Identification of 987P Protein Receptors for Enterotoxigenic Escherichia coli

CHINESE JOURNAL OF BIOTECHNOLOGY Volume 24, Issue 3, March 2008 Online English edition of the Chinese language journal RESEARCH PAPER

Cite this article as: Chin J Biotech, 2008, 24(3), 363−367.

Identification of 987P Protein Receptors for Enterotoxigenic Escherichia coli Guoqiang Zhu1, Jianye Wang1, and Xiaofang Zhu2 1

College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China

2

Northern Jiangsu People Hospital, Yangzhou 225001, China

Abstract: The 987P fimbriae of enterotoxigenic Escherichia coli (ETEC) mediate adhesive interactions with brush border vesicle (BBV) of the intestinal epithelial cells from the neonatal piglets. By adhering to intestinal epithelial cells, producing localized multiplication, the 987P ETEC can progress to mucosal surface colonization and concomitant effective enterotoxin delivery. To identify the receptors for the 987P, BBV proteins from piglet intestinal villous epithelial cells were separated by SDS-PAGE and analyzed by ligand blot, protein bands with a set of 32–35 kD recognized by the 987P fimbriae were subjected to in gel proteolysis with trypsin. The tryptic fragments were separated by microbore reversed phase HPLC (RP-HPLC), samples shown to contain one major peak by MALDI-MS were submitted to Edman sequencing, three peptides were sequenced successfully and the all of three peptides matched the sequence of human or porcine histone H1 proteins. Porcine histone H1 proteins isolated from both piglet intestinal epithelial cells and BBV demonstrated the same SDS-PAGE migration pattern and 987P-binding properties as the 987P-specific protein receptors from piglet intestinal brush border did. The results indicated that the 987P protein receptors are piglet BBV-derived Histone H1 proteins. Keywords: enterotoxigenic Escherichia coli; 987P; brush border vesicle (BBV); histone H1 proteins; receptor

Introduction 987P-fimbriated Enterotoxigenic Escherichia coli (ETEC) is one of the most important etiologic agents of neonatal diarrhea in piglets in China[1,2]. The pathogen causes diarrhea by expressing both enteroad hesive fimbria and enterotoxin, by adhering to the intestinal epithelial cells mediated by the enteroadhesive fimbria, which is an initial step and also the most important process for the interaction between the pathogen and host cells, localized multiplication of an 987P-fimbriated ETEC strain can progress to mucosal surface colonization and concomitant effective enterotoxin delivery[3]. The fimbriae consists of the helical arrangement of protein subunits along a filamentous

axis, in contrast to most fimbriae, 987P do not agglutinate mammalian red blood cells[4], but bind to the relevant piglet intestinal cells and piglet brush border proteins of 32–35 kD which were described to act as 987P protein receptors in ligand blotting assays. In this study, the porcine intestinal protein receptors for the 987P fimbriae were isolated, identified, and characterized.

1

Materials and methods

1.1 Animal: one-day-old piglets 1.2 Bacterial strains, media and reagents Diarrheagenic E. coli 987P strain and quaternary structurespecific monoclonal antibody (E11) against 987p fimbriae

Received: May 16, 2007; Accepted: August 8, 2007 Supported by: the National Natural Science Foundation of China (No. 30571374), Jiangsu National Science Foundation (No. BK2005049), the projects-sponsored by SRF for ROCS, SEM and USDA project (No. 2002-35204-12216) Corresponding author: Guoqiang Zhu. +86-514-87972590; Fax: 86-514-87972218; E-mail: [email protected], [email protected] Copyright © 2008, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.

Guoqiang Zhu et al. / Chinese Journal of Biotechnology, 2008, 24(3): 363–367

were kindly provided by Dr. Schifferli[5]. Trypsin and horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG) were purchased from Sigma (St. Louis, Mo). Enhanced chemiluminescence, biotinconjugated anti-HRP, nitrocellulose membrane were from Bio-Rad Laboratories (Richmond, Calif). Unless specified otherwise, all reagents and chemicals including media MME and LB were from Difco (Detroit, Mich). 1.3 Preparation of 987P fimbriae and brush borders of intestinal epithelial cells Fimbriae expressed on the bacterial surface were isolated from 987P fimbriae strain and prepared by heat extraction and precipitation with final 20% concentration of ammonium sulfate[6], separation and analysis of fimbrial preparation were undertaken by SDS-PAGE and Western blotting. Brush border vesicles (BBV) were prepared from small intestinal epithelial cells of one-day-old piglet as described earlier[7]. Purity of the BBV was assessed by microscopy, protein concentration were determined and protein aliquots were frozen at –70oC for latter use. 1.4 SDS-PAGE and ligand blotting assays for BBV It was described by Choi et al[8]. Briefly, purified BBV (30 μg) were separated by SDS-PAGE in the absence of reducing agents and electroblotted onto a nitrocellulose membrane. After being blocked with TBS containing 3% bovine serum albumin overnight at 4°C, blots were incubated with purified fimbriae (10 μg/mL) for 1 h at room temperature; fimbriae were detected using Mab E11, HRP-conjugated goat anti-mouse antibody, and enhanced chemiluminescence. 1.5 Amino acid sequencing BBV 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). Samples specific for the brush borders and showing only one distinct peak by MALDI-MS were submitted to sequencing by Edman degradation (Applied Biosystems, Procise Protein Sequencing system, Model 494). 1.6 Isolation of porcine histone H1 proteins and ligand blotting assays Histone H1 proteins were isolated from the intestinal epithelial cells of neonatal piglets using a standard protocol[6]. The quality and purity of H1 proteins was tested

Fig. 1 Piglet intestinal brush border vesicle proteins separated by SDS-PAGE and recognized by 987P

by SDS-PAGE to confirm. Ligand blotting assays for H1 proteins were described previously[8]. 1.7 Biotinylation of fimbriae and 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) were coated with porcine histone H1 proteins and incubated at 37°C for 3 h. The plates were blocked with 5% BSA in PBS at 37°C for 1.5 h, and incubated with biotinylated 987P fimbriae for 2 h at room temperature. Streptavidin-HRP conjugate was added to each well and incubated for 30 min at room temperature. After the final washing step, bound fimbriae were detected using o-phenylenediamine as the chromogenic substrate analogue and reading the absorbance at 450 nm[7].

2

Results

2.1 Binding of 987P fimbriae to brush border vesicles BBV proteins from piglet intestinal villous epithelial cells were separated by SDS-PAGE (Fig. 1, left) and analyzed by ligand blot, protein bands with a set of 32–35 kD were recognized by the 987P fimbriae as ligand (Fig. 1, right). The proteinaceous nature of these proteins was confirmed by their susceptibility to the proteolytic activities of trypsin or proteinase K (data not shown). 2.2 Identification of 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 separated by microbore reversed phase HPLC showed a similar peak profile. Samples of separate and distinctly unique peaks were analyzed by MALDI-MS. The samples containing a single peak by MALDI-MS were submitted for protein sequencing. The peptide sequences (AETAP, ALAAAGYDVEK and LGLK)

Guoqiang Zhu et al. / Chinese Journal of Biotechnology, 2008, 24(3): 363–367

Fig. 2 Three sequenced peptides aligned to 3 human, 2 murine and 1 rat histone H1 proteins

were shown by BLAST to match the sequences of several human and murine histones H1 (Fig. 2) and of the only sequenced porcine. Histone H1 (the testis-specific H1 variant) as well as two histone segments found in the pig expressed sequence tags (ESTs) data base. 2.3 Binding of 987P to histone H1 proteins purified from porcine Intestinal epithelial cells─The purity of the isolated Histone H1 proteins from porcine intestinal epithelial cells were evaluated by SDS-PAGE (Fig. 3). Purified porcine histone H1 proteins (Fig. 3, lanes 3 and 6) co-migrated with porcine brush border vesicle (BBV) (Fig. 3, lanes 4 and 7) and the rat histone H1 (Fig. 3, lane 1) as 32–35 kD proteins, matching the previously described migration properties of the 987P protein receptors recognized by the 987P fimbriae as ligand. But the porcine histone H1-depleted BBV extract did not have the set of proteins (Fig. 3, lanes 2 and5). The identity of the isolated porcine histone H1 proteins was confirmed by Western blotting analysis using anti-calf histone H1 polyclonal antibodies (Fig. 3, lanes 5–7). The antibody reacted only with purified porcine histone H1 proteins (Fig. 3, lane 6) and the corresponding bands in the total porcine BBV extract (Fig. 2, lane 7) but not with the porcine histone H1-depleted BBV extract (Fig. 2, lane 5),

confirming the identity of the purified proteins and the specificity of the antibodies. To verify that the porcine histone H1 proteins behave like the 987P protein receptors, the binding of 987P fimbriae to porcine histone H1 was ligand concentration-dependent, as shown in solid-phase binding (Fig. 3B, filled circles).

Fig. 3 Histone H1 proteins and the BBV receptor proteins for 987P Coomassie blue stained gels (lanes 1 to 4), and ligand blot (lanes 5 and 7) of BBV or purified histone H1 proteins. Rat histone H1 proteins (lane 1), porcine histone H1-depleted BBV extract (lanes 2 and 5), porcine histone H1 proteins purified from BBV (lanes 3 and 6) and total piglet BBV proteins (lanes 4 and 7)

Guoqiang Zhu et al. / Chinese Journal of Biotechnology, 2008, 24(3): 363–367

Moreover, unlike the 987P fimbriae, the 987P-like CS18 fimbriae, which mediated the binding of human ETEC to enterocytes, did not bind significantly to histone H1 proteins (Fig. 3B, filled squares). Because histone H1 molecules have a high content of lysine residues, it is possible that the 987P fimbriae interact non-specifically with these residues through salt bridges. To evaluate the affinity of 987P for arrays of positively charged residues, parallel binding assays were undertaken with histone H1 and poly-L-lysine molecules of 15–30 kD (Fig. 3B, open circles). Although 987P fimbriae bound to the latter molecules, binding was significantly better to histone H1 when both lysine-rich molecules were fixed to a solid phase.

3

Discussion

Brush border vesicle (BBV) of the intestinal epithelial cells from the neonatal piglets were separated by SDSPAGE and analyzed by ligand blot analysis for the 987P fimbriae of ETEC as a ligand; a set of 32–35 kD recognized by the 987P fimbriae were originally detected on intestinal brush borders and mucus of pigs[9]. The proteinaceous nature of these proteins was confirmed by their susceptibility to the proteolytic activities of trypsin or proteinase K. Later, the 987P minor subunit FasG was identified as the specific ligand molecule for the porcine protein receptors[7]. The observed interaction involved at least two continuous stretches of the FasG sequence[10]. Here, studies were aimed at characterizing the set of 32–35 kD protein receptors subjected to in gel proteolysis with trypsin. The tryptic fragments were separated by microbore reversed phase HPLC (RP-HPLC), samples shown to contain one major peak by MALDI-MS were submitted to Edman sequencing, three peptides were sequenced successfully and the all three peptides matched the sequence of human or porcine histone H1 proteins. Porcine histone H1 proteins isolated from both piglet intestinal epithelial cells and BBV demonstrated the same SDS-PAGE migration pattern and 987P-binding properties as the 987P-specific protein receptors from piglet intestinal brush border did. Protein sequencing suggested that these receptors were histone H1 proteins and the presence of histone H1 on the intestinal mucosa of neonatal piglets was confirmed by biochemical means. The above results indicated that the 987P protein receptors are piglet BBV-derived histone H1 proteins. The proteins were non-glycosylated proteins, and highly basic and lack cysteines. This result was consistent with the findings that dithiothreitol or N-glycosidase F did not affect the structure and the binding properties of the 987P protein receptors, as evaluated by SDS-PAGE and ligand blotting[11]. In this study 987P was confirmed to bind specifically to purify porcine histone H1 in a dose-dependent manner.

Using labeled ligand, the interaction could be inhibited in a dose-dependent manner with unlabeled 987P. A porcine histone H1 gene was cloned in an expression vector to help map its binding domain (data unknown). The deduced protein sequence of the open reading frame of the gene was most similar to the human and murine histone H1.3 and H1.4 proteins. It is hoped that further studies on the 987P binding domains and their relative importance for fimbrial adhesion will help to design new therapeutic and prophylactic agents against colonizing bacterial pathogens.

Acknowledgments The authors gratefully thank Dr. Dieter M. Schifferli from University of Pennsylvania as a supervisor for the study. This study was partially supported by USDA project (No. 2002-35204- 12216). REFERENCES [1] Bertschinger HU, Nief V, Tschape H. Active oral immunization of suckling piglets to prevent colonization after weaning by enterotoxigenic Escherichia coli with fimbriae F18. Vet Microbiol, 2000, 71: 255–267. [2] Barnhart MM, Saruer FG, Pinkner JS, et al. Chaperonesubunit-usher interactions required for donor strand exchange during bacterial pilus assembly. J Bacteriol, 2003, 185: 2723–2730. [3] Edwards RA, Puente JL. Fimbrial expression in enteric bacteria: a critical step in intestinal pathogenesis. Trends Microbiol, 1998, 6: 282–287. [4] Ike K, Nakazawa M, Tsuchimoto M, et al. Hemagglutination by pilus antigen 987P of enterotoxigenic Escherichia coli. Microbiol Immun, 1987, 31: 1255–1258. [5] Schifferli DM, Abraham SN, Beachey EH. Use of monoclonal antibodies to probe subunit-and polymer-specific epitopes of 987P fimbriae of Escheria coli. Infect Immun, 1987, 55: 923–930. [6] Brix K, Summa W, Lottspeich F, et al. Extracellularly occurring histone H1 mediates the binding of thyroglobulin to the cell surface of mouse macrophages. J Clin Investig, 1998, 102: 283–293. [7] Khan AS, Schifferli DM. A minor 987P protein different from the structural fimbrial subunit is the ashesin. Infect Immun, 1994, 62: 4233–4243. [8] Choi BK, Schifferli DM. Lysine residue 117 of the FasG adhesin of enterotoxigenic Escherichia coli is essential for binding of 987P fimbriae to sulfatide. Infect Immun, 1999, 67: 5755–5761. [9] Dean EA. Comparison of receptors for 987P pili of enterotoxigenic Escherichia coli in the small intestines of neonatal and older pig. Infect Immun, 1990, 58: 4030–4035. [10] Choi BK, Schifferli DM. Characterization of FasG

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[11] Zhu GQ, Chen H, Choi BK, et al. Histone H1 proteins act as receptors for the 987P fimbriae of enterotoxigenic Escherichia coli. J Biol Chem, 2005, 280: 23057–23065.