kanamycin (60 ,ug/ml) in a 24-well multidish plate containing a plastic coverslip (diameter, 13.5 mm; Sumitomo Bakelite,. Tokyo, Japan) at the bottom of each ...
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Copyright X 1994, American Society for Microbiology
Actin Accumulation Associated with Clustered and Localized Adherence in Escherichia coli Isolated from Patients with Diarrhea TATSUO YAMAMOTO,`* MAYUMI KANEKO,1 SUCHITRA CHANGCHAWALIT,2 ORALAK SERICHANTALERGS,2 SATOSHI IJUIN,1 AND PETER ECHEVERRIA2 Department of Bacteriology, School of Medicine, Juntendo University, Tokyo, Japan,1 and Department of Bacteriology, Immunology, and Molecular Genetics, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand2 Received 3 December 1993/Returned for modilication 2 February 1994/Accepted 12 April 1994
Escherfihia coli D2 (serotype 07:H-) that was isolated from a child with diarrhea hybridized with an F1845 DNA probe used to detect diffuse adherence. Strain D2 adhered to tissue culture cells (HeLa and HEp-2 cells) in a clustered pattern but did not autoagglutinate on the cell surface and induced the elongation of microvilli after 3 h of incubation. After 6 h of incubation, the infected cells were positive for fluorescent-actin staining at the site of clustered adherence. When analyzed with a confocal laser scanning microscope, each D2 cell was surrounded by accumulated actin in a capsule-like formation. Capsule-like, accumulated actin was also observed with enteropathogenic E. coli (EPEC), although in this case, actin accumulation was associated with EPEC microcolonies in a localized pattern. Four other strains of F1845 DNA probe-positive, diffusely adhering E. coli were negative for actin accumulation. Strain D2 did not hybridize with EPEC attaching and effacing DNA or EPEC adherence factor DNA probes. In addition, clustered D2 cells were found inside tissue culture cells. The data suggest a novel infectious mechanism as well as genetic heterogeneity of F1845 DNA probe-positive E. coli. Capsule-like, accumulated actin may protect the bacteria from host defense mechanisms.
On the basis of distinct virulence properties and syndromes, diarrheagenic Escherichia coli has been classified into five categories (2, 16, 33, 40): enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli, enterohemorrhagic E. coli, and enteroaggregative E. coli. Among these, EPEC and enteroaggregative E. coli have been diagnosed on the basis of their unique patterns of adherence to tissue culture cells called, respectively, localized adherence and aggregative adherence (40, 41, 46). EPEC cells autoagglutinate in tissue culture medium because of bundle-forming pili (12, 21, 53) and form microcolonies on tissue culture cells (41, 46) as well as plastic coverslips (55) in a localized pattern. Such localized adherence is associated with the presence of plasmids called EPEC adherence factor (EAF) plasmids (13, 22, 41). EPEC adhering to tissue culture cells or intestinal mucosa destroy the microvilli or induce elongation of the microvilli at the site of adherence, intimately attach to cup-like projections of the cell membrane (16, 31, 38, 45, 51, 55), and accumulate filamentous actin (F-actin) beneath the areas of EPEC adherence (13, 29, 30). At this stage, EPEC-infected cells become positive in a fluorescent-actin staining (FAS) test (29, 30). Those cytoskeletal alterations are called attaching and effacing lesions and have been demonstrated to be encoded by EPEC chromosomal genes (eae) (14, 26, 27). EPEC also invade tissue culture cells (11, 20). DNA sequences homologous to eae gene sequences have also been found in enterohemorrhagic E. coli (3, 56). By use of an adherence assay with tissue culture cells, the
third type of adherence pattern (in addition to localized adherence and aggregative adherence) has been recognized and termed diffuse adherence (41, 46). Diffuse adherence was first reported for strains with EPEC serotypes (41, 46). The adherence factor of one such strain was shown to be a plasmid-encoded outer membrane protein of 100 kDa called afimbrial adhesin involved in diffuse adherence (AIDA) I (4). Diffuse adherence of diarrhea-associated strains with nonEPEC serotypes has also been identified (50). The adherence factor of one such strain was shown to be a chromosomeencoded fimbrial adhesin called F1845 (5). F1845 is a member of a family of adhesin (Dr) which recognize the Dr(a) blood group antigen as a receptor; this family includes uropathogenic E. coli adhesins such as afimbrial adhesin I, afimbrial adhesin III, and Dr hemagglutinin (43, 49). In other diffusely adhering E. coli (DAEC) strains, which did not hybridize with F1845 or AIDA DNA probes, the genes for diffuse adherence were located on conjugative drug resistance plasmids (24). In contrast to EPEC (34) or enteroaggregative E. coli (strain 221 [37]), F1845 DNA probe-positive DAEC (50) is not significantly pathogenic for volunteers (50). The association of this type of adherence with diarrhea has remained unclear (10, 22, 24, 33, 35). In this study, we investigated the characteristics of an F1845 DNA probe-positive E. coli clinical isolate that displayed unique interactions with tissue culture cells and compared them with the characteristics of other F1845 DNA probe-positive E. coli strains and EPEC. MATERUILS AND METHODS Bacterial strains. E. coli D2 (W113-1-4) was isolated from a 2-month-old female with diarrhea in Thailand. Strain D2 adhered to HeLa cells in a diffuse pattern but not to plastic
Corresponding author. Mailing address: Department of BacteriolSchool of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyoku, Tokyo, Japan. Fax: 81-3-3814-9300. *
ogy,
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FIG. 1. Scanning electron micrographs showing adherence to HeLa cells (A and C) and HEp-2 cells (B and D) of E. coli D2 after 3 h of incubation. Arrowheads indicate bacterial adherence in a clustered pattern. One cluster was about 10 ,um in diameter and consisted of ca. 20 adherent bacteria. Bars are in micrometers.
coverslips and hybridized with the F1845 DNA probe (0.45kbp PstI fragment of pSLM852 which originated in a gene on the chromosomal F1845 locus [5]) in colony hybridization (55). E. coli Dl (W90-1-4; serotype 036:H4 [55]), D3 (W198-1-4; serotype O11:H15 [55]), D4 (WC20-1-1; serotype 020:H34), and D6 (WC43-1-4; serotype 020:H34) were isolated from patients with diarrhea in Thailand, adhered to HeLa cells in a diffuse pattern but not to plastic coverslips, and hybridized with the F1845 DNA probe. EPEC strains W105-4-1 (serotype 0119:H6) and L06 (EI441-5; serotype 0119:H6 [55]) were isolated from patients with diarrhea in Thailand, adhered to HeLa cells in a localized pattern, and hybridized with the EAF DNA probe (39) and the EPEC attaching and effacing (EAE) DNA probe (27). E. coli HB101 is a hybrid between E. coli K-12 and E. coli B and lacks restriction ability. E. coli Xac is a K-12 derivative strain. Media and bacterial growth. For bacterial growth, L broth (32) was used as a liquid medium. Growth was followed by
incubation at 37°C for 14 to 18 h with agitation (unless otherwise noted). Brain heart infusion broth (Difco Laboratories, Detroit, Mich.) was also used. MacConkey agar (Eiken Chemical, Tokyo, Japan), Trypticase soy agar (Difco), L agar (32), and Mueller-Hinton agar (Difco) were used as solid media. Incubations were done for 18 to 20 h at 37°C. Serotyping. 0 and H serotypes of E. coli D2 were determined with 0- and H-specific antisera (from collections in the Department of Bacteriology, National Institute of Health, Tokyo, Japan) essentially by standard methods (44). For the detection of 0 serotypes, bacterial cells were heated to 100°C for 1 h or autoclaved and then tested by tube (or slide) agglutination. For the detection of H serotypes, bacterial cells were fixed with Formalin and then tested as described above. Adherence test with tissue culture cells. The adherence of E. coli to HeLa and HEp-2 cells was examined essentially by a method previously described (40, 46, 52), except that samples were finally examined with a scanning electron microscope
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FIG. 2. Transmission electron micrograph showing adherence to HeLa cells of E. coli D2 after 3 h of incubation. Arrowheads indicate elongated microvilli surrounding adherent bacteria. The bar is in micrometers.
(55) instead of a light microscope. HeLa or HEp-2 cells were grown for 24 h at 37°C in Eagle MEM (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 5% fetal calf serum and kanamycin (60 ,ug/ml) in a 24-well multidish plate containing a plastic coverslip (diameter, 13.5 mm; Sumitomo Bakelite, Tokyo, Japan) at the bottom of each well. HeLa or HEp-2 cells adhered to the plastic coverslip at -50% confluence. The cells were then washed with Eagle MEM. Subsequently, 1 ml of Eagle MEM supplemented with 5% fetal calf serum and 1% (wt/vol) D-mannose was added to each well. This step was followed by the addition of 5 or 40 ,ul of bacterial culture per well and by incubation for 3 h at 37°C. In some experiments, the tissue culture medium was replaced with fresh medium and then incubation was done at 37°C for an additional 3 h. After being washed four times with phosphate-buffered saline (PBS; pH 7.4), cells bound to bacteria on the plastic coverslip were fixed with 2.5% (vol/vol) glutaraldehyde in PBS for 1 h at room temperature and subsequently postfixed with 1% (wt/vol) osmium tetroxide for 15 min at room temperature and then for 45 min at 4°C or for 2 h at 4°C. Scanning electron microscopy. The osmium tetroxide-fixed samples were dehydrated with acetone and critical-point dried. The samples were then coated with gold-palladium and analyzed by scanning electron microscopy. Transmission electron microscopy. The osmium tetroxidefixed samples described above were dehydrated with acetone and embedded in EPOK 812 (Oukenn Inc., Tokyo, Japan). The embedded block was cut with an ultramicrotome (MT500) with a diamond knife and stained with uranyl acetate and
lead citrate. The stained samples were analyzed by transmission electron microscopy. FAS test. Actin accumulation in tissue culture cells was examined as previously described (29, 30). HeLa or HEp-2 cells bound on a glass coverslip were infected with bacteria as described above. After incubation, cells on the glass coverslip were washed three times with PBS (throughout) and fixed with 3% Formalin for 20 min at room temperature. After being washed, the samples were treated with 0.1% Triton X-100 (in PBS) for 4 min at room temperature and washed. Fluorescein isothiocyanate-phalloidin (Sigma Chemical Co., St. Louis, Mo.) was then added for 20 min at room temperature in the dark. After being washed, the samples were analyzed with MRC-600 and MRC-1000 series laser scanning confocal imaging systems (Bio-Rad, Tokyo, Japan). Gentamicin protection assay. Bacterial entry into HEp-2 cells was determined as previously described (48). In brief, bacteria were grown on Trypticase soy agar overnight at 37°C, inoculated into brain heart infusion broth, and grown with shaking for 2 h at 37°C. The bacterial culture was diluted 10-fold in Eagle MEM, and 1 ml of the dilution was added to HEp-2 cell monolayers which had been washed three times with PBS. The samples were incubated for 2 h at 37°C. The medium was diluted, and the extracellular bacterial cell number was counted by plating on L agar plates. Infected monolayers were washed three times with PBS and covered with a medium containing gentamicin (100 ,ug/ml) for 1.5 h at 37°C. The monolayers were washed six times with PBS and flooded with 1% Triton X-100 in 10 mM Tris-HCl (pH 7.4) containing
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FIG. 3. Scanning electron micrographs showing adherence to HeLa cells (A and C) and HEp-2 cells (B) of E. coli D2 after 6 h of incubation. Panels A and B show bacterial adherence in a diffuse pattern. Bars are in micrometers.
1 mM EDTA for 15 min at room temperature to release intracellular bacteria. Dilutions were made, and the intracellular bacterial cell number was counted by plating on L agar plates. Experiments were run five times per strain. Susceptibility testing. Susceptibility testing of bacterial strains was done by the standard agar dilution method with Mueller-Hinton agar (25). When susceptibility to sulfamethoxazole or trimethoprim was tested, Mueller-Hinton agar supplemented with 7.5% (vol/vol) defibrinated horse blood (frozen and thawed) was used instead of Mueller-Hinton agar alone. The MIC was determined as described previously (25). E. coli NIHJ was used as a reference strain for quality control (25). Plasmid analysis and transformation with plasmid DNA. Plasmids in E. coli were analyzed (or isolated) essentially by a previously described method (28) with the following modifications (55). Bacterial cells grown in L broth (5 ml) were suspended in 100 RI of 40 mM Tris-acetate (pH 7.9) containing 2 mM EDTA (pH 7.9) in a 1.5-ml microcentrifuge tube. This step was followed by the addition of 200 RI of lysis solution (3% sodium dodecyl sulfate [SDS], 50 mM Tris, 0.128 N
NaOH) at room temperature. After being mixed by brief agitation, the solution was heated to 55°C for 20 min and then mixed with 600 ,ul of phenol-chloroform (1:1 [vol/vol]) by brief shaking. The solution was then centrifuged at room temperature, and the resultant upper aqueous phase (-160 RI) was retained. Plasmid DNA thus prepared (10 ,ul) was electrophoresed in 0.3 or 0.5% agarose with reference plasmid DNAs of known molecular sizes (including plasmid NR1, 94.5 kbp in size [54]). The remaining plasmid DNA (100 [lI) was precipitated with ethanol and suspended in 50 RI of 10 mM Tris-HCl (pH 7.5) containing 0.1 mM EDTA. This suspension was mixed with 200 RI of 50 mM CaCl2-treated competent cells of E. coli HB101, and transformants were selected on MacConkey agar plates containing antimicrobial agents. Colony hybridization. E. coli strains grown on MacConkey agar plates were transferred to Whatman 541 filters and processed as described previously (17, 36). Hybridization was performed with 106 cpm of DNA fragments (probes) labeled with a-32P (by random priming [19]) in a mixture containing 2x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 5x Denhardt solution, 0.1% SDS, and 100 ,g of denatured
ACTIN ACCUMULATION ASSOCIATED WITH E. COLI ADHERENCE
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FIG. 4. Transmission electron micrograph showing adherence to HeLa cells of E. coli D2 after 6 h of incubation. The bar is in micrometers. sonicated calf thymus DNA per ml overnight at 42°C. Filters then washed twice in 2x SSC-0.1% SDS for 10 min at 25°C, three times in 2x SSC-0.1% SDS for 10 min at 65°C, twice in 0.1 x SSC-0.1% SDS for 15 min at 65°C, and once in 2x SSC for 10 min at 25°C. Filters were exposed to X-Omat AR film (Eastman Kodak Co., Rochester, N.Y.) at -70°C with an intensifying screen. DNA fragments used as probes were an 0.45-kbp PstI fragment of pSLM852 (5) for the F1845 fimbrial adhesin, a 1-kbp BamHI-SalI fragment of pMAR22 (39) for EAF, a 1-kbp KpnI-SalI fragment of pCVD434 (27) for EAE, a 17-kbp EcoRI fragment of pRM17 (47) for the EIEC plasmid, a 1.154-kbp BamHI fragment of pJN37-19 (42) for Shiga-like toxin I, and an 0.842-kbp PstI-SmaI fragment of pNN110-18 (6) for Shiga-like toxin II. were
RESULTS Characterization of E. coli D2. The serotype of E. coli D2 was 07:H-. In colony hybridization, strain D2 was positive for F1845 but negative for EAF, EAE, the EIEC plasmid, and Shiga-like toxin I or II. Strain D2 had two large plasmids of 95 and 150 kbp. Strain D2 was cultured with HeLa or HEp-2 cells for 3 or 6 h, and its abilities to adhere to tissue culture cells, accumulate F-actin, and invade cells were tested. Clustered adherence and elongation of microvilli at 3 h of incubation. Strain D2 adhered to HeLa and HEp-2 cells in a clustered pattern, as shown in Fig. 1A and B. Autoagglutination of bacterial cells was not observed. The adherent bacteria strikingly induced the elongation of microvilli at the site of adherence, and the elongated microvilli (sometimes fused to each other) totally enwrapped the adherent bacteria (Fig. 1C
and D). The elongated microvilli were found even beneath the adherent bacteria, as shown in Fig. 2. The infected cells were negative in the FAS test, and no bacteria were found inside. Diffuse adherence at 6 h of incubation. After 6 h of incubation, strain D2 adhered to HeLa and HEp-2 cells in a diffuse pattern (Fig. 3A and B). The adherent bacteria had marked surface structures and were not enwrapped by the microvilli (Fig. 3C and 4). Accumulation of F-actin around clustered bacteria. HeLa and HEp-2 cells infected with strain D2 for 6 h were positive in the FAS test, as determined with a confocal laser scanning microscope (Fig. 5A and B). The fluorescence spots (sites of accumulation of actin) observed were clustered in a manner similar to the bacterial adherence pattern at 3 h of incubation. The accumulated actin uniformly surrounded the bacterial cells like a capsule (Fig. SC) and was also found inside the cells (Fig. SD). Invasion of tissue culture cells. Strain D2 was found inside HeLa and HEp-2 cells after 6 h of incubation. Close adherence of strain D2 to tissue culture cells was observed (Fig. 6A). Some infected cells had bacteria (D2) inside, and areas near the bacteria were destroyed (Fig. 6B). Clustered bacteria were also found inside HeLa and HEp-2 cells; however, such cells appeared to be dead (Fig. 6C). Invasion by strain D2 was also examined with a gentamicin protection assay (Table 1). Strain D2 invaded HEp-2 cells to an extent comparable to invasion by an EPEC strain (W1054-1).
Adherence of E. coli HB101 carrying large plasmids derived from strain D2. The 95- and 150-kbp plasmids of strain D2
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FIG. 5. FAS test of HeLa cells (A) and HEp-2 cells (B to D) infected with E. coli D2 for 6 h. Samples were analyzed with a confocal laser scanning microscope. In panels A and B, arrowheads indicate actin accumulation in a clustered pattern (bright spots). One cluster was about 10 jxm in diameter and consisted of ca. 20 fluorescence spots. These parameters are very similar to those of clustered adherence at 3 h of incubation (Fig. 1A and B). In panel C, the bacterial cell (dark spot indicated by an arrowhead) is surrounded by polymerized actin. Panel D represents a section image of panel C at the x and z axes; an arrowhead and an arrow, respectively, indicate the bottom of HEp-2 cells (attached to a glass coverslip) and polymerized actin. In panels A to C, many stress fibers (actin filaments) of the cells can be seen in the background. Bars are in micrometers.
encode resistance to tetracycline (MIC, 64 ,ug/ml, when carried by E. coli HB101) and to ampicillin, chloramphenicol, kanamycin, and trimethoprim (MIC, .256 ,ug/ml, when carried by E. coli HB101), respectively. E. coli HB101 carrying those plasmids displayed no significant adherence to HeLa or HEp-2 cells. Lack of actin accumulation in other F1845 DNA probepositive DAEC strains. DAEC strains Dl, D3, D4, and D6 (which hybridized with the F1845 DNA probe) adhered to HeLa and HEp-2 cells in a diffuse pattern after 3 and 6 h of incubation, as shown in Fig. 7A. No adherent bacteria were enwrapped by the microvilli. The four DAEC strains yielded negative results in FAS tests, as determined with a confocal laser scanning microscope, after 3 and 6 h of incubation, as shown in Fig. 7B.
Actin accumulation associated with EPEC microcolonies. EPEC strain L06 adhered to HeLa and HEp-2 cells in a localized pattern after 3 and 6 h of incubation, as shown in Fig. 8A. At the bottom of the bacterial microcolonies (Fig. 8A, arrowheads), bacteria were caught or enwrapped by the elongated microvilli, and some bacteria appeared to be inserted into the HeLa and HEp-2 cell membranes. Actin accumulation was observed after 3 and 6 h of incubation at the site of attachment of bacterial microcolonies, as shown in Fig. 8B (arrow 1), although some fluorescence spots were associated with bacteria (in microcolonies) located slightly away from the tissue culture cells (Fig. 8B, arrow 2). The bacterial cells were uniformly surrounded by accumulated actin, which looked like a capsule (Fig. 8C, arrowhead). The capsule-like actin structures were aggregated but not
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FIG. 5-Continued.
fused to each other (Fig. 8C). Most capsule-like actin was located immediately beneath the cell surface (Fig. 8D and E, arrows).
DISCUSSION In addition to localized adherence, diffuse adherence, and aggregative adherence (40, 41, 46), this study demonstrated a new type of adherence (clustered adherence) to tissue culture cells (HeLa and HEp-2 cells). The unique features of clustered adherence are that bacterial cells did not autoagglutinate, and single bacterial cells (typically about 20) adhered to "restricted areas" (typically areas 10 ,um in diameter) on the tissue culture cells, forming a cluster. Therefore, clustered adherence is distinctly different from localized adherence, in which bacterial cells autoagglutinate and form microcolonies; thus, adherent bacteria are found almost exclusively in microcolonies (12, 21, 41, 46, 53), as shown in Fig. 8A. In addition, unlike localized adherence (of EPEC), in which microcolonies are found even on plastic coverslips (55), clustered adherence (of strain D2) is
observed only on tissue culture cells. Clustered adherence may not be as clear-cut as localized adherence, because bacterial cells are found in groups varying in size (as in Fig. 1A and B, with groups of 1 to 20 or more bacteria). The possibility exists that in clustered adherence, adherent bacteria change the surface of the tissue culture cells at and around the site of TABLE 1. Invasion by E. coli D2 of HEp-2 cells E. coli strain
D2
W105-4-1b
CFU of bacterial cells/wella Extracellular
Intracellular
1.6 x io8 8.0 x 107 1.3 X 107
2.8 x 103 4.0 x 103
Xacc 3.0 X 102 a HEp-2 cell cultures were incubated for 2 h with bacteria. The intracellular bacterial cell number was determined with a gentamicin protection assay (see Materials and Methods). Experiments were run five times per strain. b An EPEC strain which hybridized with EAF and EAE DNA probes. c K-12 derivative strain.
2924
YAMAMOTO ET AL.
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FIG. 6. Transmission electron micrographs showing adherence to (A) and invasion of (B and C) HEp-2 cells by E. coli D2 after 6 h of incubation. In panel C, arrowheads indicate bacterial invasion in a clustered pattern. Bars are in micrometers.
ACI1N ACCUMULATION ASSOCIATED WITH E. COLI ADHERENCE
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adherence, allowing more bacteria to adhere and making the cluster larger. Indeed, adherent bacterial cells had a striking ability to induce the elongation of microvilli and were enwrapped by the elongated and fused microvilli. Still another possibility is that bacteria recognize "receptors" present on the tissue culture cells and adhere.
Strain D2 hybridized with the F1845 DNA probe (5) used to detect diffuse adherence and changed its adherence pattern from clustered adherence (at 3 h of incubation) to diffuse adherence (at 6 h of incubation). In accordance with this phenomenon, adherent bacteria at 6 h of incubation had marked surface structures (Fig. 4). Since F1845 (fimbrial
FIG. 7. Adherence (A) and lack of actin accumulation (B) with F1845 DNA probe-positive E. coli Dl after 6 h of incubation. HEp-2 cells were used. In panel A, samples were analyzed by scanning electron microscopy; bacterial adherence in a diffuse pattern can be seen. In panel B, samples were analyzed by confocal laser scanning microscopy; no bright fluorescence spots were seen on HEp-2 cells. Bars are in micrometers.
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FIG. 8. Localized adherence (A) and microcolony-associated actin accumulation (B to E) with EPEC strain L06 after 3 h of incubation. HEp-2 cells were used. In panel A, samples were analyzed by scanning electron microscopy; bacterial adherence in a localized pattern can be seen (arrowheads indicate bacterial microcolonies). In panels B to E, samples were analyzed by confocal laser scanning microscopy. Panel B represents a merged image of a laser transmission microscopic image and a confocal laser scanning microscopic image obtained in the same area; an arrowhead indicates the laser transmission microscopic image of a bacterial microcolony, and arrows indicate confocal laser scanning microscopic images of polymerized actin-arrow 1 indicates polymerized actin associated with a microcolony on an HEp-2 cell, and arrow 2 indicates polymerized actin associated with a microcolony located slightly away from an HEp-2 cell. In panel C, the bacterial cell (dark spot indicated by an arrowhead) is surrounded by polymerized actin, and such capsule-like actin structures are aggregated (but not fused). Panels D and E represent section images of panel C at the x and z axes; arrowheads and arrows, respectively, indicate the bottom of HEp-2 cells (attached to a glass coverslip) and polymerized actin immediately beneath the cell surface. In panel C, many stress fibers (actin filaments) of the cells can be seen in the background. Bars are in micrometers.
adhesin (5) is a member of the Dr family of adhesins (43, 49) and the F1845 DNA probe (5), which we used in this study, may not be specific for F1845 and detect the F1845 determinant as well as determinants of other, related adhesins of this family, the marked surface structures observed at 6 h of incubation do not necessarily indicate fimbrial adhesin F1845. The bacterial surface structures observed at 6 h of incubation should be examined with antisera specific for F1845, or bacteria should be examined with DNA probes more specific for F1845. This study demonstrated an example of F1845 DNA probepositive E. coli (DAEC) that caused the accumulation of actin. For a detailed analysis, we used a confocal laser scanning microscope rather than a conventional fluorescence microscope. A unique feature of the actin accumulation in strain D2 is that it appeared as a cluster (at 6 h of incubation). In addition, the accumulated actin was observed as a capsule uniformly surrounding each bacterial cell. Confocal laser scanning microscopic analysis of four other F1845 DNA probepositive DAEC strains produced negative results, indicating that not all F1845 DNA probe-positive E. coli strains induce actin accumulation. The gene sequence for actin accumulation in strain D2 was distinct from that in EPEC. For strain D2, the typical sizes of a cluster of adherent bacteria (at 3 h of incubation), a cluster of capsule-like actin structures (at 6 h of incubation), and a cluster of intracellular bacteria (at 6 h of incubation) were similar to each other; about 10 p,m in diameter, and consisted of about 20 bacteria (or structures of capsule-like actin). These results strongly suggest that D2 cells adhered to the tissue culture cells in a clustered pattern and then invaded the cells as a cluster, causing the accumulation of actin. Cantey and Moseley (7) introduced the F1845 gene into
enterohemorrhagic E. coli [which possesses the chromosomal gene(s) homologous to the EPEC eae gene(s) and causes attaching and effacing lesions, like EPEC (3, 30, 56)] and demonstrated that such bacteria caused the accumulation of actin in a distribution consistent with diffuse adherence of the bacteria. This information may indicate that the adherence factor of strain D2 at 3 h of incubation is distinct from F1845 or that an additional virulence factor(s) is involved in clustered adherence at 3 h of incubation. Such factor(s) may subsequently contribute to actin rearrangement, i.e., induction of the elongation of microvilli (probably by actin polymerization) and the formation of capsule-like actin. Previous studies of EPEC (13, 30) showed that F-actin proliferates beneath areas of intimate bacterial attachment. However, confocal laser scanning microscopic analysis clearly demonstrated that EPEC cells were uniformly surrounded by capsule-like actin. Moreover, bacteria surrounded by accumulated actin were found located even slightly away from tissue culture cells (Fig. 8B). Such capsule-like actin may protect the bacteria from host defense mechanisms and contribute to EPEC-associated chronic diarrhea (9, 13, 16, 18, 23, 33). The formation of capsule-like actin in strain D2 may also fall under this category. Attaching and effacing lesions caused by bacteria have increasingly been reported, e.g., EAF-positive E. coli of nonEPEC serotypes (1) and Helicobacterpyloni (15). The incidence of FAS-positive F1845 DNA probe-positive DAEC in Thai patients with diarrhea is not known. Francis et al. (20) reported that EPEC invasion does not appear to be as destructive to eukaryotic cells as invasion by shigellae or EIEC and speculated that an intracellular location may protect the bacteria from clearance from the intestine and from immunoglobulin A of the immune system. In this study,
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FIG 8-Continued.
invasion by strain D2 was observed in a gentamicin protection assay at levels comparable to invasion by EPEC. For strain D2, bacterial invasion may cause the death of tissue culture cells. An association of the large plasmids (95 and 150 kbp) of strain D2 with adherence was not demonstrated in this study. However, since it is known that plasmids can contain regulatory factors which control chromosomal structural genes (e.g., the ms gene of enterotoxigenic E. coli, which controls E. coli surface antigens 1 and 2 [8]), it may be concluded that these plasmids (of strain D2) do not contain all of the information necessary for adherence. Diarrhea-associated DAEC is a group with genetic heterogeneity (4, 5, 22, 24, 41, 43, 46, 49; this study). Further studies on DAEC epidemiology as well as DAEC infectious mechanisms are required. ACKNOWLEDGMENTS We thank Kazumichi Tamura for serotyping of strain D2. This work was supported by a grant from Ohyama Health Foundation Inc. and by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan.
REFERENCES 1. Albert, M. J., K. Alam, M. Ansaruzzaman, J. Montanaro, M. Islam, S. M. Faruque, K. Haider, K. Bettelheim, and S. Tzipori. 1991. Localized adherence and attaching-effacing properties of nonenteropathogenic serotypes of Escherichia coli. Infect. Immun. 59:1864-1868. 2. Baudy, B., S. J. Savarino, P. Vial, J. B. Kaper, and M. M. Levine. 1990. A sensitive and specific DNA probe to identify enteroaggregative Escherichia coli: a recently discovered diarrheal pathogen. J. Infect. Dis. 161:1249-1251. 3. Beebakhee, G., M. Louie, J. D. Azavedo, and J. Brunton. 1992. Cloning and nucleotide sequence of the eae gene homologue from enterohemorrhagic Escherichia coli serotype 0157:H7. FEMS Microbiol. Lett. 91:63-68. 4. Benz, I., and M. A. Schmidt. 1992. Isolation and serologic characterization of AIDA-I, the adhesin mediating the diffuse adherence phenotype of the diarrhea-associated Escherichia coli strain 2787 (0126:H27). Infect. Immun. 60:13-18. 5. Bilge, S. S., C. R. Clausen, W. Lau, and S. L. Moseley. 1989. Molecular characterization of a fimbrial adhesin, F1845, mediating diffuse adherence of diarrhea-associated Escherichia coli to HEp-2 cells. J. Bacteriol. 171:4281-4289. 6. Brown, J. E., P. Echeverria, D. N. Taylor, J. Seriwatana, V.
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