title: Isolation and characterization of mini-Tn5Km2 insertion mutants of enterohemorrhagic Escherichia coli O157:H7 deficient in adherence to Caco-2 cells.
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 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.
authors.affiliation: Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
authors.forename: I
authors.initials: I
authors.lastname: Tatsuno
authors.forename: H
authors.initials: H
authors.lastname: Kimura
authors.forename: A
authors.initials: A
authors.lastname: Okutani
authors.forename: K
authors.initials: K
authors.lastname: Kanamaru
authors.forename: H
authors.initials: H
authors.lastname: Abe
authors.forename: S
authors.initials: S
authors.lastname: Nagai
authors.forename: K
authors.initials: K
authors.lastname: Makino
authors.forename: H
authors.initials: H
authors.lastname: Shinagawa
authors.forename: M
authors.initials: M
authors.lastname: Yoshida
authors.forename: K
authors.initials: K
authors.lastname: Sato
authors.forename: J
authors.initials: J
authors.lastname: Nakamoto
authors.forename: T
authors.initials: T
authors.lastname: Tobe
authors.forename: C
authors.initials: C
authors.lastname: Sasakawa
mesh_terms: D018829:Adhesins, Bacterial; D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D018938:Caco-2 Cells; D002352:Carrier Proteins; D004251:DNA Transposable Elements; D004927:Escherichia coli Infections / Q000382:microbiology; D019453:Escherichia coli O157 / Q000187:drug effects / Q000235:genetics* / Q000472:pathogenicity*; D029968:Escherichia coli Proteins; D006801:Humans; D007613:Kanamycin Resistance / Q000235:genetics; D016254:Mutagenesis, Insertional; D014774:Virulence
chemical_list: D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; C102051:EspA protein, E coli; C073819:eaeA protein, E coli