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MOLECULAR AND CELLULAR PATHOGENESIS

Isolation and Characterization of Mini-Tn5Km2 Insertion Mutants of Enterohemorrhagic Escherichia coliO157:H7 Deficient in Adherence to Caco-2 Cells

Ichiro Tatsuno, Hiroshi Kimura, Akiko Okutani, Kyoko Kanamaru, Hiroyuki Abe, Shinya Nagai, Kozo Makino, Hideo Shinagawa, Mitsutaka Yoshida, Katsuhiro Sato, Jyunichi Nakamoto, Toru Tobe, Chihiro Sasakawa
Ichiro Tatsuno
Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639,
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Hiroshi Kimura
Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639,
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Akiko Okutani
Laboratory of Veterinary Public Health, Graduate School of Agriculture and Life Science, University of Tokyo, Bunkyo-ku, Tokyo 113-8657,
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Kyoko Kanamaru
Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639,
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Hiroyuki Abe
Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639,
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Shinya Nagai
Nippon Institute for Biological Science, 9-2221-1 Shinmachi, Ome, Tokyo 198-0024,
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Kozo Makino
Department of Molecular Microbiology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, and
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Hideo Shinagawa
Department of Molecular Microbiology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, and
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Mitsutaka Yoshida
Central Laboratory of Medical Science, Division of Electron Microscopy, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
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Katsuhiro Sato
Central Laboratory of Medical Science, Division of Electron Microscopy, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
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Jyunichi Nakamoto
Central Laboratory of Medical Science, Division of Electron Microscopy, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
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Toru Tobe
Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639,
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Chihiro Sasakawa
Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639,
Department of Molecular Microbiology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, and
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DOI: 10.1128/IAI.68.10.5943-5952.2000
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ABSTRACT

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 nonpolareae (encoding intimin) mutant. Examination of theeae 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.

  • Copyright © 2000 American Society for Microbiology
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Isolation and Characterization of Mini-Tn5Km2 Insertion Mutants of Enterohemorrhagic Escherichia coliO157:H7 Deficient in Adherence to Caco-2 Cells
Ichiro Tatsuno, Hiroshi Kimura, Akiko Okutani, Kyoko Kanamaru, Hiroyuki Abe, Shinya Nagai, Kozo Makino, Hideo Shinagawa, Mitsutaka Yoshida, Katsuhiro Sato, Jyunichi Nakamoto, Toru Tobe, Chihiro Sasakawa
Infection and Immunity Oct 2000, 68 (10) 5943-5952; DOI: 10.1128/IAI.68.10.5943-5952.2000

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Isolation and Characterization of Mini-Tn5Km2 Insertion Mutants of Enterohemorrhagic Escherichia coliO157:H7 Deficient in Adherence to Caco-2 Cells
Ichiro Tatsuno, Hiroshi Kimura, Akiko Okutani, Kyoko Kanamaru, Hiroyuki Abe, Shinya Nagai, Kozo Makino, Hideo Shinagawa, Mitsutaka Yoshida, Katsuhiro Sato, Jyunichi Nakamoto, Toru Tobe, Chihiro Sasakawa
Infection and Immunity Oct 2000, 68 (10) 5943-5952; DOI: 10.1128/IAI.68.10.5943-5952.2000
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KEYWORDS

Adhesins, Bacterial
Bacterial Adhesion
Carrier Proteins
DNA Transposable Elements
Escherichia coli O157
Escherichia coli Proteins

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