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Infection and Immunity, October 2000, p. 5943-5952, Vol. 68, No. 10
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.
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
*
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.
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