This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tatsuno, I.
Right arrow Articles by Sasakawa, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tatsuno, I.
Right arrow Articles by Sasakawa, C.

 Previous Article  |  Next Article 

Infection and Immunity, October 2000, p. 5943-5952, Vol. 68, No. 10
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Isolation and Characterization of Mini-Tn5Km2 Insertion Mutants of Enterohemorrhagic Escherichia coli O157:H7 Deficient in Adherence to Caco-2 Cells

Ichiro Tatsuno,1 Hiroshi Kimura,1 Akiko Okutani,2 Kyoko Kanamaru,1 Hiroyuki Abe,1 Shinya Nagai,3 Kozo Makino,4 Hideo Shinagawa,4 Mitsutaka Yoshida,5 Katsuhiro Sato,5 Jyunichi Nakamoto,5 Toru Tobe,1 and Chihiro Sasakawa1,4,*

Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639,1 Laboratory of Veterinary Public Health, Graduate School of Agriculture and Life Science, University of Tokyo, Bunkyo-ku, Tokyo 113-8657,2 Nippon Institute for Biological Science, 9-2221-1 Shinmachi, Ome, Tokyo 198-0024,3 Department of Molecular Microbiology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871,4 and Central Laboratory of Medical Science, Division of Electron Microscopy, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421,5 Japan

Received 20 March 2000/Returned for modification 20 June 2000/Accepted 9 July 2000

Adherence of enterohemorrhagic Escherichia coli (EHEC) to intestinal epithelium is essential for initiation of the infection. To identify genes involved in adherence, an EHEC O157:H7 strain (O157Sakai) was mutagenized by mini-Tn5Km2, where Km refers to kanamycin resistance, and 4,677 insertion mutants were screened for their ability to form microcolonies (MC) on Caco-2 cells. The less adherent mutants were divided into three groups: those with no adherent ability (designated as class 1 mutants, n = 10), those less adherent than the wild type (class 2 mutants, n = 16), and those unable to form MC but which adhered in a diffuse manner (class 3 mutants, n = 1). The sites of insertion in class 1 mutants were all found within genes of the locus for enterocyte effacement (LEE) thought to be required for type III protein secretion. Indeed, the class 1 mutants failed to secrete type III secreted proteins such as EspA and Tir into the culture medium. The insertions in class 2 mutants were outside the LEE, and all the mutants except one were able to secrete type III proteins into the culture medium. The class 3 mutant had the insertion in the tir gene in the LEE and was deficient in Tir and intimin expression, suggesting that in the absence of intimin-Tir, O157Sakai can still adhere to Caco-2 cells but in a diffused manner. This was confirmed by construction of a nonpolar eae (encoding intimin) mutant. Examination of the eae mutant together with O157Sakai and one of the class 1 mutants for the ability to form MC revealed that EHEC initially adhered diffusely at 1.5 h after infection. Following washing out of the nonadherent bacteria, while wild-type EHEC bacteria developed MC for another 2 to 3 h on Caco-2 cells, the eae mutant diffusely adhered throughout the infection without forming MC. MC with O157Sakai but not the diffusely adherent eae mutant could evoke F-actin condensation beneath the bacterium. Our results suggest that EHEC encodes additional adherence-associated loci and that the type III secreted proteins are involved in the initial diffuse adherence, while the intimin-Tir interaction is required for the subsequent development of MC.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5252. Fax: 81-3-5449-5405. E-mail: sasakawa{at}ims.u-tokyo.ac.jp.

Infection and Immunity, October 2000, p. 5943-5952, Vol. 68, No. 10
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Nakanishi, N., Tashiro, K., Kuhara, S., Hayashi, T., Sugimoto, N., Tobe, T. (2009). Regulation of virulence by butyrate sensing in enterohaemorrhagic Escherichia coli. Microbiology 155: 521-530 [Abstract] [Full Text]  
  • Ho, T. D., Davis, B. M., Ritchie, J. M., Waldor, M. K. (2008). Type 2 Secretion Promotes Enterohemorrhagic Escherichia coli Adherence and Intestinal Colonization. Infect. Immun. 76: 1858-1865 [Abstract] [Full Text]  
  • Rendon, M. A., Saldana, Z., Erdem, A. L., Monteiro-Neto, V., Vazquez, A., Kaper, J. B., Puente, J. L., Giron, J. A. (2007). Commensal and pathogenic Escherichia coli use a common pilus adherence factor for epithelial cell colonization. Proc. Natl. Acad. Sci. USA 104: 10637-10642 [Abstract] [Full Text]  
  • Li, J., Hovde, C. J. (2007). Expression Profiles of Bovine Genes in the Rectoanal Junction Mucosa during Colonization with Escherichia coli O157:H7. Appl. Environ. Microbiol. 73: 2380-2385 [Abstract] [Full Text]  
  • Ando, H., Abe, H., Sugimoto, N., Tobe, T. (2007). Maturation of functional type III secretion machinery by activation of anaerobic respiration in enterohaemorrhagic Escherichia coli. Microbiology 153: 464-473 [Abstract] [Full Text]  
  • Tatsuno, I., Mundy, R., Frankel, G., Chong, Y., Phillips, A. D., Torres, A. G., Kaper, J. B. (2006). The lpf Gene Cluster for Long Polar Fimbriae Is Not Involved in Adherence of Enteropathogenic Escherichia coli or Virulence of Citrobacter rodentium. Infect. Immun. 74: 265-272 [Abstract] [Full Text]  
  • Tozzoli, R., Caprioli, A., Morabito, S. (2005). Detection of toxB, a Plasmid Virulence Gene of Escherichia coli O157, in Enterohemorrhagic and Enteropathogenic E. coli. J. Clin. Microbiol. 43: 4052-4056 [Abstract] [Full Text]  
  • Nagai, T., Abe, A., Sasakawa, C. (2005). Targeting of Enteropathogenic Escherichia coli EspF to Host Mitochondria Is Essential for Bacterial Pathogenesis: CRITICAL ROLE OF THE 16TH LEUCINE RESIDUE IN EspF. J. Biol. Chem. 280: 2998-3011 [Abstract] [Full Text]  
  • Torres, A. G., Zhou, X., Kaper, J. B. (2005). Adherence of Diarrheagenic Escherichia coli Strains to Epithelial Cells. Infect. Immun. 73: 18-29 [Full Text]  
  • Toma, C., Martinez Espinosa, E., Song, T., Miliwebsky, E., Chinen, I., Iyoda, S., Iwanaga, M., Rivas, M. (2004). Distribution of Putative Adhesins in Different Seropathotypes of Shiga Toxin-Producing Escherichia coli. J. Clin. Microbiol. 42: 4937-4946 [Abstract] [Full Text]  
  • Dziva, F., van Diemen, P. M., Stevens, M. P., Smith, A. J., Wallis, T. S. (2004). Identification of Escherichia coli O157 : H7 genes influencing colonization of the bovine gastrointestinal tract using signature-tagged mutagenesis. Microbiology 150: 3631-3645 [Abstract] [Full Text]  
  • Stevens, M. P., Roe, A. J., Vlisidou, I., van Diemen, P. M., La Ragione, R. M., Best, A., Woodward, M. J., Gally, D. L., Wallis, T. S. (2004). Mutation of toxB and a Truncated Version of the efa-1 Gene in Escherichia coli O157:H7 Influences the Expression and Secretion of Locus of Enterocyte Effacement-Encoded Proteins but not Intestinal Colonization in Calves or Sheep. Infect. Immun. 72: 5402-5411 [Abstract] [Full Text]  
  • Torres, A. G., Kaper, J. B. (2003). Multiple Elements Controlling Adherence of Enterohemorrhagic Escherichia coli O157:H7 to HeLa Cells. Infect. Immun. 71: 4985-4995 [Abstract] [Full Text]  
  • Kim, S., Watarai, M., Kondo, Y., Erdenebaatar, J., Makino, S.-i., Shirahata, T. (2003). Isolation and Characterization of Mini-Tn5Km2 Insertion Mutants of Brucella abortus Deficient in Internalization and Intracellular Growth in HeLa Cells. Infect. Immun. 71: 3020-3027 [Abstract] [Full Text]  
  • Morabito, S., Tozzoli, R., Oswald, E., Caprioli, A. (2003). A Mosaic Pathogenicity Island Made Up of the Locus of Enterocyte Effacement and a Pathogenicity Island of Escherichiacoli O157:H7 Is Frequently Present in Attaching and Effacing E. coli. Infect. Immun. 71: 3343-3348 [Abstract] [Full Text]  
  • Tatsuno, I., Nagano, K., Taguchi, K., Rong, L., Mori, H., Sasakawa, C. (2003). Increased Adherence to Caco-2 Cells Caused by Disruption of the yhiE and yhiF Genes in Enterohemorrhagic Escherichia coli O157:H7. Infect. Immun. 71: 2598-2606 [Abstract] [Full Text]  
  • Stevens, M. P., van Diemen, P. M., Dziva, F., Jones, P. W., Wallis, T. S. (2002). Options for the control of enterohaemorrhagic Escherichia coli in ruminants. Microbiology 148: 3767-3778 [Full Text]  
  • Torres, A. G., Giron, J. A., Perna, N. T., Burland, V., Blattner, F. R., Avelino-Flores, F., Kaper, J. B. (2002). Identification and Characterization of lpfABCC'DE, a Fimbrial Operon of Enterohemorrhagic Escherichia coli O157:H7. Infect. Immun. 70: 5416-5427 [Abstract] [Full Text]  
  • Stevens, M. P., van Diemen, P. M., Frankel, G., Phillips, A. D., Wallis, T. S. (2002). Efa1 Influences Colonization of the Bovine Intestine by Shiga Toxin-Producing Escherichia coli Serotypes O5 and O111. Infect. Immun. 70: 5158-5166 [Abstract] [Full Text]  
  • Abe, H., Tatsuno, I., Tobe, T., Okutani, A., Sasakawa, C. (2002). Bicarbonate Ion Stimulates the Expression of Locus of Enterocyte Effacement-Encoded Genes in Enterohemorrhagic Escherichia coli O157:H7. Infect. Immun. 70: 3500-3509 [Abstract] [Full Text]  
  • Pradel, N., Leroy-Setrin, S., Joly, B., Livrelli, V. (2002). Genomic Subtraction To Identify and Characterize Sequences of Shiga Toxin-Producing Escherichia coli O91:H21. Appl. Environ. Microbiol. 68: 2316-2325 [Abstract] [Full Text]  
  • Stevens, M. P., Marches, O., Campbell, J., Huter, V., Frankel, G., Phillips, A. D., Oswald, E., Wallis, T. S. (2002). Intimin, Tir, and Shiga Toxin 1 Do Not Influence Enteropathogenic Responses to Shiga Toxin-Producing Escherichia coli in Bovine Ligated Intestinal Loops. Infect. Immun. 70: 945-952 [Abstract] [Full Text]  
  • Tatsuno, I., Horie, M., Abe, H., Miki, T., Makino, K., Shinagawa, H., Taguchi, H., Kamiya, S., Hayashi, T., Sasakawa, C. (2001). toxB Gene on pO157 of Enterohemorrhagic Escherichiacoli O157:H7 Is Required for Full Epithelial Cell Adherence Phenotype. Infect. Immun. 69: 6660-6669 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»