Article Figures & Data
Figures
- 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.
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.
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.
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.
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. 4legend 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.
Tables
- Table 1.
Adherence and other characteristics of strains
Strains Adherencea EspA secretionb Intiminb FASc Growthd MCe WTf ++++ + + + N + Class 1 A3-C10 − − + ND N − A3-E9 − − + ND N − A5-E4 − − + ND N − A6-E7 − − + ND N − B4-B2 − − + ND N − B6-H7 − − + ND N − B8-E7 − − + ND N − B8-F12 − − + ND N − B9-F9 − − + ND N − D5-B12 − − + ND N − Class 2 A3-H2 + +/− + + N + C6-E9 ++ + + + N + C6-F9 + + + + N + C6-H10 ++ + + + N + C7-A10 + + + + N + C8-A2 + + + + N + F4-H4 + + + + N + F5-F4 + + + + N + F6-B9 ++ + + + N + F6-D11 ++ + + + N + G2-B12 ++ + + + N + G4-B6 + + + + N + G4-B12 + + + + N + G4-C10 ++ + + + N + G4-E10 + + + + N + H7-C4 + + + + N + Class 3 G1-E11 + + − − N − ↵a Adherence was divided by the mean adherence (100%) of the O157Sakai-T parental strain. ++++, >80%; +++, 80 to 50%; ++, 50 to 25%; +, 25 to 1%; −, <1%. Data are the means of triplicate determinations, which differed by <15%, from representative experiments.
↵b EspA in culture supernatant and intimin in whole bacterial cell were detected by anti-EspA and anti-intimin antibody, respectively. +, detected; −, not detected; +/−, detected at a reduced level.
↵c FAS assays of infected Caco-2 cells were performed by using rhodamine-phalloidin. ND, not determined because nonadherent to Caco-2 cells.
↵d Growth rates of each mutant and the parental wild type were essentially the same. N, normal.
↵e Ability to form microcolonies; +, positive; −, negative.
↵f WT, wild type.
- Table 2.
Products encoded by the gene withaor in the vicinity of mini-Tn5Km2
Strain Gene product Site (min) onE. coli K-12 genomeb Accession no. or reference A3-H2 Hypothetical 17.8-kDa protein (yfjG) 59.2 P52121 (1) C6-E9 Putative membrane protein – AE000234 C6-F9 Putative membrane protein – AE000234 C6-H10 Putative membrane protein – AE000234 C7-A10 Putative membrane protein – AE000234 C8-A2 Hypothetical 8-membrane-spanning 37.6-kDa protein (yiaH) 80.2 P37669 F4-H4 Putative NAGC-like transcriptional regulator (yphHI) 57.8 AE000341 ABC transporter-periplasmic binding protein YPHF precursor P77269 Hypothetical 127.3-kDa protein P76585 Serine hydroxy transferase (glyA) 38 F5-F4 Putative potassium channel (kch) 28.3 P31069 (32) F6-B9 Hypothetical 10-membrane-spanning 58.4-kDa protein 27.2 P40877 F6-D11 Hypothetical 40.2-kDa protein 64.7 Q46803 Hypothetical sigma 54-dependent transcriptional regulator Q46802 G2-B12 Putative adherence factor (efa1) – AF159462 (34) G4-B6 Arsenite (efflux) pump membrane protein (arsF) 78.59 7 Origopeptidase A (prlC) 3 G4-B12 Hypothetical 27.7-kDa protein 14.6 P77627 G4-C10 (orf3 of EPEC pathogenicity island LIM) – 46 G4-E10 Lysine-specific permease (lysP) 48.5 43 Hypothetical transcriptional regulator P32484 H7-C4 Hypothetical 10-membrane-spanning 43.6-kDa protein 87.5 P32135 ↵a Products encoded by the gene with mini-Tn5Km2 are in bold type.
↵b The insertion sites placed in the DNA sequences specific to the O157Sakai genome sequence are indicated by a dash (–). The sites of C6-E9, C6-F9, C6-H10, and C7-A10 were in the same ∼3-kb regions encoding an open reading frame partially homologous to the b1372 gene, which encodes a putative membrane protein of 1,122 amino acids of E. coli K-12. F6-D11 was placed in a region containing genes encoding a hypothetical sigma 54-dependent transcriptional regulator and a hypothetical 40.2-kDa protein homologous to ornithine carbamoyltransferase. G4-C10 was placed in orf3 in a region homologous to the EPEC pathogenicity island named LIM, encoding a chaperon-like protein involved in the stable expression of BfpA and intimin (47).
