N-Acetyllactosamine Conjugated to Gold Nanoparticles Inhibits Enteropa
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《感染与免疫杂志》
Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, AB T2N 4N1, Canada
Division of Gastroenterology, University of Calgary, Calgary, AB T2N 4N1, Canada
Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada
ABSTRACT
We previously reported that the bundle-forming pilus-mediated localized adherence of enteropathogenic Escherichia coli to HEp-2, T84, and Caco-2 cells is inhibited by N-acetyllactosamine neoglycoconjugates. The results presented here extend this observation to the epithelium of biopsy specimens obtained from the human adult duodenum, terminal ileum, and colon.
TEXT
Enteropathogenic Escherichia coli (EPEC) is a common cause of diarrhea in neonates in developing nations and in adults who travel to regions of the world where the organism is endemic (17). EPEC strains infect and colonize their hosts through a three-step interactive process that involves initial binding, signal transduction, and intimate adherence (4, 6, 8, 9, 14). In the first stage of this process, EPEC forms microcolonies on the host cell surface, a phenotype called localized adherence (7, 10) which is dependent on bundle-forming pili (BFP) expressed by the EPEC (7, 10). These BFP are also thought to be required for efficient colonization of the host during infection (3, 5).
We previously reported that N-acetyllactosamine (LacNAc) neoglycoconjugates inhibit EPEC localized adherence in laryngeal HEp-2 cells (21), as well as in the intestinal Caco-2 and T84 cell lines (13, 21). More recently, we demonstrated that BFP likely act as a LacNAc-specific adhesin in this processes (13). Here, we extended these tissue culture cell observations to human intestinal biopsy specimens to further explore the feasibility of using LacNAc-based glycoconjugate preparations as a potential treatment strategy in EPEC gastrointestinal infections.
After informed consent was obtained, tissue samples were obtained during colonoscopy procedures performed for colon cancer screening and/or during upper endoscopy procedures using standard large biopsy forceps. Biopsies from adjacent regions were used for pathological assessment. Only biopsy specimens with normal morphology as determined by histological review were used in this study. The method and process used for obtaining the specimens were reviewed and approved by the Ethics Review Panel of the University of Calgary. The ages and sexes of the subjects from whom biopsies were obtained are shown in Table 1. The mean age of the seven male and nine female subjects was 57 years (range, 51 years). Once removed, the biopsy samples were placed into ice-chilled phosphate-buffered physiological saline (pH 7.2) and used in the experiments within 1 h.
Polystyrene biopsy holders (Fig. 1) were custom designed so that EPEC binding could be physically restricted to the epithelial surface of the biopsy. The biopsy specimens were oriented and mounted with the luminal side up using a dissecting microscope. Thus, the mucosal biopsy involved a two-chamber system, with a bottom submucosal side and an upper luminal side. Both chambers were filled with Dulbecco modified Eagle medium during the experiments.
Prior to each experiment, BFP expression by the wild-type E2348/69 EPEC strain (13, 21) was induced by culturing the bacteria in Dulbecco modified Eagle medium as described previously (20, 21). The cultures were added to 48-well plates containing either 0.8 mg/ml LacNAc-Au or Pk-Au (13) and incubated for 30 min at 37°C in a CO2 incubator. The method used for preparation of the LacNAc and Pk-Au compounds has been described previously (11). The medium was removed from the luminal upper compartment of a biopsy chamber, and 100 μl of the LacNAc-Au-E2348/69 or Pk-Au-E2348/69 mixture was added to the epithelial surface of a biopsy sample. After 30 min of incubation at 37°C in the CO2 incubator, the biopsies were washed five times with ice-chilled phosphate-buffered saline to remove unbound bacteria.
To determine the number of adherent bacteria, whole genomic DNA was extracted from the intestinal biopsies using a QIAamp DNA Micro kit (QIAGEN, Mississauga, Ontario, Canada). Bacteria were quantified by performing real-time PCR of the intimin (eaeA) gene using the oligonucleotide primers described elsewhere (16). Real-time PCR was performed with a Rotor-Gene 3000 instrument (Corbett Research, Sydney, Australia) using a QuantiTect SYBR Green PCR kit (QIAGEN, Mississauga, Ontario, Canada) according to the manufacturer's instructions. The thermal cycling conditions used for amplifying the eaeA gene are described elsewhere (16). Five 10-fold dilutions of whole genomic DNA that was isolated from E2348/69 using a standard protocol (1) were used as an internal control for the PCR.
Using this approach, we observed significant variability in the number of EPEC cells attached to the individual biopsy specimens (Table 1). In the transverse colon biopsies alone, we observed a >5-log range in EPEC adherence; the values ranged from 480 adherent bacteria for patient 35 to 1.2 x 107 adherent bacteria for patient 94 (Table 1). For two patients, no EPEC bound to the biopsy samples (data not shown). There was no correlation between subject age or sex and the number of adherent bacteria (P = 0.655 and P = 0.952, respectively, as determined by the Pearson product-moment correlation test).
Regardless of the variability in EPEC binding, LacNAc-Au at a concentration of 0.8 mg/ml inhibited (compared to the negative Pk-Au control) EPEC adherence to the epithelial surface in all biopsies obtained from all subjects (Fig. 2) (13). The inhibitory effect was significant in the duodenal (P = 0.002), the terminal ileum (P = 0.002), and the transverse colon (P < 0.001) biopsy specimens (as determined by the Wilcoxon signed-rank test). The residual EPEC binding to the intestinal biopsy specimens when LacNAc-Au was present may be attributed to other potential EPEC adhesins, including a phosphatidylethanolamine-specific BFP-associated adhesin (2, 22), flagella (11), EspA (15), and intimin (18, 19).
This study allowed us to extend our previous observation that EPEC may exploit host cell LacNAc or LacNAc-related glycan receptor sequences during colonization of HEp-2, Caco-2, and T84 cells in culture to the adult human intestinal epithelium. This suggests that a BFP-associated lectin-like adhesin is likely involved in the initial attachment of EPEC to LacNAc or LacNAc-related receptors on intestinal epithelial cells in adults at least. This suggestion is further supported by studies which showed that mutant EPEC strains that lack the capacity to express BFP were less virulent in human adult volunteer challenge studies (3). In contrast to these results, Hicks and colleagues (12) reported that BFP were not important mediators of EPEC early adherence to pediatric intestinal biopsy specimens when the in vitro organ culture system was used. The discrepancy between our results and those of Hicks and colleagues may be attributed to differences between EPEC attachment to adult tissues and EPEC attachment to pediatric tissues. However, in a more recent paper, Cleary and colleagues (5) reported that BFP are required for efficient colonization of pediatric intestinal biopsy tissue at early times, a conclusion that is consistent with the results reported here.
Since LacNAc-Au reduced EPEC binding to the epithelial surface of all the biopsy specimens examined, such a glycoconjugate preparation may offer a new therapeutic modality for managing patients with EPEC infections and also may have prophylactic value for subjects traveling to regions where the organism is endemic. Our results also support investigation of the therapeutic or prophylactic potential of Au-based glycoconjugates bearing specific glycan sequences implicated in the colonization strategies of other important microbial pathogens.
ACKNOWLEDGMENTS
Financial support was provided by the Canadian Bacterial Diseases Network (G.D.A.), the Alberta Ingenuity Centre for Carbohydrate Science (G.D.A. and P.I.K.), and the Canadian Institutes for Health Research (G.D.A.). R.M.H. was the recipient of a studentship from NSERC.
G.D.A. is a coinventor on U.S. patent 6,291,435 for the treatment of diarrhea caused by enteropathogenic E. coli.
FOOTNOTES
Corresponding author. Mailing address: Department of Microbiology & Infectious Diseases, Faculty of Medicine, University of Calgary, 3330 Hospital Drive, NW, Calgary, AB T2N 4N1, Canada. Phone: (403) 220-6885. Fax: (403) 270-2772. E-mail: glen.armstrong@ucalgary.ca.
REFERENCES
1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1999. Short protocols in molecular biology, 4th ed. John Wiley & Sons, New York, NY.
2. Barnett Foster, D., D. Philpott, M. Abul-Milh, M. Huesca, P. M. Sherman, and C. A. Lingwood. 1999. Phosphatidylethanolamine recognition promotes enteropathogenic E. coli and enterohemorrhagic E. coli host cell attachment. Microb. Pathog. 27:289-301.
3. Bieber, D., S. W. Ramer, C. Y. Wu, W. J. Murray, T. Tobe, R. Fernandez, and G. K. Schoolnik. 1998. Type IV pili, transient bacterial aggregates, and virulence of enteropathogenic Escherichia coli. Science 280:2114-2118.
4. Campellone, K. G., and J. M. Leong. 2003. Tails of two Tirs: actin pedestal formation by enteropathogenic E. coli and enterohemorrhagic E. coli O157:H7. Curr. Opin. Microbiol. 6:82-90.
5. Cleary, J., L. C. Lai, R. K. Shaw, A. Straatman-Iwanowska, M. S. Donnenberg, G. Frankel, and S. Knutton. 2004. Enteropathogenic Escherichia coli (EPEC) adhesion to intestinal epithelial cells: role of bundle-forming pili (BFP), EspA filaments and intimin. Microbiology 150:527-538.
6. Delahay, R. M., G. Frankel, and S. Knutton. 2001. Intimate interactions of enteropathogenic Escherichia coli at the host cell surface. Curr. Opin. Infect. Dis. 14:559-565.
7. Donnenberg, M. S., J. A. Giron, J. P. Nataro, and J. B. Kaper. 1992. A plasmid-encoded type IV fimbrial gene of enteropathogenic Escherichia coli associated with localized adherence. Mol. Microbiol. 6:3427-3437.
8. Donnenberg, M. S., and J. B. Kaper. 1992. Enteropathogenic Escherichia coli. Infect. Immun. 60:3953-3961.
9. Donnenberg, M. S., and T. S. Whittam. 2001. Pathogenesis and evolution of virulence in enteropathogenic and enterohemorrhagic Escherichia coli. J. Clin. Investig. 107:539-548.
10. Giron, J. A., A. S. Ho, and G. K. Schoolnik. 1993. Characterization of fimbriae produced by enteropathogenic Escherichia coli. J. Bacteriol. 175:7391-7403.
11. Giron, J. A., A. G. Torres, E. Freer, and J. B. Kaper. 2002. The flagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells. Mol. Microbiol. 44:361-379.
12. Hicks, S., G. Frankel, J. B. Kaper, G. Dougan, and A. D. Phillips. 1998. Role of intimin and bundle-forming pili in enteropathogenic Escherichia coli adhesion to pediatric intestinal tissue in vitro. Infect. Immun. 66:1570-1578.
13. Hyland, R., T. P. Griener, G. L. Mulvey, P. I. Kitov, O. P. Srivastava, P. Marcato, and G. D. Armstrong. 2006. Basis for N-acetyllactosamine-mediated inhibition of enteropathogenic Escherichia coli localized adherence. J. Med. Microbiol. 55:669-675.
14. Kenny, B. 2002. Mechanism of action of EPEC type III effector molecules. Int. J. Med. Microbiol. 291:469-477.
15. Knutton, S., I. Rosenshine, M. J. Pallen, I. Nisan, B. C. Neves, C. Bain, C. Wolff, G. Dougan, and G. Frankel. 1998. A novel EspA-associated surface organelle of enteropathogenic Escherichia coli involved in protein translocation into epithelial cells. EMBO J. 17:2166-2176.
16. Leverton, L. Q., and J. B. Kaper. 2005. Temporal expression of enteropathogenic Escherichia coli virulence genes in an in vitro model of infection. Infect. Immun. 73:1034-1043.
17. Nataro, J. P., and J. B. Kaper. 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11:142-201.
18. Phillips, A. D., and G. Frankel. 2000. Intimin-mediated tissue specificity in enteropathogenic Escherichia coli interaction with human intestinal organ cultures. J. Infect. Dis. 181:1496-1500.
19. Sinclair, J. F., and A. D. O'Brien. 2002. Cell surface-localized nucleolin is a eukaryotic receptor for the adhesin intimin-gamma of enterohemorrhagic Escherichia coli O157:H7. J. Biol. Chem. 277:2876-2885.
20. Vanmaele, R. P., M. C. Finlayson, and G. D. Armstrong. 1995. Effect of enteropathogenic Escherichia coli on adherent properties of Chinese hamster ovary cells. Infect. Immun. 63:191-198.
21. Vanmaele, R. P., L. D. Heerze, and G. D. Armstrong. 1999. Role of lactosyl glycan sequences in inhibiting enteropathogenic Escherichia coli attachment. Infect. Immun. 67:3302-3307.
22. Wu, Y., B. Lau, S. Smith, K. Troyan, and D. E. Barnett Foster. 2004. Enteropathogenic Escherichia coli infection triggers host phospholipid metabolism perturbations. Infect. Immun. 72:6764-6772.(Romney M. Hyland, Paul Beck, George L. M)
Division of Gastroenterology, University of Calgary, Calgary, AB T2N 4N1, Canada
Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada
ABSTRACT
We previously reported that the bundle-forming pilus-mediated localized adherence of enteropathogenic Escherichia coli to HEp-2, T84, and Caco-2 cells is inhibited by N-acetyllactosamine neoglycoconjugates. The results presented here extend this observation to the epithelium of biopsy specimens obtained from the human adult duodenum, terminal ileum, and colon.
TEXT
Enteropathogenic Escherichia coli (EPEC) is a common cause of diarrhea in neonates in developing nations and in adults who travel to regions of the world where the organism is endemic (17). EPEC strains infect and colonize their hosts through a three-step interactive process that involves initial binding, signal transduction, and intimate adherence (4, 6, 8, 9, 14). In the first stage of this process, EPEC forms microcolonies on the host cell surface, a phenotype called localized adherence (7, 10) which is dependent on bundle-forming pili (BFP) expressed by the EPEC (7, 10). These BFP are also thought to be required for efficient colonization of the host during infection (3, 5).
We previously reported that N-acetyllactosamine (LacNAc) neoglycoconjugates inhibit EPEC localized adherence in laryngeal HEp-2 cells (21), as well as in the intestinal Caco-2 and T84 cell lines (13, 21). More recently, we demonstrated that BFP likely act as a LacNAc-specific adhesin in this processes (13). Here, we extended these tissue culture cell observations to human intestinal biopsy specimens to further explore the feasibility of using LacNAc-based glycoconjugate preparations as a potential treatment strategy in EPEC gastrointestinal infections.
After informed consent was obtained, tissue samples were obtained during colonoscopy procedures performed for colon cancer screening and/or during upper endoscopy procedures using standard large biopsy forceps. Biopsies from adjacent regions were used for pathological assessment. Only biopsy specimens with normal morphology as determined by histological review were used in this study. The method and process used for obtaining the specimens were reviewed and approved by the Ethics Review Panel of the University of Calgary. The ages and sexes of the subjects from whom biopsies were obtained are shown in Table 1. The mean age of the seven male and nine female subjects was 57 years (range, 51 years). Once removed, the biopsy samples were placed into ice-chilled phosphate-buffered physiological saline (pH 7.2) and used in the experiments within 1 h.
Polystyrene biopsy holders (Fig. 1) were custom designed so that EPEC binding could be physically restricted to the epithelial surface of the biopsy. The biopsy specimens were oriented and mounted with the luminal side up using a dissecting microscope. Thus, the mucosal biopsy involved a two-chamber system, with a bottom submucosal side and an upper luminal side. Both chambers were filled with Dulbecco modified Eagle medium during the experiments.
Prior to each experiment, BFP expression by the wild-type E2348/69 EPEC strain (13, 21) was induced by culturing the bacteria in Dulbecco modified Eagle medium as described previously (20, 21). The cultures were added to 48-well plates containing either 0.8 mg/ml LacNAc-Au or Pk-Au (13) and incubated for 30 min at 37°C in a CO2 incubator. The method used for preparation of the LacNAc and Pk-Au compounds has been described previously (11). The medium was removed from the luminal upper compartment of a biopsy chamber, and 100 μl of the LacNAc-Au-E2348/69 or Pk-Au-E2348/69 mixture was added to the epithelial surface of a biopsy sample. After 30 min of incubation at 37°C in the CO2 incubator, the biopsies were washed five times with ice-chilled phosphate-buffered saline to remove unbound bacteria.
To determine the number of adherent bacteria, whole genomic DNA was extracted from the intestinal biopsies using a QIAamp DNA Micro kit (QIAGEN, Mississauga, Ontario, Canada). Bacteria were quantified by performing real-time PCR of the intimin (eaeA) gene using the oligonucleotide primers described elsewhere (16). Real-time PCR was performed with a Rotor-Gene 3000 instrument (Corbett Research, Sydney, Australia) using a QuantiTect SYBR Green PCR kit (QIAGEN, Mississauga, Ontario, Canada) according to the manufacturer's instructions. The thermal cycling conditions used for amplifying the eaeA gene are described elsewhere (16). Five 10-fold dilutions of whole genomic DNA that was isolated from E2348/69 using a standard protocol (1) were used as an internal control for the PCR.
Using this approach, we observed significant variability in the number of EPEC cells attached to the individual biopsy specimens (Table 1). In the transverse colon biopsies alone, we observed a >5-log range in EPEC adherence; the values ranged from 480 adherent bacteria for patient 35 to 1.2 x 107 adherent bacteria for patient 94 (Table 1). For two patients, no EPEC bound to the biopsy samples (data not shown). There was no correlation between subject age or sex and the number of adherent bacteria (P = 0.655 and P = 0.952, respectively, as determined by the Pearson product-moment correlation test).
Regardless of the variability in EPEC binding, LacNAc-Au at a concentration of 0.8 mg/ml inhibited (compared to the negative Pk-Au control) EPEC adherence to the epithelial surface in all biopsies obtained from all subjects (Fig. 2) (13). The inhibitory effect was significant in the duodenal (P = 0.002), the terminal ileum (P = 0.002), and the transverse colon (P < 0.001) biopsy specimens (as determined by the Wilcoxon signed-rank test). The residual EPEC binding to the intestinal biopsy specimens when LacNAc-Au was present may be attributed to other potential EPEC adhesins, including a phosphatidylethanolamine-specific BFP-associated adhesin (2, 22), flagella (11), EspA (15), and intimin (18, 19).
This study allowed us to extend our previous observation that EPEC may exploit host cell LacNAc or LacNAc-related glycan receptor sequences during colonization of HEp-2, Caco-2, and T84 cells in culture to the adult human intestinal epithelium. This suggests that a BFP-associated lectin-like adhesin is likely involved in the initial attachment of EPEC to LacNAc or LacNAc-related receptors on intestinal epithelial cells in adults at least. This suggestion is further supported by studies which showed that mutant EPEC strains that lack the capacity to express BFP were less virulent in human adult volunteer challenge studies (3). In contrast to these results, Hicks and colleagues (12) reported that BFP were not important mediators of EPEC early adherence to pediatric intestinal biopsy specimens when the in vitro organ culture system was used. The discrepancy between our results and those of Hicks and colleagues may be attributed to differences between EPEC attachment to adult tissues and EPEC attachment to pediatric tissues. However, in a more recent paper, Cleary and colleagues (5) reported that BFP are required for efficient colonization of pediatric intestinal biopsy tissue at early times, a conclusion that is consistent with the results reported here.
Since LacNAc-Au reduced EPEC binding to the epithelial surface of all the biopsy specimens examined, such a glycoconjugate preparation may offer a new therapeutic modality for managing patients with EPEC infections and also may have prophylactic value for subjects traveling to regions where the organism is endemic. Our results also support investigation of the therapeutic or prophylactic potential of Au-based glycoconjugates bearing specific glycan sequences implicated in the colonization strategies of other important microbial pathogens.
ACKNOWLEDGMENTS
Financial support was provided by the Canadian Bacterial Diseases Network (G.D.A.), the Alberta Ingenuity Centre for Carbohydrate Science (G.D.A. and P.I.K.), and the Canadian Institutes for Health Research (G.D.A.). R.M.H. was the recipient of a studentship from NSERC.
G.D.A. is a coinventor on U.S. patent 6,291,435 for the treatment of diarrhea caused by enteropathogenic E. coli.
FOOTNOTES
Corresponding author. Mailing address: Department of Microbiology & Infectious Diseases, Faculty of Medicine, University of Calgary, 3330 Hospital Drive, NW, Calgary, AB T2N 4N1, Canada. Phone: (403) 220-6885. Fax: (403) 270-2772. E-mail: glen.armstrong@ucalgary.ca.
REFERENCES
1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1999. Short protocols in molecular biology, 4th ed. John Wiley & Sons, New York, NY.
2. Barnett Foster, D., D. Philpott, M. Abul-Milh, M. Huesca, P. M. Sherman, and C. A. Lingwood. 1999. Phosphatidylethanolamine recognition promotes enteropathogenic E. coli and enterohemorrhagic E. coli host cell attachment. Microb. Pathog. 27:289-301.
3. Bieber, D., S. W. Ramer, C. Y. Wu, W. J. Murray, T. Tobe, R. Fernandez, and G. K. Schoolnik. 1998. Type IV pili, transient bacterial aggregates, and virulence of enteropathogenic Escherichia coli. Science 280:2114-2118.
4. Campellone, K. G., and J. M. Leong. 2003. Tails of two Tirs: actin pedestal formation by enteropathogenic E. coli and enterohemorrhagic E. coli O157:H7. Curr. Opin. Microbiol. 6:82-90.
5. Cleary, J., L. C. Lai, R. K. Shaw, A. Straatman-Iwanowska, M. S. Donnenberg, G. Frankel, and S. Knutton. 2004. Enteropathogenic Escherichia coli (EPEC) adhesion to intestinal epithelial cells: role of bundle-forming pili (BFP), EspA filaments and intimin. Microbiology 150:527-538.
6. Delahay, R. M., G. Frankel, and S. Knutton. 2001. Intimate interactions of enteropathogenic Escherichia coli at the host cell surface. Curr. Opin. Infect. Dis. 14:559-565.
7. Donnenberg, M. S., J. A. Giron, J. P. Nataro, and J. B. Kaper. 1992. A plasmid-encoded type IV fimbrial gene of enteropathogenic Escherichia coli associated with localized adherence. Mol. Microbiol. 6:3427-3437.
8. Donnenberg, M. S., and J. B. Kaper. 1992. Enteropathogenic Escherichia coli. Infect. Immun. 60:3953-3961.
9. Donnenberg, M. S., and T. S. Whittam. 2001. Pathogenesis and evolution of virulence in enteropathogenic and enterohemorrhagic Escherichia coli. J. Clin. Investig. 107:539-548.
10. Giron, J. A., A. S. Ho, and G. K. Schoolnik. 1993. Characterization of fimbriae produced by enteropathogenic Escherichia coli. J. Bacteriol. 175:7391-7403.
11. Giron, J. A., A. G. Torres, E. Freer, and J. B. Kaper. 2002. The flagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells. Mol. Microbiol. 44:361-379.
12. Hicks, S., G. Frankel, J. B. Kaper, G. Dougan, and A. D. Phillips. 1998. Role of intimin and bundle-forming pili in enteropathogenic Escherichia coli adhesion to pediatric intestinal tissue in vitro. Infect. Immun. 66:1570-1578.
13. Hyland, R., T. P. Griener, G. L. Mulvey, P. I. Kitov, O. P. Srivastava, P. Marcato, and G. D. Armstrong. 2006. Basis for N-acetyllactosamine-mediated inhibition of enteropathogenic Escherichia coli localized adherence. J. Med. Microbiol. 55:669-675.
14. Kenny, B. 2002. Mechanism of action of EPEC type III effector molecules. Int. J. Med. Microbiol. 291:469-477.
15. Knutton, S., I. Rosenshine, M. J. Pallen, I. Nisan, B. C. Neves, C. Bain, C. Wolff, G. Dougan, and G. Frankel. 1998. A novel EspA-associated surface organelle of enteropathogenic Escherichia coli involved in protein translocation into epithelial cells. EMBO J. 17:2166-2176.
16. Leverton, L. Q., and J. B. Kaper. 2005. Temporal expression of enteropathogenic Escherichia coli virulence genes in an in vitro model of infection. Infect. Immun. 73:1034-1043.
17. Nataro, J. P., and J. B. Kaper. 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11:142-201.
18. Phillips, A. D., and G. Frankel. 2000. Intimin-mediated tissue specificity in enteropathogenic Escherichia coli interaction with human intestinal organ cultures. J. Infect. Dis. 181:1496-1500.
19. Sinclair, J. F., and A. D. O'Brien. 2002. Cell surface-localized nucleolin is a eukaryotic receptor for the adhesin intimin-gamma of enterohemorrhagic Escherichia coli O157:H7. J. Biol. Chem. 277:2876-2885.
20. Vanmaele, R. P., M. C. Finlayson, and G. D. Armstrong. 1995. Effect of enteropathogenic Escherichia coli on adherent properties of Chinese hamster ovary cells. Infect. Immun. 63:191-198.
21. Vanmaele, R. P., L. D. Heerze, and G. D. Armstrong. 1999. Role of lactosyl glycan sequences in inhibiting enteropathogenic Escherichia coli attachment. Infect. Immun. 67:3302-3307.
22. Wu, Y., B. Lau, S. Smith, K. Troyan, and D. E. Barnett Foster. 2004. Enteropathogenic Escherichia coli infection triggers host phospholipid metabolism perturbations. Infect. Immun. 72:6764-6772.(Romney M. Hyland, Paul Beck, George L. M)