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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.



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