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• W2079031703 abstract "Many enteric pathogens, including enterotoxigenic Escherichia coli (ETEC), produce one or more serine proteases that are secreted via the autotransporter (or type V) bacterial secretion pathway. These molecules have collectively been referred to as SPATE proteins (serine protease autotransporter of the Enterobacteriaceae). EatA, an autotransporter previously identified in ETEC, possesses a functional serine protease motif within its secreted amino-terminal passenger domain. Although this protein is expressed by many ETEC strains and is highly immunogenic, its precise function is unknown. Here, we demonstrate that EatA degrades a recently characterized adhesin, EtpA, resulting in modulation of bacterial adhesion and accelerated delivery of the heat-labile toxin, a principal ETEC virulence determinant. Antibodies raised against the passenger domain of EatA impair ETEC delivery of labile toxin to epithelial cells suggesting that EatA may be an effective target for vaccine development. Many enteric pathogens, including enterotoxigenic Escherichia coli (ETEC), produce one or more serine proteases that are secreted via the autotransporter (or type V) bacterial secretion pathway. These molecules have collectively been referred to as SPATE proteins (serine protease autotransporter of the Enterobacteriaceae). EatA, an autotransporter previously identified in ETEC, possesses a functional serine protease motif within its secreted amino-terminal passenger domain. Although this protein is expressed by many ETEC strains and is highly immunogenic, its precise function is unknown. Here, we demonstrate that EatA degrades a recently characterized adhesin, EtpA, resulting in modulation of bacterial adhesion and accelerated delivery of the heat-labile toxin, a principal ETEC virulence determinant. Antibodies raised against the passenger domain of EatA impair ETEC delivery of labile toxin to epithelial cells suggesting that EatA may be an effective target for vaccine development. Infectious diarrheal disease caused by the enterotoxigenic Escherichia coli (ETEC) 2The abbreviations used are: ETECenterotoxigenic E. coliLTlabile toxinKmkanamycinCmchloramphenicol. accounts for hundreds of thousands of deaths each year in areas where sanitation and clean water remain scarce (1Qadri F. Svennerholm A.M. Faruque A.S. Sack R.B. Clin. Microbiol. Rev. 2005; 18: 465-483Crossref PubMed Scopus (692) Google Scholar). ETEC are a diverse group of pathogens that have in common the ability to colonize the small intestine where they deliver heat-labile (LT) and/or heat-stable toxins. These enterotoxins activate production of host cell cyclic nucleotides (cAMP and cGMP, respectively) in turn stimulating cellular kinases that phosphorylate and activate the cystic fibrosis transmembrane regulator chloride channel (2Fleckenstein J.M. Hardwidge P.R. Munson G.P. Rasko D.A. Sommerfelt H. Steinsland H. Microbes Infect. 2010; 12: 89-98Crossref PubMed Scopus (214) Google Scholar). The ensuing salt and water losses in the intestinal lumen are ultimately responsible for the diarrheal illness. enterotoxigenic E. coli labile toxin kanamycin chloramphenicol. ETEC have recently been shown to produce a number of secreted proteins in addition to the established enterotoxins (3Patel S.K. Dotson J. Allen K.P. Fleckenstein J.M. Infect. Immun. 2004; 72: 1786-1794Crossref PubMed Scopus (104) Google Scholar, 4Pilonieta M.C. Bodero M.D. Munson G.P. J. Bacteriol. 2007; 189: 5060-5067Crossref PubMed Scopus (38) Google Scholar, 5Fleckenstein J.M. Roy K. Fischer J.F. Burkitt M. Infect. Immun. 2006; 74: 2245-2258Crossref PubMed Scopus (97) Google Scholar). However, the precise role of these exoproteins in pathogenesis is still being established. Like many pathogenic bacteria, ETEC produce putative virulence proteins that are secreted via the autotransporter mechanism. Autotransporters are contained in a single protein composed of three essential domains as follows: a signal peptide, amino-terminal passenger region, and a carboxyl-terminal β-barrel domain. Although the term autotransporter was devised to convey the notion that these proteins possessed all of the elements required for secretion of the passenger, it appears increasingly likely that additional conserved periplasmic chaperones or outer membrane proteins may be required for autotransporter biogenesis (6Ruiz-Perez F. Henderson I.R. Leyton D.L. Rossiter A.E. Zhang Y. Nataro J.P. J. Bacteriol. 2009; 191: 6571-6583Crossref PubMed Scopus (111) Google Scholar, 7Ieva R. Bernstein H.D. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 19120-19125Crossref PubMed Scopus (156) Google Scholar). The passenger region typically serves as the functional region of the molecule in autotransporters described to date. In a variety of Gram-negative pathogens, many passenger domains contain serine protease motifs (8Maroncle N.M. Sivick K.E. Brady R. Stokes F.E. Mobley H.L. Infect. Immun. 2006; 74: 6124-6134Crossref PubMed Scopus (50) Google Scholar, 9Dutta P.R. Cappello R. Navarro-García F. Nataro J.P. Infect. Immun. 2002; 70: 7105-7113Crossref PubMed Scopus (163) Google Scholar, 10Boisen N. Ruiz-Perez F. Scheutz F. Krogfelt K.A. Nataro J.P. Am. J. Trop. Med. Hyg. 2009; 80: 294-301Crossref PubMed Scopus (104) Google Scholar) and are therefore referred to as serine protease autotransporter of the Enterobacteriaceae (SPATE) proteins (11Henderson I.R. Nataro J.P. Infect. Immun. 2001; 69: 1231-1243Crossref PubMed Scopus (350) Google Scholar). The pathogenic role played by the majority of SPATE proteins remains uncertain (9Dutta P.R. Cappello R. Navarro-García F. Nataro J.P. Infect. Immun. 2002; 70: 7105-7113Crossref PubMed Scopus (163) Google Scholar). Recent studies suggest that EatA, a SPATE protein previously described in ETEC (Fig. 1a) (3Patel S.K. Dotson J. Allen K.P. Fleckenstein J.M. Infect. Immun. 2004; 72: 1786-1794Crossref PubMed Scopus (104) Google Scholar), is immunologically recognized following both experimental murine and natural human infections with ETEC (13Roy K. Bartels S. Qadri F. Fleckenstein J.M. Infect. Immun. 2010; 78: 3027-3035Crossref PubMed Scopus (60) Google Scholar). In vivo expression of EatA and the recent identification of eatA genes in most of the recently sequenced ETEC strains (14Rasko D.A. Rosovitz M.J. Myers G.S. Mongodin E.F. Fricke W.F. Gajer P. Crabtree J. Sebaihia M. Thomson N.R. Chaudhuri R. Henderson I.R. Sperandio V. Ravel J. J. Bacteriol. 2008; 190: 6881-6893Crossref PubMed Scopus (612) Google Scholar, 15Crossman L.C. Chaudhuri R.R. Beatson S.A. Wells T.J. Desvaux M. Cunningham A.F. Petty N.K. Mahon V. Brinkley C. Hobman J.L. Savarino S.J. Turner S.M. Pallen M.J. Penn C.W. Parkhill J. Turner A.K. Johnson T.J. Thomson N.R. Smith S.G. Henderson I.R. J. Bacteriol. 2010; 192: 5822-5831Crossref PubMed Scopus (141) Google Scholar, 16Froehlich B. Parkhill J. Sanders M. Quail M.A. Scott J.R. J. Bacteriol. 2005; 187: 6509-6516Crossref PubMed Scopus (51) Google Scholar), including the prototype H10407 strain in which it was originally discovered (3Patel S.K. Dotson J. Allen K.P. Fleckenstein J.M. Infect. Immun. 2004; 72: 1786-1794Crossref PubMed Scopus (104) Google Scholar), suggest that it likely plays an important role in virulence of this pathovar. Similar to SepA, its close Shigella homologue (17Benjelloun-Touimi Z. Sansonetti P.J. Parsot C. Mol. Microbiol. 1995; 17: 123-135Crossref PubMed Scopus (158) Google Scholar), EatA has been associated with accelerated virulence in a rabbit ileal loop model (3Patel S.K. Dotson J. Allen K.P. Fleckenstein J.M. Infect. Immun. 2004; 72: 1786-1794Crossref PubMed Scopus (104) Google Scholar). However, the precise functions of EatA as well as SepA remain unknown. To date, the majority of ETEC virulence studies have focused specifically on the role of plasmid-encoded fimbrial colonization factors, or the established enterotoxins. However, more recent data suggest that many elements of ETEC virulence, specifically processes pertaining to bacterial adhesion and intestinal colonization, are actually quite complex (2Fleckenstein J.M. Hardwidge P.R. Munson G.P. Rasko D.A. Sommerfelt H. Steinsland H. Microbes Infect. 2010; 12: 89-98Crossref PubMed Scopus (214) Google Scholar, 18Croxen M.A. Finlay B.B. Nat. Rev. Microbiol. 2010; 8: 26-38Crossref PubMed Scopus (735) Google Scholar) and likely involve multiple factors. These include integral outer membrane proteins (19Fleckenstein J.M. Kopecko D.J. Warren R.L. Elsinghorst E.A. Infect. Immun. 1996; 64: 2256-2265Crossref PubMed Google Scholar), the TibA autotransporter protein (20Elsinghorst E.A. Weitz J.A. Infect. Immun. 1994; 62: 3463-3471Crossref PubMed Google Scholar), the secreted EtpA adhesin molecule (5Fleckenstein J.M. Roy K. Fischer J.F. Burkitt M. Infect. Immun. 2006; 74: 2245-2258Crossref PubMed Scopus (97) Google Scholar, 21Roy K. Hilliard G.M. Hamilton D.J. Luo J. Ostmann M.M. Fleckenstein J.M. Nature. 2009; 457: 594-598Crossref PubMed Scopus (150) Google Scholar), and flagella (21Roy K. Hilliard G.M. Hamilton D.J. Luo J. Ostmann M.M. Fleckenstein J.M. Nature. 2009; 457: 594-598Crossref PubMed Scopus (150) Google Scholar), as well as the heat-labile toxin (22Johnson A.M. Kaushik R.S. Francis D.H. Fleckenstein J.M. Hardwidge P.R. J. Bacteriol. 2009; 191: 178-186Crossref PubMed Scopus (78) Google Scholar). Here, we further examine the contribution of eatA to virulence and demonstrate that it plays a significant but unanticipated role in modulating adherence of ETEC by degrading the EtpA adhesin. Importantly, the presence of EatA was shown to accelerate delivery of the heat-labile toxin to target epithelial cells. A complete list of bacterial strains and plasmids used in these experiments is provided in Table 1. LMG194ΔfliC was constructed as described previously (21Roy K. Hilliard G.M. Hamilton D.J. Luo J. Ostmann M.M. Fleckenstein J.M. Nature. 2009; 457: 594-598Crossref PubMed Scopus (150) Google Scholar) using λ red recombination (23Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 6640-6645Crossref PubMed Scopus (11304) Google Scholar) to interrupt the flagellin (fliC) gene. Briefly, a kanamycin resistance cassette was amplified from pKD4 using primers jf121905.1 (5′-ggaaacccaatacgtaatcaacgacttgcaatataggataacgaatcGTGTAGGCTGGAGCTGCTTC-3′) and jf121905.2 (5′-tgccaacacggagttaccggcctgctggatgatctgcgctttcgaCATATGAATATCCTCCTTA-3′) (lowercase regions of primers denote gene homology regions, and uppercase regions indicate pKD4 sequence). Following digestion with DpnI to remove the remaining template plasmid, the PCR product was introduced into LMG194 carrying the pKD46 (23Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 6640-6645Crossref PubMed Scopus (11304) Google Scholar) λ recombinase helper plasmid. Recombinants selected on kanamycin plates at 37 °C were then screened for motility and flagellin production by immunoblotting (21Roy K. Hilliard G.M. Hamilton D.J. Luo J. Ostmann M.M. Fleckenstein J.M. Nature. 2009; 457: 594-598Crossref PubMed Scopus (150) Google Scholar).TABLE 1Bacterial strains and plasmidsE. coli strain/plasmidRelevant genotype or descriptionRef./sourceStrains H10407ETEC serotype O78:H11, LT+,ST+, eatA+,etpBAC+3Patel S.K. Dotson J. Allen K.P. Fleckenstein J.M. Infect. Immun. 2004; 72: 1786-1794Crossref PubMed Scopus (104) Google Scholar, 5Fleckenstein J.M. Roy K. Fischer J.F. Burkitt M. Infect. Immun. 2006; 74: 2245-2258Crossref PubMed Scopus (97) Google Scholar, 39Evans D.G. Silver R.P. Evans Jr., D.J. Chase D.G. Gorbach S.L. Infect. Immun. 1975; 12: 656-667Crossref PubMed Google Scholar jf571Deletion of eltA encoding A subunit of LT24Dorsey F.C. Fischer J.F. Fleckenstein J.M. Cell. Microbiol. 2006; 8: 1516-1527Crossref PubMed Scopus (72) Google Scholar jf946Derivative of H10407 lacZ::KmR24Dorsey F.C. Fischer J.F. Fleckenstein J.M. Cell. Microbiol. 2006; 8: 1516-1527Crossref PubMed Scopus (72) Google Scholar jf904H10407 eatA::CmR mutant3Patel S.K. Dotson J. Allen K.P. Fleckenstein J.M. Infect. Immun. 2004; 72: 1786-1794Crossref PubMed Scopus (104) Google Scholar LMG194F− ΔlacX74 galE thi rpsL ΔphoA (Pvull) Δara-714 leu::Tn10Invitrogen LMG194ΔfliCLMG194 with fliC::KmRThis study TOP10F-mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ (ara leu) 7697 galU galK rpsL (StrR)Invitrogen jf1443ΔeltAB (pJL001)This study jf1446ΔeltAB (pBADmycHisA)This studyPlasmids pBAD-TOPO/lacZ/V5-HispBAD-TOPO vector containing lacZ control insert3Patel S.K. Dotson J. Allen K.P. Fleckenstein J.M. Infect. Immun. 2004; 72: 1786-1794Crossref PubMed Scopus (104) Google Scholar; Invitrogen pSB0017064-bp BamHI Δlac subclone from pBAD-TOPO/lacZ/V5-HisThis study pSP0144185-bp EatA amplicon cloned into pBAD-TOPO3Patel S.K. Dotson J. Allen K.P. Fleckenstein J.M. Infect. Immun. 2004; 72: 1786-1794Crossref PubMed Scopus (104) Google Scholar pSP019pSP014 altered by SDM for His-134 →Arg-134 substitution of EatA3Patel S.K. Dotson J. Allen K.P. Fleckenstein J.M. Infect. Immun. 2004; 72: 1786-1794Crossref PubMed Scopus (104) Google Scholar pJL017etpBA cloned on pBAD-myc His plasmid with etpA in-frame with C-terminal myc-His6 tags21Roy K. Hilliard G.M. Hamilton D.J. Luo J. Ostmann M.M. Fleckenstein J.M. Nature. 2009; 457: 594-598Crossref PubMed Scopus (150) Google Scholar, 26Fleckenstein J.M. Roy K. Nat. Protoc. 2009; 4: 1083-1092Crossref PubMed Scopus (13) Google Scholar pJL030etpC cloned on pACYC18421Roy K. Hilliard G.M. Hamilton D.J. Luo J. Ostmann M.M. Fleckenstein J.M. Nature. 2009; 457: 594-598Crossref PubMed Scopus (150) Google Scholar, 26Fleckenstein J.M. Roy K. Nat. Protoc. 2009; 4: 1083-1092Crossref PubMed Scopus (13) Google Scholar Open table in a new tab To create a negative control vector plasmid for complementation studies, pBAD-TOPO/lacZ/V5-His was digested with BamHI to remove a 119-bp fragment spanning the end of the promoter region and the start of lacZ. The remaining 7064-bp fragment was then religated on itself and re-introduced into E. coli Top10 to select white (lac−) colonies on Luria agar plates containing ampicillin, arabinose (0.02%), and 5-bromo-4-chloro-3-indolyl β-d-galactoside (X-gal). The resulting plasmid pSB001 was then isolated and used to transform the eatA mutant jf904 to ampicillin and chloramphenicol resistance. Adherence assays for these studies were performed as described previously (21Roy K. Hilliard G.M. Hamilton D.J. Luo J. Ostmann M.M. Fleckenstein J.M. Nature. 2009; 457: 594-598Crossref PubMed Scopus (150) Google Scholar). Briefly, bacteria were grown overnight from frozen glycerol stocks in 2 ml of Luria broth containing antibiotics where appropriate at 37 °C, 225 rpm. The following morning strains were diluted 1:100 in fresh media and grown to mid-logarithmic growth phase (∼90 min of incubation at 37 °C). For strains complemented with arabinose-inducible plasmids, arabinose was added to a final concentration of 0.02% for the last 30 min of incubation. Bacteria were then added (at an multiplicity of infection (bacteria/target epithelial cell) of ∼100:1) to CaCo-2 cell monolayers seeded the previous evening in 96-well tissue culture plates. After 1 h, at 37 °C, 5% CO2, monolayers were washed with RPMI 1640 medium and lysed in 0.01% Triton X-100 to release both adherent and intracellular bacteria. Dilutions were then plated onto Luria agar plates and incubated overnight at 37 °C. To identify intracellular bacteria, monolayers were washed at 3 h following infection and the media replaced with fresh pre-warmed media containing gentamicin (100 μg/ml) and incubated for 1 additional hour. After washing to remove remaining antibiotics, monolayers were lysed with Triton X-100 to release intracellular organisms and plated as above. To examine the pattern of bacterial attachment to the cell surface, organisms were added to Caco-2 epithelial cells grown on sterile glass coverslips pretreated with poly-l-lysine and incubated in 5% CO2 atmosphere at 37 °C for 1–2 h. After washing three times with tissue culture media, cells were fixed with paraformaldehyde, washed with PBS, and blocked with PBS containing 1% BSA. Anti-O78 rabbit polyclonal antibody followed by AlexaFluor-488-labeled anti-rabbit antibody were used to identify the attached organisms, and cells were stained with both diamidino-2-phenylindole (DAPI) and membrane stain (CellMask, Red, Invitrogen). After acquisition of images on a Zeiss LSM510 confocal microscope, the number of individual bacteria adherent to cells was examined by importing TIFF image files into ImageJ (version 1.44) and tracking the number of adherent bacteria/cell using the cell counter plugin module (ImageJ > plugins > analyze > cell counter). jf946, a derivative of ETEC H10407 containing a kanamycin resistance marker in the lacZYA locus (24Dorsey F.C. Fischer J.F. Fleckenstein J.M. Cell. Microbiol. 2006; 8: 1516-1527Crossref PubMed Scopus (72) Google Scholar), and the jf904 eatA mutant (3Patel S.K. Dotson J. Allen K.P. Fleckenstein J.M. Infect. Immun. 2004; 72: 1786-1794Crossref PubMed Scopus (104) Google Scholar) were tested in the streptomycin-treated murine colonization model as described previously (25Allen K.P. Randolph M.M. Fleckenstein J.M. Infect. Immun. 2006; 74: 869-875Crossref PubMed Scopus (103) Google Scholar). Briefly, mice (CD-1, Charles River) were treated with streptomycin in drinking water (5 g/liter) for 48 h prior to inoculation, which was changed to antibiotic-free sterile water the evening before challenge. Cimetidine (50 mg/kg) was administered intraperitoneally 1.5 h prior to gavage challenge with bacteria in a final volume of 400 μl. Mice were euthanized 24 h later, and bacterial colonization was assessed by plating saponin (5% solution in PBS) lysates of small intestinal segments onto Luria agar plates containing kanamycin (Km, 25 μg/ml) or chloramphenicol (Cm, 25 μg/ml). To assess the number of ETEC organisms shed in stool, fecal pellets were collected from individual mice, resuspended in PBS, and dilutions plated onto replicate Luria agar plates. Competition studies were carried out as described previously (21Roy K. Hilliard G.M. Hamilton D.J. Luo J. Ostmann M.M. Fleckenstein J.M. Nature. 2009; 457: 594-598Crossref PubMed Scopus (150) Google Scholar), with co-administration of ≈1 × 104 of both the jf946 and jf904 stains in a final volume of 400 μl. Intestinal lysates were used to separately inoculate both Cm- and Km-containing agar plates. The competitive index for each colonized mouse was then calculated as follows: competitive index = ((mutant (CmR)/wild type (KmR))output cfu/(mutant/wild type) input CFU), where the input fraction was determined directly by colony counting (cfu) prior to preparation of the inoculum. All experimental procedures involving animals were reviewed and approved by the University of Tennessee Health Science Center Institutional Animal Care and Use Committee. Animals were housed, cared for, and used in compliance with the Guide for the Care and Use of Laboratory Animals in an AAALAC International-accredited program. Production and purification of recombinant Myc-polyhistidine-tagged EtpA glycoprotein (rEtpA-myc-His) was carried out as described previously in detail (26Fleckenstein J.M. Roy K. Nat. Protoc. 2009; 4: 1083-1092Crossref PubMed Scopus (13) Google Scholar). Briefly, E. coli Top10 carrying both pJL017 and pJL030 was induced with arabinose and the secreted rEtpA-myc-His protein recovered from concentrated supernatants by batch metal affinity chromatography (Talon, Clontech). To produce recombinant EatA passenger protein (rEatAp), the arabinose-inducible EatA expression plasmid pSP014 (3Patel S.K. Dotson J. Allen K.P. Fleckenstein J.M. Infect. Immun. 2004; 72: 1786-1794Crossref PubMed Scopus (104) Google Scholar) was first introduced into LMG194ΔfliC, selecting for ampicillin- and kanamycin-resistant transformants. LMG194ΔfliC(pSP014) was then grown in 250 ml of Luria broth containing kanamycin (25 μg/ml) and ampicillin (100 μg/ml) to an A600 of ∼0.5 then induced for 2 h with arabinose at a final concentration 0.02%. Culture supernatants containing rEatAp were then concentrated ∼50-fold by ultrafiltration using a 100-kDa MWCO filter. After buffer exchange using column loading buffer (50 mm sodium phosphate, 150 mm NaCl, pH 7.2) and further concentration to a final volume of 0.5 ml, rEatAp was purified to homogeneity by gel filtration chromatography on a Sephacryl S-300 high resolution column (26Fleckenstein J.M. Roy K. Nat. Protoc. 2009; 4: 1083-1092Crossref PubMed Scopus (13) Google Scholar). Mutant rEatp(H134R) was prepared in the same fashion after introduction of pSP019 into LMG194ΔfliC. After growth of bacteria in Luria broth for the indicated time, cultures were centrifuged at 10,000 × g for 20 min to pellet bacteria, and supernatants of either H10407 or the jf904 eatA mutant were recovered, sterile-filtered through a 0.45-μm filter (Millipore), and concentrated by ultrafiltration as above. Equal amounts of protein were then loaded onto adjacent wells, separated by SDS-PAGE (10%), the gel stained with Sypro Ruby (Invitrogen) and imaged on a Typhoon variable-mode imager (GE Healthcare). To detect EtpA in culture supernatants, proteins were first concentrated by TCA precipitation as described previously (5Fleckenstein J.M. Roy K. Fischer J.F. Burkitt M. Infect. Immun. 2006; 74: 2245-2258Crossref PubMed Scopus (97) Google Scholar), separated by SDS-PAGE, and immunoblotted using pre-absorbed anti-EtpA polyclonal rabbit sera (1:2000) and goat anti-rabbit immunoglobulin G (Fc) horseradish peroxidase-labeled secondary antibody (1:60,000, Pierce). All blocking and incubation steps were carried out at room temperature in Tris-buffered saline (TBS, pH 7.4) containing 0.05% (v/v) Tween 20 and milk (5% w/v). Luminal-based chemiluminescent substrate (SuperSignal, Pierce) was used for detection. Antibody against the passenger domain of EatA was affinity-purified as described previously (27Harlow E. Lane D. Harlow E. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1999: 77-80Google Scholar). Briefly, ∼100 μg of purified mutant EatA passenger domain was transferred onto nitrocellulose, and the membrane was blocked with 1% BSA in TBS-T for 30 min at room temperature. Next, the membrane was incubated with polyclonal rabbit antisera raised against rEatA passenger domain (r6H-EatA(88–581)) (3Patel S.K. Dotson J. Allen K.P. Fleckenstein J.M. Infect. Immun. 2004; 72: 1786-1794Crossref PubMed Scopus (104) Google Scholar) diluted 1:10 in TBS-T for 2 h. Antibodies were then eluted with 100 mm glycine, pH 3.0, neutralized with 1 m Tris-HCl, pH 8.8, dialyzed against PBS, concentrated, and then sterile-filtered. To detect interaction of EatA with EtpA, 5 μg of recombinant EtpA.myc.His was electrophoretically transferred onto nitrocellulose and incubated with an equimolar amount of mutant protease activity-deficient EatA (H134R) for 2 h at room temperature. Membrane was washed and blocked in TBS, 3% milk for 30 min and probed with polyclonal affinity-purified primary antibody raised against r6H-EatA(88–581) (3Patel S.K. Dotson J. Allen K.P. Fleckenstein J.M. Infect. Immun. 2004; 72: 1786-1794Crossref PubMed Scopus (104) Google Scholar) followed by detection with HRP-labeled anti-rabbit antibody (1:5000) and chemiluminescent substrate. Varying amounts of rEatAp or rEatAp(H134R) were mixed with 1 μg of rEatA-myc-His and incubated at 37 °C. To inhibit protease activity, 4-amidinophenylmethanesulfonyl fluoride hydrochloride was added at a final concentration of 25 μm. The contents of the reaction was mixed with loading dye (six times) and separated by SDS-PAGE prior to immunoblotting for EtpA. To examine the efficiency of LT delivery to target host cells, bacteria from overnight cultures of the wild type H10407 or the ΔeatA strain were added to Caco-2 epithelial cell monolayers. Following incubation of the bacteria with monolayers for 1–3 h, cells were lysed, and intracellular concentration of cAMP was determined by competitive enzyme immunoassay (GE Healthcare). To measure production of LT by wild type and eatA mutant, bacteria were grown at 37 °C, 225 rpm in 2 ml of casamino acids/yeast extract medium (CAYE, 2% casamino acids, 0.15% yeast extract, 0.25% NaCl, 0.871% K2HPO4, 0.25% glucose, and 0.1% (v/v) trace salts solution consisting of 5% MgSO4, 0.5% MnCl2, 0.5% FeCl3) (28Mundell D.H. Anselmo C.R. Wishnow R.M. Infect. Immun. 1976; 14: 383-388Crossref PubMed Google Scholar) Supernatants were then tested in mixed ganglioside ELISAs as described previously (29Clements J.D. Infect. Immun. 1990; 58: 1159-1166Crossref PubMed Google Scholar). To assess specific gene transcripts, total RNA was first isolated from bacteria (RNeasy, Qiagen) and quantified spectrophotometrically. RNA was freed of contaminating DNA by repeated DNase treatments. Total RNA from bacteria was converted to cDNA by use of reverse transcriptase and random hexamers. Gene expression was quantified by use of an ABI-PRISM 7900HT sequence detection system with gene-specific primers in PCR buffer containing SYBR Green. Gene-specific transcripts were normalized to the housekeeping gene arcA. To examine the activity of EatA in cAMP assays, the purified recombinant passenger domain of EatA (rEatAp) was added exogenously to Caco-2 target epithelial monolayers with or without purified LT holotoxin. After 3 h, cells were lysed, and cAMP concentrations were determined as above. To examine proteolytic cleavage of the A subunit of LT by EatA, the purified passenger domain (∼9 pmol) was mixed with LT holotoxin (116 pmol), and after incubation for 1 h at 37 °C, proteins were separated by SDS-PAGE and stained with Coomassie Blue. Trypsin (∼2 pmol) was used as a positive control for proteolytic cleavage of the A subunit of LT. LT holotoxin used in these experiments was provided by Dr. John D. Clements of Tulane University School of Medicine. To date, most studies of ETEC adherence to the intestinal epithelium have focused specifically on the role of plasmid-encoded fimbrial colonization factors. However, more recent data suggest that the process of ETEC adhesion is complex and likely involves multiple virulence factors, including the secreted adhesin molecule, EtpA (5Fleckenstein J.M. Roy K. Fischer J.F. Burkitt M. Infect. Immun. 2006; 74: 2245-2258Crossref PubMed Scopus (97) Google Scholar), flagella (21Roy K. Hilliard G.M. Hamilton D.J. Luo J. Ostmann M.M. Fleckenstein J.M. Nature. 2009; 457: 594-598Crossref PubMed Scopus (150) Google Scholar), as well as the heat-labile toxin (22Johnson A.M. Kaushik R.S. Francis D.H. Fleckenstein J.M. Hardwidge P.R. J. Bacteriol. 2009; 191: 178-186Crossref PubMed Scopus (78) Google Scholar). Each of these factors has been shown to promote adhesion of ETEC to epithelial cells and to promote intestinal colonization. Curiously, by contrast, we observed that a mutant strain deficient in the production of another putative virulence protein, the EatA autotransporter (diagramed in Fig. 1a), appeared more adherent than wild type bacteria using in vitro intestinal epithelial cell monolayer adhesion assays (Fig. 1b), suggesting that this molecule might negatively modulate interactions between ETEC and the host cell. This inhibitory effect appeared to require protease activity of the EatA passenger domain as complementation of the mutant with a plasmid expressing wild type recombinant protein restored adherence to levels similar to those observed with the parent strain, whereas complementation with a plasmid-expressing protein bearing a H134R mutation in the EatA serine protease catalytic domain had no effect (Fig. 1b). Similarly, we were able to restore adherence of the eatA mutant to WT levels by addition of exogenous recombinant EatA passenger protein (rEatAp), although this was not true for the mutant rEatAp(H134R) molecule (Fig. 1c). Interestingly, antibodies raised against the passenger domain also enhanced adherence of the WT strain but had no appreciable effect on the mutant (Fig. 1d), further suggesting that EatA normally modulates the interaction of ETEC with target host cells. Because molecules influencing bacterial adherence in vitro also tend to participate in intestinal colonization, an essential virulence trait for enteric pathogens, we examined the relative ability of WT and eatA mutant ETEC strains to colonize small intestine. The results of these studies paralleled those obtained in vitro, with the eatA mutant exhibiting enhanced intestinal colonization at either 24 or 72 h following infection (Fig. 2, a and b). Similarly, the eatA mutant was shed with greater abundance in stool (Fig. 2, d and e). However, the eatA mutant did not out-compete the wild type ETEC strain when these organisms were introduced simultaneously (Fig. 2c), perhaps suggesting that levels of EatA secreted in vivo are sufficient to reverse the hyper-colonization phenotype exhibited by the mutant when introduced alone. We next questioned whether EatA might normally suppress bacterial adhesion by targeting one or more bacterial adhesins for proteolytic degradation. Because the passenger domain of EatA is released from the bacteria and freely secreted into the surrounding media (3Patel S.K. Dotson J. Allen K.P. Fleckenstein J.M. Infect. Immun. 2004; 72: 1786-1794Crossref PubMed Scopus (104) Google Scholar), we first examined culture supernatants from WT bacteria or the eatA mutant. As predicted, these demonstrated that the EatA passenger was absent from the ΔeatA mutant (jf904) strain. However, we also noted an abundance of EtpA glycoprotein in culture supernatants of the jf904 strain relative to the WT (Fig. 3a). Immunoblots of culture supernatants demonstrated significant accumulation of EtpA in the eatA mutant cultures, whereas complementation of the eatA" @default.
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• W2079031703 date "2011-08-01" @default.
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• W2079031703 title "Adhesin Degradation Accelerates Delivery of Heat-labile Toxin by Enterotoxigenic Escherichia coli" @default.
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