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. 2000 Oct;68(10):5943-52.
doi: 10.1128/IAI.68.10.5943-5952.2000.

Isolation and characterization of mini-Tn5Km2 insertion mutants of enterohemorrhagic Escherichia coli O157:H7 deficient in adherence to Caco-2 cells

Affiliations

Isolation and characterization of mini-Tn5Km2 insertion mutants of enterohemorrhagic Escherichia coli O157:H7 deficient in adherence to Caco-2 cells

I Tatsuno et al. Infect Immun. 2000 Oct.

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.

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Figures

FIG. 1
FIG. 1
Adherence phenotypes of class 2 mutants and class 3 mutants are shown. The C8-A2 and C6-H10 mutants are representatives of class 2 mutants. The G1-E11 mutant shows a diffused adherence phenotype. The bacteria grown in DMEM-glycerol for 2 h were used to infect Caco-2 cell monolayers. The infected monolayers were incubated for 1.5 h and washed five times with PBS. After another 2.5 h of incubation, the monolayers were again washed three times, fixed with methanol, and stained with Giemsa solution to visualize the adherent bacterial colonies. WT, wild type.
FIG. 2
FIG. 2
Genetic organization of the EHEC O157:H7 LEE adapted from Perna et al. (38). Insertion sites of all class 1 mutants and the class 3 mutant (G1-E11) are shown. Genes with a mini-Tn5Km2 insertion are shown by black arrows. Operons LEE1 to LEE4 were those of the EPEC LEE (30).
FIG. 3
FIG. 3
Levels of secretion and expression of EspA and intimin of class 1 to 3 mutants. Trichloroacetic acid-precipitated culture supernatants (sup) and bacterial cell lysates (whole) derived from equal amounts of wild-type strain O157T, class 1 mutants (A), class 3 mutants (B), and class 2 mutants (C) were resolved by SDS-12% PAGE, transferred to nitrocellulose membrane, and probed with polyclonal rabbit antisera specific to each protein indicated at the right of the photos.
FIG. 4
FIG. 4
Adherence behavior of O157Sakai (WT), the intimin mutant (Δeae), type III secretion mutant (B9-F9), and EPEC B171-8 (EPEC). B9-F9 is representative of class 1 mutants defective in the type III secretion system. (A) The bacteria were grown at 37°C for 2 h in DMEM-glycerol and then used to infect Caco-2 cell monolayers. Then these infected monolayers were incubated for 1.5 h and washed five times with PBS. After another 0, 1, 2, or 3 h of incubation at 37°C in DMEM-glycerol, the monolayers were again washed three times with PBS, fixed with methanol, and stained with Giemsa solution to visualize the adherent bacterial colonies. Strains and incubation times are shown above and on the left side of photos, respectively. (B) The black bars represent the total number of sites containing adherent EHEC, having either a single bacterium or a cluster of multiple bacteria. The white bars represent the number of clusters containing at least eight bacteria. The data shown are the means and standard errors of the means for 20 microscopic fields. The representative results were obtained from three independent experiments.
FIG. 5
FIG. 5
Photomicrographs of the intimin mutant and parental wild-type strain in a rhodamine-phalloidin assay for FAS in Caco-2 cells. (A) Caco-2 cells infected by bacteria as described in the Fig. 4 legend are shown as follows: in fluorescent views after treatment with anti-O157 LPS rabbit antibody followed by anti-rabbit goat antibody conjugated with fluorescein isothiocyanate (Bacteria), in fluorescent views of actin stained by rhodamine-phalloidin (Actin), and in a superimposed view of bacteria (green) and actin (red) (Super impose). Strains and incubation times are indicated at the left of photos. (B) Quantitative FAS assay of the O157Sakai wild type (WT) and the intimin mutant (Δeae), derived from the experiments whose results are shown in panel A.

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