses about newly identified host proteins and their potential role in diarrhea. Bacterial Strains—The bacterial strains used in this study were wild-type EPEC E2348/69 (6Levine M.M. Bergquist E.J. Nalin D.R. Waterman D.H. Hornick R.B. Young C.R. Sotman S. Lancet. 1978; 1: 1119-1122Abstract PubMed Scopus (496) Google Scholar) and EPEC E2348/69ΔescN (7Gauthier A. Puente J.L. Finlay B.B. Infect. Immun. 2003; 71: 3310-3319Crossref PubMed Scopus (127) Google Scholar). Cell Culture—Human Caco-2 cells (8Rousset M. Biochimie (Paris). 1986; 68: 1035-1040Crossref PubMed Scopus (379) Google Scholar) were grown at 37 °C, 5% CO2, in Dulbecco's modified Eagle's medium supplemented with 10% decomplemented fetal calf serum, and 1% non-essential amino acids. Cells were cultured in 24-mm diameter polyester Transwell plates (Costar) for at least 21 days prior to infection. Infections and Protein Preparation—Bacterial cultures grown overnight were subcultured 1:50 into Dulbecco's modified Eagle's medium and grown for 3 h at 37 °C, 5% CO2, without shaking. Bacteria were applied to Caco-2 cells for 4 h at a multiplicity of infection of 50:1. Following infection, the media was aspirated and cells were washed 8 times with PBS. Cells were lysed in 500 μl of 1% Triton X-100 in PBS and centrifuged at 12,000 × g for 5 min. The supernatant was transferred to 5 volumes of cold acetone, precipitated, and resuspended in 0.1% SDS, 6 m urea. ICAT Labeling and Analysis—Caco-2 lysates were labeled as described in the Supplementary Materials. SEQUEST™ (Thermo Finnigan) was used to sequence the peptides and XPRESS (9Han D.K. Eng J. Zhou H. Aebersold R. Nat. Biotechnol. 2001; 19: 946-951Crossref PubMed Scopus (828) Google Scholar) software was used to perform relative quantitation between light and heavy ICAT-tagged peptides. PeptideProphet™ (10Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3897) Google Scholar) was used to verify correctness of peptide assignments. Western Blot Analysis—Samples for Western blot analysis were resolved by SDS-PAGE and analyzed as described in Ref. 7Gauthier A. Puente J.L. Finlay B.B. Infect. Immun. 2003; 71: 3310-3319Crossref PubMed Scopus (127) Google Scholar. The following antibodies were utilized at a dilution of 1:1000: calpain-5 (BD Biosciences), caspase-7 (New England Biolabs), dynactin (BD Biosciences), dynamin (BD Biosciences), espin (BD Biosciences), integrin-linked protein kinase (New England Biolabs), Nod2 (Immunologicalsdirect.com), Rac1 (New England Biolabs), and talin (Sigma). Immunofluorescence—HeLa cells were grown on glass coverslips in 24-well tissue culture plates and infected for 4 h with 5.0 μl of bacterial overnight culture. After infection, cells were washed 3 times in PBS containing Ca2+ and Mg2+ and fixed in 2.5% paraformaldehyde in PBS for 10 min at room temperature. Cells were permeabilized in 0.1% saponin in PBS, blocked in 5% goat serum in PBS + 0.1% saponin, and incubated with the following primary antibodies diluted 1:1000 in blocking solution for 1 h at room temperature: integrin-linked protein kinase, talin, and calpain-5. We utilized the mammalian Caco-2 cell culture line as a model for EPEC pathogenesis in the small bowel. Caco-2 cells provide an ideal infection model, as they are derived from the human intestine, able to polarize, develop microvilli, form tight junctions, and are infected by EPEC (11Canil C. Rosenshine I. Ruschkowski S. Donnenberg M.S. Kaper J.B. Finlay B.B. Infect. Immun. 1993; 61: 2755-2762Crossref PubMed Google Scholar). To study host responses specific to the EPEC TTSS, we compared the effect of a well characterized wild-type strain (E2348/69; wt) to a strain deficient in the TTSS (E2348/69ΔescN; N-). Caco-2 lysates prepared after 4 h infection were differentially ICAT labeled (Fig. 1 and supporting text), and analyzed by microcapillary high performance liquid chromatography-tandem mass spectrometry (μLC-MS/MS) (9Han D.K. Eng J. Zhou H. Aebersold R. Nat. Biotechnol. 2001; 19: 946-951Crossref PubMed Scopus (828) Google Scholar). Relative protein abundance was determined by the ratio of signal intensities of peptide pairs using the XPRESS software tool (9Han D.K. Eng J. Zhou H. Aebersold R. Nat. Biotechnol. 2001; 19: 946-951Crossref PubMed Scopus (828) Google Scholar). The sequence identity of the proteins in the sample was determined by correlating collision-induced dissociation mass spectra with the NCBI protein data base using the SEQUEST algorithm (12Eng J. McCormack A.L. Yates J.R. J. Am. Soc. Mass. Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5443) Google Scholar). 2,090 proteins were identified from 10,921 tandem mass spectra of peptides matched to peptide sequences in a data base using SEQUEST (see Supplementary Materials Fig. S1 for the complete dataset). 264 proteins with an annotated biological function (∼13%) displayed at least 2-fold expression differences between wt- and N--infected cells (Table I). Approximately equal numbers of proteins were up- versus down-regulated. We used the bioinformatics tool GoMiner (13Zeeberg B.R. Feng W. Wang G. Wang M.D. Fojo A.T. Sunshine M. Narasimhan S. Kane D.W. Reinhold W.C. Lababidi S. Bussey K.J. Riss J. Barrett J.C. Weinstein J.N. Genome Biol. 2003; 4: R28Crossref PubMed Google Scholar) to classify the differentially regulated Caco-2 proteins into functional categories based on the functional annotations assigned to these proteins (14Ashburner M. Ball C.A. Blake J.A. Botstein D. Butler H. Cherry J.M. Davis A.P. Dolinski K. Dwight S.S. Eppig J.T. Harris M.A. Hill D.P. Issel-Tarver L. Kasarskis A. Lewis S. Matese J.C. Richardson J.E. Ringwald M. Rubin G.M. Sherlock G. Nat. Genet. 2000; 25: 25-29Crossref PubMed Scopus (27248) Google Scholar) (Table II).Table IICAT summary statisticswtaIdentified only in Caco-2 cells infected with wtN-bIdentified only in Caco-2 cells infected with N-UpcExpression ratios in which [wt > N-] is greater than 2-fold. Hypothetical proteins are shown in parenthesesDowndExpression ratios in which [wt < N-] is greater than 2-fold. Hypothetical proteins are shown in parenthesesUnchanged37125 (138)139 (141)1486a Identified only in Caco-2 cells infected with wtb Identified only in Caco-2 cells infected with N-c Expression ratios in which [wt > N-] is greater than 2-fold. Hypothetical proteins are shown in parenthesesd Expression ratios in which [wt < N-] is greater than 2-fold. Hypothetical proteins are shown in parentheses Open table in a new tab Table IIDifferentially regulated Caco-2 proteins (>2-fold) from ICAT experimentation Data are sorted primarily by biological function as annotated by GoMiner (13Zeeberg B.R. Feng W. Wang G. Wang M.D. Fojo A.T. Sunshine M. Narasimhan S. Kane D.W. Reinhold W.C. Lababidi S. Bussey K.J. Riss J. Barrett J.C. Weinstein J.N. Genome Biol. 2003; 4: R28Crossref PubMed Google Scholar) and secondarily by average wt/N; expression ratio. Bolded proteins are discussed in text and previously implicated in bacterial pathogenesis.a Average ratio of all quantified peptides for each protein representing fold increase in protein abundance in Caco-2 cells during infection with wt relative to N-b Identified only in Caco-2 cells infected with N-c Identified only in Caco-2 cells infected with wt a Average ratio of all quantified peptides for each protein representing fold increase in protein abundance in Caco-2 cells during infection with wt relative to N- b Identified only in Caco-2 cells infected with N- c Identified only in Caco-2 cells infected with wt Research into EPEC interactions with epithelial cells has focused primarily on host cytoskeletal rearrangements and signaling pathways mediating such rearrangements. Indeed, the major functional categories contributing most to the total of differentially regulated proteins are those involved in the cytoskeleton, cell adhesion, and G-protein signaling (Fig. 2). Proteins involved in ion transport and ion channel function were in the of proteins, potentially contributing to diarrhea. We validated in silico our results by all differentially expressed proteins all in bacterial The of the proteins previously implicated in bacterial pathogenesis and to involved in is in Table and discussed in The most in the host epithelial cell during EPEC infection is the formation of which EPEC The primary components of the EPEC pedestal have been characterized with R. Finlay B.B. Infect. Immun. 2001; 69: PubMed Scopus Google Scholar). We identified of the 20 proteins of which are in a TTSS-dependent the expression of an integrin-linked kinase (see and confirmed expression of the host kinase potentially responsible for A. B. W. D. of The for Cell Scholar) Protein abundance and are performed by each a data base of human protein sequences using Supplementary Materials Fig. data derived from the of a peptide ion unique to the calpain-5. is for effacement during EPEC pathogenesis and to the A. K. D. J. C. M. D. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). were made in PeptideProphet™ based on how the mass spectra matched the E. S. K. A. Nesvizhskii A.I. Eng J. D.R. Aebersold R. Mol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). XPRESS software the of the and peptides and a peptide relative abundance ratio based on the abundance ratio was Materials Fig. To the of these data with of protein we performed Western blot analysis of numerous host proteins for which we TTSS-dependent expression (Fig. Caco-2 monolayers were infected with either wt or N- for h and subjected to expression between and 4 h in cells infected with wt, expression low in cells infected with N- (Fig. The relative in expression was to to the abundance ratio derived from ICAT We the expression of a involved in K. H. H. M. H. K. 2000; 4: Scopus Google Scholar). expression was not in wt cells by was expressed in N--infected cells (Fig. We this analysis to a of TTSS-dependent differentially expressed proteins using cell lysates prepared from We analyzed the expression of proteins and ICAT abundance ratios versus expression ratios from Western (Fig. was between the results were with immunofluorescence and in ICAT J.A. D.R. Eng J. Aebersold R. Nat. Genet. 2003; PubMed Scopus Google Scholar). We used immunofluorescence to the expression of Caco-2 proteins by ICAT to differentially regulated during infection. cells were infected with either wt or N-, and integrin-linked kinase and talin Materials Fig. After as calpain-5 expression was in to N--infected we of with this signaling as of the EPEC We the expression and of talin with We present the first proteomic analysis of the TTSS-specific human response to a pathogen. We used ICAT technology to changes in the host proteome in response to wt- and TTSS-deficient EPEC and with The of ICAT to mammalian has over analyses (5Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Nat. 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Spectrom. 2003; 17: PubMed Scopus Google Scholar). data that a TTSS-delivered EPEC effector the expression of this proteomic analysis of the Caco-2 host response to EPEC has novel testable hypotheses about how this enteric human pathogen The of quantitative mass spectrometry the for cell and greatly the of proteins that results with analyses of protein expression, as well as of the host response to enteric is to that our expression ratios in cells infected with versus EPEC quantitative of host protein expression between infection for epithelial interactions with although differences in and infection to a of the identified epithelial of protein function through ICAT of the host response among both diverse pathogens and specific bacterial effector We are to the of the differentially regulated Caco-2 proteins to EPEC We J. L. and for with

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