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• W2090696636 abstract "The tip adhesin FasG of the 987P fimbriae of enterotoxigenic Escherichia coli mediates two distinct adhesive interactions with brush border molecules of the intestinal epithelial cells of neonatal piglets. First, FasG attaches strongly to sulfatide with hydroxylated fatty acyl chains. This interaction involves lysine 117 and other lysine residues of FasG. Second, FasG recognizes specific intestinal brush border proteins that migrate on a sodium-dodecyl sulfate-polyacrylamide gel like a distinct set of 32–35-kDa proteins, as shown by ligand blotting assays. The protein sequence of high performance liquid chromatography-purified tryptic fragments of the major protein band matched sequences of human and murine histone H1 proteins. Porcine histone H1 proteins isolated from piglet intestinal epithelial cells demonstrated the same SDS-PAGE migration pattern and 987P binding properties as the 987P-specific protein receptors from porcine intestinal brush borders. Binding was dose-dependent and shown to be specific in adhesion inhibition and gel migration shift assays. Moreover, mapping of the histone H1 binding domain suggested that it is located in their lysine-rich C-terminal domains. Histone H1 molecules were visualized on the microvilli of intestinal epithelial cells by immunohistochemistry and electron microscopy. Taken together these results indicated that the intestinal protein receptors for 987P are histone H1 proteins. It is suggested that histones are released into the intestinal lumen by the high turnover of the intestinal epithelium. Their strong cationic properties can explain their association with the negatively charged brush border surfaces. There, the histone H1 molecules stabilize the sulfatide-fimbriae interaction by simultaneously binding to the membrane and to 987P. The tip adhesin FasG of the 987P fimbriae of enterotoxigenic Escherichia coli mediates two distinct adhesive interactions with brush border molecules of the intestinal epithelial cells of neonatal piglets. First, FasG attaches strongly to sulfatide with hydroxylated fatty acyl chains. This interaction involves lysine 117 and other lysine residues of FasG. Second, FasG recognizes specific intestinal brush border proteins that migrate on a sodium-dodecyl sulfate-polyacrylamide gel like a distinct set of 32–35-kDa proteins, as shown by ligand blotting assays. The protein sequence of high performance liquid chromatography-purified tryptic fragments of the major protein band matched sequences of human and murine histone H1 proteins. Porcine histone H1 proteins isolated from piglet intestinal epithelial cells demonstrated the same SDS-PAGE migration pattern and 987P binding properties as the 987P-specific protein receptors from porcine intestinal brush borders. Binding was dose-dependent and shown to be specific in adhesion inhibition and gel migration shift assays. Moreover, mapping of the histone H1 binding domain suggested that it is located in their lysine-rich C-terminal domains. Histone H1 molecules were visualized on the microvilli of intestinal epithelial cells by immunohistochemistry and electron microscopy. Taken together these results indicated that the intestinal protein receptors for 987P are histone H1 proteins. It is suggested that histones are released into the intestinal lumen by the high turnover of the intestinal epithelium. Their strong cationic properties can explain their association with the negatively charged brush border surfaces. There, the histone H1 molecules stabilize the sulfatide-fimbriae interaction by simultaneously binding to the membrane and to 987P. Enterotoxigenic Escherichia coli (ETEC) 1The abbreviations used are: ETEC, enterotoxigenic Escherichia coli; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; HPLC, high performance liquid chromatography; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry; ORF, open reading frame; EST, Expressed Sequence Tag; BBV, brush border vesicle; TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone. cause diarrhea in mammals by expressing at least one type of enteroadhesive fimbria and one type of enterotoxin. By adhering to intestinal epithelial cells, localized multiplication of an ETEC strain can progress to mucosal surface colonization and concomitant effective enterotoxin delivery. This was illustrated with host-specific ETEC strains in both animals and human volunteers (1Rutter J.M. Jones G.W. Nature. 1973; 242: 531-532Crossref PubMed Scopus (95) Google Scholar, 2Satterwhite T.K. Evans D.G. DuPont H.L. Evans Jr., D.J. Lancet. 1978; 2: 181-184Abstract PubMed Scopus (95) Google Scholar, 3Jones G.W. Rutter J.M. Infect. Immun. 1972; 6: 918-927Crossref PubMed Google Scholar, 4Smith H.W. Linggood M.L. J. Med. Microbiol. 1971; 4: 467-485Crossref PubMed Scopus (244) Google Scholar, 5Casey T.A. Schneider R.A. Dean-Nystrom E.A. Infect. Immun. 1993; 61: 2249-2252Crossref PubMed Google Scholar). ETEC are the most important etiologic agents of both neonatal and postweaning diarrhea in pigs (6Fairbrother J.M. Straw B.E. D'Allaire S. Mengeling W.L. Taylor D.J. Diseases of Swine. 8th Ed. Iowa State University Press, Ames, Iowa1999: 433-441Google Scholar, 7Moon H.W. Bunn T.O. Vaccine. 1993; 11: 200-213Crossref Scopus (173) Google Scholar). 987P-fimbriated ETEC cause diarrhea in neonatal piglets in the United States (8Shin S.J. Chang Y-F. Timour M. Lauderdale T.-L. Lein D.H. Vet. Microbiol. 1994; 38: 217-225Crossref PubMed Scopus (15) Google Scholar), Europe (9Garabal J.I. Vazquez F. Blanco J. Blanco M. Gonzalez E.A. Vet. Microbiol. 1997; 54: 321-328Crossref PubMed Scopus (30) Google Scholar, 10Blanco M. Blanco J.E. Gonzalez E.A. Mora A. Jansen W. Gomes T.A. Zerbini L.F. Yano T. de Castro A.F. Blanco J. J. Clin. Microbiol. 1997; 35: 2958-2963Crossref PubMed Google Scholar), Asia (11Supar Hirst R.G. Patten B.E. Vet. Microbiol. 1991; 26: 393-400Crossref PubMed Scopus (6) Google Scholar, 12Yamamoto T. Nakazawa M. J. Clin. Microbiol. 1997; 35: 223-227Crossref PubMed Google Scholar, 13Chen C. Kume T. Nakazawa M. Tsubaki H. Kitasato Arch. Exp. Med. 1984; 57: 211-220PubMed Google Scholar, 14Gao S. Liu X. Zhang R. Wei Sheng Wu Xue Bao. 1994; 34: 220-225PubMed Google Scholar, 15Khai L.T.L. Khoa Hoc Ky Thuat Thu Y. 2001; 8: 13-18Google Scholar), and Central and South America (16Flores-Abuxapqui J.J. Suarez-Hoil G.J. Puc-Franco M.A. Heredia-Navarrete M.R. Vivas-Rosel M.L. Oberhelman R.A. Rev. Latinoam Microbiol. 1997; 39: 145-151PubMed Google Scholar, 17García D. Cavazza M.E. Botero L. Gorziglia M. Urbina G. Liprandi F. Esparza J. Vet. Microbiol. 1987; 13: 47-56Crossref PubMed Scopus (5) Google Scholar, 18Fuentes Aguilar F. Campal Espinosa A.C. Casas Suarez S. Arteaga More N. Castro Santana M.D. Barranco J.A. Leon Barreras L. Revista Salud Anim. 2001; 23: 84-90Google Scholar). In addition to this widespread distribution, the recent identification of human ETEC strains with 987P-like fimbriae (19Viboud G.I. Jonson G. Dean-Nystrom E. Svennerholm A-M. Infect. Immun. 1996; 64: 1233-1239Crossref PubMed Google Scholar, 20Valvatne H. Sommerfelt H. Gaastra W. Bhan M.K. Grewal H.M.S. Infect. Immun. 1996; 64: 2635-2642Crossref PubMed Google Scholar) on various continents highlights the evolutionary adaptability of these fimbriae (21Honarvar S. Choi B-K. Schifferli D.M. Mol. Microbiol. 2003; 48: 157-171Crossref PubMed Scopus (27) Google Scholar). The 987P fimbria consists of the helical arrangement of protein subunits along a filamentous axis (22Isaacson R.E. Richter P. J. Bacteriol. 1981; 146: 784-789Crossref PubMed Google Scholar, 23Schifferli D.M. Abraham S.N. Beachey E.H. Infect. Immun. 1987; 55: 923-930Crossref PubMed Google Scholar). It is a heteropolymeric structure that is made up of one major subunit, FasA, and two minor subunits, FasF and FasG (24Khan A.S. Schifferli D.M. Infect. Immun. 1994; 62: 4233-4243Crossref PubMed Google Scholar). Fimbriae are not produced in the absence of any of these subunits (25Schifferli D.M. Beachey E.H. Taylor R.K. J. Bacteriol. 1991; 173: 1230-1240Crossref PubMed Google Scholar). Electron microscopy and export studies indicated that FasG is the first exported subunit followed by FasF and FasA (26Cao J. Khan A.S. Bayer M.E. Schifferli D.M. J. Bacteriol. 1995; 177: 3704-3713Crossref PubMed Google Scholar). Because fimbriae grow from the base, FasG was proposed to be the tip subunit and FasF, a linker molecule. The 987P system has one outer membrane protein (FasD) proposed to fold in a β-barrel structure and act as the 987P usher molecule (27Schifferli D.M. Alrutz M. J. Bacteriol. 1994; 176: 1099-1110Crossref PubMed Google Scholar). 987P was the first fimbrial system found to utilize two different subunit-specific periplasmic chaperones. The FasB chaperone interacts with the major fimbrial subunit, FasA (28Edwards R.A. Cao J. Schifferli D.M. J. Bacteriol. 1996; 178: 3426-3433Crossref PubMed Google Scholar), but not with FasG, the adhesin subunit. Conversely, FasC interacts only with FasG, but not FasA. A third chaperone-like protein, FasE, was also located in the periplasm and was shown to be required for optimal export of FasG. In contrast to most fimbriae, 987P do not agglutinate mammalian red blood cells but bind to receptors only found on the relevant piglet intestinal cells (29Dean E.A. Infect. Immun. 1990; 58: 4030-4035Crossref PubMed Google Scholar). Age-related resistance to 987P-fimbriated ETEC was related to the appearance of new receptors in the mucus of post-neonatal pigs (29Dean E.A. Infect. Immun. 1990; 58: 4030-4035Crossref PubMed Google Scholar, 30Dean E.A. Whipp S.C. Moon H.W. Infect. Immun. 1989; 57: 82-87Crossref PubMed Google Scholar). These receptors were proposed to inhibit 987P-mediated bacterial colonization by competing with membrane-anchored receptors. Glycolipid receptors were identified and further characterized as sulfatide and hydroxylated ceramide monohexoside (31Khan A.S. Johnston N.H. Goldfine H. Schifferli D.M. Infect. Immun. 1996; 64: 3688-3693Crossref PubMed Google Scholar, 32Dean-Nystrom E.A. Samuel J.E. Infect. Immun. 1994; 62: 4789-4794Crossref PubMed Google Scholar). FasG was shown to be responsible for 987P fimbrial binding to the former molecule, whereas FasA mediated 987P binding to the latter receptor (31Khan A.S. Johnston N.H. Goldfine H. Schifferli D.M. Infect. Immun. 1996; 64: 3688-3693Crossref PubMed Google Scholar). Alanine-scanning mutagenesis of fasG identified several positive-charged residues as involved in sulfatide recognition (33Choi B-K. Schifferli D.M. Infect. Immun. 1999; 67: 5755-5761Crossref PubMed Google Scholar). One fasG mutant (K117A) did not bind to sulfatide, suggesting that the substituted lysine residue and possibly the other charged residues communicate with the sulfate group of sulfatide by hydrogen bonding and/or salt bridges. In addition to sulfatide, a group of piglet brush border proteins of ∼32–35 kDa were described to act as 987P protein receptors in ligand blotting assays (29Dean E.A. Infect. Immun. 1990; 58: 4030-4035Crossref PubMed Google Scholar). This was confirmed by showing that FasG was the 987P adhesin responsible for this interaction (24Khan A.S. Schifferli D.M. Infect. Immun. 1994; 62: 4233-4243Crossref PubMed Google Scholar). All the allelic FasG adhesins with reduced binding to sulfatide still interacted like wild-type FasG with the protein receptors (33Choi B-K. Schifferli D.M. Infect. Immun. 1999; 67: 5755-5761Crossref PubMed Google Scholar). Studies with truncated FasG molecules indicated that at least two FasG segments (Glu-211—Ser-220 and Asp-20 —Ser-41) were involved in protein receptor recognition (34Choi B.K. Schifferli D.M. Infect. Immun. 2001; 69: 6625-6632Crossref PubMed Scopus (12) Google Scholar). Different FasG residues participated in sulfatide and protein receptor binding. Thus, the FasG adhesin was proposed to harbor two different binding domains for its two types of receptors. In this study the porcine intestinal protein receptor for the 987P fimbriae was identified, and the domain most important for the interaction with 987P was characterized. Bacterial Strains and Plasmid Constructs—E. coli strains and plasmids used in this study are listed in Table I. Standard procedures were used to prepare new constructs.Table IBacterial strains and plasmidsStrain or plasmidGenotype or relevant characteristicsReferenceE. coli987O9:K103:987P (prototype F6)71Isaacson R.E. Nagy B. Moon H.W. J. Infect. Dis. 1977; 135: 531-539Crossref PubMed Scopus (46) Google Scholar,72Nagy B. Moon H.W. Isaacson R.E. Infect. Immun. 1977; 16: 344-352Crossref PubMed Google ScholarSE5000MC4100 recA56 (Fim-)73Silhavy T.J. Berman M.L. Enquist L.W. Experiments with Gene Fusion. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1984: XIIGoogle ScholarBL21(DE3)B F- ompT hsdSB(rB-mB-) dcm gal (DE3)NovagenB834(DE3)F ompT hsdSB(rB-mB-) dcm gal met (DE3)NovagenPlasmidspBKC1fasA-fasF in pACYC18433Choi B-K. Schifferli D.M. Infect. Immun. 1999; 67: 5755-5761Crossref PubMed Google ScholarpBKC2fasG in pALTER-133Choi B-K. Schifferli D.M. Infect. Immun. 1999; 67: 5755-5761Crossref PubMed Google ScholarpZSH1Porcine histone H1.3 ORF (phH1.3) in pGEM-TThis studypZS669phH1.3 in pET22bThis studypZS384phH1.3 (Met-1 to Pro-129) in pET22bThis studypZS444phH1.3 (Met-1 to Pro-148) in pET22bThis studypZS212phH1.3 (Ala-36 to Leu-108) in pET22bThis studypZS468phH1.3 (deletion of Lys-33 to Asn-109) in pET22bThis study Open table in a new tab Media and Reagents—Wild-type E. coli strain 987 was grown in minimal medium E supplemented with pantothenic acid and glycerol (35Edwards R.A. Schifferli D.M. Mol. Microbiol. 1997; 25: 797-809Crossref PubMed Scopus (44) Google Scholar). Strain JM109 was used for recombinant DNA work. Strain B834(DE3) (Novagen, Madison, WI) was used for 35S-labeling of the 987P fimbriae. Strain BL21(DE3) was used for the expression of a porcine histone H1 protein and partial fragments of it. Cultures for colony isolation or plasmid purification were grown in LB medium. When appropriate, media were supplemented with the following antibiotics: ampicillin (200 μg/ml), chloramphenicol (30 μg/ml), tetracycline (10 μg/ml). Culture media were purchased from Difco. Restriction and modification enzymes were from New England Biolabs, Inc. (Beverly, MA). Unless specified, reagents were purchased from Sigma. Preparation of 987P Fimbriae and Brush Borders of Intestinal Epithelial Cells—Fimbriae expressed on the bacterial surface were prepared by heat extraction, and brush border vesicles were prepared from piglet enterocytes as described earlier (24Khan A.S. Schifferli D.M. Infect. Immun. 1994; 62: 4233-4243Crossref PubMed Google Scholar). Polyacrylamide Gel Electrophoresis, Western and Ligand Blotting— Bacterial pellets, isolated fimbriae, purified brush border vesicles, or histone H1 proteins were separated by SDS-PAGE in the absence of reducing agents. Gel migration shift assays were undertaken with 987P-histone H1 or 987P-poly-l-lysine mixtures (4.5:1 concentration ratios) in native 5% polyacrylamide gels (0.75 mm thick, 8 cm long) by using a phosphate buffer (0.1 m, pH 7) and reversing the electrodes at the power supply so that the positively charged histone H1 proteins migrated to the negative cathode (30 mA constant current, 4–8 h) (36Gallagher S.R. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 2. John Wiley & Sons, Inc., 1999: 1-11Google Scholar). For Western blotting, the histone H1 proteins were probed with a sheep anti-calf histone H1 polyclonal antibody (Fizgerald, Concord, MA) or with a rabbit anti-porcine histone polyclonal antibody (prepared by Cocalico Biologicals Inc., Reamstown, PA, using purified pig histone H1 proteins as antigen) using horseradish peroxidase (HRP)-conjugated secondary antibodies and enhanced chemiluminescence (ECL) for detection (27Schifferli D.M. Alrutz M. J. Bacteriol. 1994; 176: 1099-1110Crossref PubMed Google Scholar). For the ligand blotting assays, the blots were probed with isolated 987P fimbriae (10 μg in PBS, 0.5% BSA) or with HRP-conjugated concanavalin A. Bound fimbriae were detected with 987P quaternary structure-specific monoclonal antibody E11 (23Schifferli D.M. Abraham S.N. Beachey E.H. Infect. Immun. 1987; 55: 923-930Crossref PubMed Google Scholar), HRP-conjugated secondary antibodies, and ECL. For some experiments, isolated 35S-labeled 987P fimbriae were prepared from E. coli B834(DE3)/pBKC1, pBKC2, and used for fluorography, as described previously (34Choi B.K. Schifferli D.M. Infect. Immun. 2001; 69: 6625-6632Crossref PubMed Scopus (12) Google Scholar). For the ligand blotting inhibition assay, nitrocellulose strips with SDS-PAGE separated histone H1 (5 μg) were incubated with different concentrations of inhibitors for 1 h at room temperature. After several washing steps, the strips were incubated with 35S-labeled fimbriae. Amino Acid Sequencing—Brush border vesicle proteins were separated by SDS-PAGE. Protein bands recognized by the 987P fimbriae were mapped by a ligand blotting assay of one of two gels run in parallel. The two major protein bands labeled by ligand blotting were cut out of the gel and separately subjected to in-gel proteolysis with trypsin. A blank control band from a parallel gel run without brush border proteins was subjected to the same treatment. Tryptic fragments of the two proteins and the mock control were separated by microbore reversed phase HPLC. Of the two analyzed protein bands giving the same HPLC profile, only samples from HPLC peaks of the top major band were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) (Proteonomics Facility, Dr. D. W. Speicher, The Wistar Institute, University of Pennsylvania). Several samples specific for the brush borders (i.e. not present in the HPLC profile of the mock control band) and showing only one distinct peak by MALDI-MS were submitted to sequencing by Edman degradation (Applied Biosystems, Procise Protein Sequencing system, Model 494). Isolation of Porcine Histone H1 Proteins—Histone H1 proteins were isolated from intestinal epithelial cells of neonatal piglets by using a standard protocol (37Ofek I. Courtney H.S. Schifferli D.M. Beachey E.H. J. Clin. Microbiol. 1986; 24: 512-516Crossref PubMed Google Scholar, 38Brix K. Summa W. Lottspeich F. Herzog V. J. Clin. Investig. 1998; 102: 283-293Crossref PubMed Scopus (72) Google Scholar). Briefly, epithelial cells were obtained by gently scraping the mucosa of washed segments of the small intestines of 1–3-days-old piglets and placing the material in iced-cold PBS, pH 7.2, containing a mixture of protease inhibitors (Roche Applied Science). The mucosal cells were washed 3 times, and histone H1 proteins were extracted from the mucosa with 5% HClO4 at 4 °C for 30 min. The treated cells were pelleted by centrifugation (14,000 × g, 10 min) at 4 °C. The histone H1 proteins were obtained by precipitation in trichloroacetic acid (20%, final concentration) at 4 °C overnight. The precipitated histone H1 proteins were concentrated by centrifugation (14,000 × g, 20 min, 4 °C), washed with cold acetone three times, dried in a vacuum desiccator and dissolved in deonized H2O to determine the protein concentration (39Markwell M.A. Haas S.M. Bieber L.L. Tolbert N.E. Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5347) Google Scholar). Rat histones were from a rat nuclear extract. The quality of each batch was tested by SDS-PAGE to confirm at least 90% purity. Protein aliquots were stored at –70 °C for later use. Binding Assay in Microtiter Plates—Fimbriae (1 mg in 1 ml of PBS) were biotinylated with 2 μl of 10 mg/ml NHS-LC-LC-Biotin (Pierce) in deionized H2O for 2 h at room temperature. The fimbriae were dialyzed in PBS to remove unreacted biotin. Ninety-six-well enzyme-linked immunosorbent assay plates (polyvinyl chloride; Falcon, BD Biosciences) were coated with porcine histone H1 proteins or BSA as a control (5 μg/ml 0.1 m PBS, pH 7.2, 100 μl/well) and incubated at 37 °C for 3 h. The plates were blocked with 5% BSA in PBS at 37 °C for 1.5 h, washed 4 times with PBS, and incubated with serial dilutions of biotinylated 987P fimbriae in PBS, 0.5% BSA, 0.01% Tween 20 for 2 h at room temperature. After four washes with PBS, 0.01% Tween 20, 100 μl of streptavidin-HRP conjugate diluted 1/2000 in PBS, 0.5% BSA was added to each well and incubated for 30 min at room temperature. After the last washing step, bound fimbriae were detected by using o-phenylenediamine as the chromogenic substrate analogue and reading the absorbance at 450 nm (37Ofek I. Courtney H.S. Schifferli D.M. Beachey E.H. J. Clin. Microbiol. 1986; 24: 512-516Crossref PubMed Google Scholar). Protease Treatments of Brush Border Vesicles or Histone H1—Isolated brush borders (10 mg protein/ml, 50 μl) were digested for 1 h at 56 °C with proteinase K (Roche Applied Science, 10 μg) in 100 μl of digestion buffer P (10 mm Tris, pH 7.8, 5 mm EDTA, 0.5% SDS) or for 2 h at 37 °C with TPCK-treated trypsin from bovine pancreas (25 μg) in 100 μl of digestion buffer TC (100 mm NH4HCO3, pH 8.0, 0.1% SDS). Histone H1 (36 μg) was digested for 4 h at 37 °C with TLCK-treated chymotrypsin from bovine pancreas (0.5 μg) in 40 μl of digestion buffer TC or for 24 h at 37 °C with Staphylococcus aureus V8 protease (0.6 unit) in 40 μl of digestion buffer V (50 mm NH4HCO3, pH 8.0, 1.25 mm CaCl2, 1.275 m urea). The digested brush border or histone H1 proteins were retaken in 2 × SDS sample buffer for SDS-PAGE, as described above. Cloning and Expression of Porcine Histone H1 in E. coli—The following steps were taken to clone a full-length porcine histone H1 cDNA. Two expressed sequence tags (ESTs) in the pig data base (AW415122 and B1185028) contained DNA that was similar to known human and murine histone H1 sequences. These EST sequences were used to design two similar pairs of oligonucleotide primers for PCR (UH1–2, 5′-CCCCCAGTGTCCGAGCTCATCAC-3′, and LH1–2, 5′-GGTCTGTACCAAGGTGCCCTTGCTCAC-3′; UH1–3, 5′CCTCCGGTGTCCGAGCTCATCAC-3′, and LH1–3, 5′-AGTCTGCACCAGGGTGCCCTTGCTCAC-3′. Using a λ ZAPII library of pig jejunal cDNA (40Halsted C.H. Ling E.H. Luthi-Carter R. Villanueva J.A. Gardner J.M. Coyle J.T. J. Biol. Chem. 1998; 273: 20417-20424Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), two PCR fragments (both 177 bp) were obtained and cloned into plasmid pGEM-T (Promega, Madison, WI). The cloned DNA fragments were sequenced and shown to be 91.6 –100% similar to the corresponding region of the EST sequences. In a second step, the two 177-bp PCR fragments were purified and labeled in one reaction mix (Random Primer fluorescein labeling kit; PerkinElmer Life Sciences) to probe the λ ZAPII library by plaque hybridization as recommended (Stratagene, La Jolla, CA). From about 105 plaques, three positive plaques could be purified successfully in the following screenings. The inserts of the corresponding in vivo excised phagemids were sequenced, and one insert was shown to harbor the 5′ end of a histone H1 ORF. This 384-bp DNA fragment was amplified with a stop codon at its 3′ end using PCR and cloned into plasmid pET22b (Novagen) to get pZS384 (from amino acid residue Met-1 to Pro-128) (Table I). Third, a full-length porcine histone H1 cDNA was cloned by reverse transcription-PCR (Marathon cDNA amplification kit; Clontech, Palo Alto, CA) using primers designed to include the start and stop codons of porcine histone H1, as suggested by the EST sequences (UH1, 5′-ATGTCGGAGACCGCTCCAGTG-3′, and LH1, 5′-CTATTACTTCTTCTTGGAGACGGCTTTCT-3′), and isolated mRNA from piglet enterocytes as template (Micro-Trak 2.0 mRNA isolation system; Invitrogen). The amplified cDNA was cloned into pGEM-T (pZSH1) and shown to represent a full-length 669-bp ORF by DNA sequencing. To express histone H1 or various continuous domains of it in E. coli, four PCR products incorporating histone H1 DNA were amplified from pZSH1 as template. For this three pairs of primers including NdeI at their 5′ end and BamHI at their 3′ end were used to amplify and clone corresponding fragments into the expression plasmid pET22b. The three resulting constructs were pZS669 (full-length histone H1), pZS444 (from residues Met-1 to Phe-148), and pZS212 (from residues Ala-36 to Leu-108). A fourth construct, pZS468, was prepared by inverse PCR (41Silver J. McPherson M.J. Quirke P. Taylor G.R. PCR. IRL Press, Oxford University Press, Oxford1991: 137-146Google Scholar) using pZS669 as template and two primers designed to delete residues Lys-30 to Asn-109 from the full-length histone H1 (Table I). The histone H1 protein and protein fragments were expressed in E. coli BL21(DE3) and analyzed by SDS-PAGE as described previously (26Cao J. Khan A.S. Bayer M.E. Schifferli D.M. J. Bacteriol. 1995; 177: 3704-3713Crossref PubMed Google Scholar, 34Choi B.K. Schifferli D.M. Infect. Immun. 2001; 69: 6625-6632Crossref PubMed Scopus (12) Google Scholar). Immunohistochemistry and Electron Microscopy—Several 1-cm-long intestinal segments were collected from neonatal piglets and fixed immediately after euthanasia following IACUC guidelines. For immunohistochemistry, tissues were fixed in 10% buffered formalin, dehydrated, and paraffin-embedded. Glass slide-mounted serial sections were prepared for indirect immunohistochemistry using mouse anti-human histone H1 monoclonal IgG2a (clone AE-4; Upstate, Charlottesville, VA). All reactions were performed at room temperature. Tissue sections were treated with H2O2 for 10 min to inactivate endogenous peroxidases and incubated with the primary antibody for 30 min followed by biotinylated anti-mouse antibody for 15 min at room temperature, HRP-conjugated streptavidin for 15 min, and 3,3′-diaminobenzidine as chromogen for 3 min. The counterstaining was obtained using Mayer's hematoxylin; the slides were then dehydrated, and coverslips were applied. The negative controls were obtained using N-Universal Negative Control (cocktail of mixture of nonspecific mouse IgG, IgM, IgG2a, IgG2b, and IgG3, DakoCytomation, Carpinteria, CA) instead of the primary antibodies. Transmission electron microscopy was undertaken at the Biomedical Imaging Core, University of Pennsylvania. Intestinal tissue was fixed in 4% paraformaldehyde, embedded in LR-White (Electron Microscopy Sciences, Hatfield, PA), and UV-cured at –20 °C. 90-nm-thick sections were picked up on nickel grids for post-embedding immunostain with the mouse anti-human histone H1 monoclonal IgG2a (clone AE-4 described above) followed by an anti-mouse antibody gold particle (10- or 15-nm diameters) conjugate. A mouse monoclonal IgG2a was used as the isotype (negative) control (Caltag Laboratories, Burlingame, CA). Images were captured in Jeol JEM 1010 by an AMT 12-HR-aided Hamamatsu CCD camera (42Smith R.M. Jarett L. De Pablo F. Scanes C.G. Weintraub B.D. Handbook of Endocrine Research Techniques. Academic Press, Inc., New York1993Google Scholar). All the supplies for electron microscopy were from Electron Microscopy Sciences (Fort Washington, PA). Binding of 987P Fimbriae to Protein Receptors of Brush Border Vesicles—As shown previously, the 987P fimbriae, used as ligand, bind to a group of intestinal brush molecules migrating like globular proteins of 32–35 kDa (Fig. 1A, lanes 1 and 4). The proteinaceous nature of these receptors was confirmed by their susceptibility to the proteolytic activities of trypsin or proteinase K (Fig. 1A, lanes 2, 3, 5, and 6). N-Glycosidase F treatment of the isolated brush border vesicles did not affect the 987P binding activity and the migration properties of the receptor bands on SDS-polyacrylamide gels (Fig. 1B, lanes 5 and 6). To control for the activity of the enzyme, a known N-glycosylated protein (RNase B) was treated with the same N-glycosidase and probed with concanavalin A, a lectin known to recognize the N-glycosylated moiety of RNase B. In contrast to the untreated RNase B, N-glycosidase F-treated RNase B appeared smaller by SDS-PAGE analysis and was not recognized by concanavalin A on a ligand blot (Fig. 1B, lanes 1–4), indicating that the enzyme was active. Moreover, dithiothreitol treatment of the isolated brush border vesicles did also not affect the 987P binding activity and the migration properties of the receptor bands (Fig. 1C, lanes 3 and 4). The activity of dithiothreitol was controlled with the major 987P fimbrial subunit which contains one disulfide bridge and migrates as an apparently larger protein when reduced (Fig. 1C, lanes 1 and 2). These results indicated that the studied intestinal 987P protein receptors do not involve N-linked carbohydrates or disulfide bridges that are important for their 987P fimbriae-specific receptor activity. Identification of the Protein Receptors for 987P—The identity of the 987P protein receptors was determined by protein sequencing. For this, brush border proteins from piglet intestinal epithelial cells were separated by SDS-PAGE, and the two major protein bands recognized by the 987P fimbriae were individually digested with trypsin. Tryptic fragments of the two protein bands separated by microbore reversed phase HPLC showed a similar peak profile. This indicated that the two protein bands represented partially degraded or post-translationally modified variants of the same protein. Thus, only the upper protein band was studied further. Samples of separate and distinctly unique peaks were analyzed by MALDI-MS. The samples containing a single peak by MALDI-MS were submitted to protein sequencing. Two samples contained one peptide each that could be sequenced successfully. The peptide sequences (ALAAAGYDVEK an" @default.
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