Characterization of Flagella Purified from Enterohemorrhagic,Vero-Cytotoxin-Producing Escherichia coli Serotype 0157:H7
PHILIP SHERMAN,* ROHINI SONI, AND HERMAN YEGER
Division of Gastroenterology, Departments of Pediatrics and Pathology, Research Institute, The Hospital forSick Children, University of Toronto, Toronto, Ontario MSG IX8, Canada
Received 25 January 1988/Accepted 21 March 1988
Escherichia coli of the serotype 0157:H7 has recently been isolated in human fecal specimens in associationwith sporadic cases and outbreaks of hemorrhagic colitis and with the hemolytic uremic syndrome. The aim ofthis study was to characterize the flagellin protein subunit constituents of flagellar filaments from E. coli0157:H7 strain CL-56. Flagellin isolated from a reference Salmonella enteritidis strain was used forcomparison. Flagella were dissociated by incubation of bacteria under acidic conditions, centrifugation, anddifferential ammonium sulfate precipitation. Reconstituted flagellar filaments were demonstrated by threecomplementary methods: transmission electron microscopy, antigenic reactivity with H7 antiserum by a dotblot immunoassay, and immunogold localization of antiserum raised to the purified antigen to intact flagella onwhole E. coli 0157:H7. On sodium dodecyl sulfate-polyacrylamide gels flagellin proteins from E. coli 0157:H7demonstrated an apparent Mr of 66,000. The isoelectric point of E. coli 0157:H7 flagellin was 5.42. Byimmunoblotting, H7 flagellin proteins were shown to be immunogenic. They induced a systemic immuneresponse both in rabbits challenged with whole bacteria and in a human previously infected with E. coli0157:H7.
Escherichia coli of the serotype 0157:H7 has been iso-lated from stool specimens in association with both sporadiccases (22) and outbreaks (23) of hemorrhagic colitis inhumans. This bacterial strain has also been associated withthe most common cause of acute renal failure in children, thehemolytic uremic syndrome (12). The organisms producehigh levels of a cytopathic toxin variously termed Verocytotoxin and Shiga-like toxin (4).
Flagella are filamentous protein appendages that protrudefrom the cell surfaces of many bacteria and confer motileproperties to the organisms (10, 25). As a result of motility,flagella may permit increased bacterial penetration of thesurface mucous gel and thus promote increased colonizationby enteric pathogens at the mucosal surface (19, 28). Flagellaare composed of repeating protein subunits termed flagellinand a proximal basal body which anchors the flagellarfilament to the bacterial cell surface by a series of hook-associated proteins (7, 10). Flagellin proteins produced bycertain members of the family Enterobacteriaceae such as E.coli and Salmonella species are antigenically heterogeneous(9, 11, 17), thus providing the basis for producing antisera foruse in serotyping classifications of enteric organisms (17). Inthis study, we isolated H7 flagellar filaments from an E. colistrain of the 0157:H7 serotype and demonstrated that thefilaments are immunogenic.
MATERIALS AND METHODS
Bacteria and growth conditions. An E. coli strain, desig-nated CL-56, of the serotype 0157:H7 originally isolatedfrom stool specimens of a child with hemorrhagic colitis andthe hemolytic uremic syndrome (12) was kindly provided tous by M. Karmali, The Hospital for Sick Children, Toronto.The isolate does not appear to elaborate fimbrial antigens invitro (24). For comparison with previously purified flagella,Salmonella enteritidis of the serotype 1,9,12:g,m: - (Salmo-nella Reference Laboratory reference number 1267; kindly
* Corresponding author.
provided to us by G. Ibrahim, University of New SouthWales, Australia) was used in these experiments (8). Stockcultures of both organisms were stored in 10% glycerol at-70°C. Each strain was inoculated into Penassay broth(Difco Laboratories, Detroit, Mich.) and grown under static,nonaerated conditions at 37°C. After overnight incubation,bacteria were harvested by centrifugation at 5,000 x g for 30min and pelleted bacteria were suspended in sterile 0.9%normal saline.
Isolation and purification of filaments from H7 flagella. Asdescribed by Ibrahim et al. (8), flagella were dissociated frombacteria by adjusting the pH of the bacterial suspension to2.0 with 1 N hydrochloric acid for 30 min. Bacteria devoid offlagella were pelleted by centrifugation at 5,000 x g for 30min. The flagellin-rich cell-free supernatant was centrifugedat 100,000 x g for 1 h at 4°C, and the supernatant was
adjusted to pH 7.2 with 1 N sodium hydroxide. Flagellin andreconstituted flagellar filaments were precipitated by incuba-tion with 2.67 M ammonium sulfate for 16 h at 4°C andcollected by centrifugation at 15,000 x g for 15 min. Thepellet was suspended in distilled water and dialyzed in tubingwith a 50,000-molecular-weight cutoff against distilled watercontaining activated charcoal at 4°C for 24 h. Purification ofH7 filaments was performed in the presence or absence of a1.0 mM final concentration of the proteinase inhibitor phen-ylmethylsulfonyl fluoride (Sigma Chemical Co., St. Louis,Mo.).
Electron microscopy. The presence of both flagella on
intact organisms and reconstituted flagellar filaments was
determined by transmission electron microscopy after neg-ative staining as described previously (8). Briefly, intactorganisms and purified flagellar filaments were placed ontoFormvar-coated 300-mesh copper grids for 1 min, excess
fluid was removed with filter paper, and the grids were
stained with phosphotungstic acid (2% [wt/vol]; Sigma) atpH 7.0. The grids were examined in a Philips 300 transmis-sion electron microscope at 80 kV.
Analytical polyacrylamide gel electrophoresis. The compo-
sition of purified flagellin was determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis in the presence of0.5 M urea after the gels were stained with Coomassie blue(8, 15). The apparent molecular weights of flagellin subunitswere calculated relative to known molecular weight stan-dards (Bio-Rad Laboratories, Richmond, Calif.). The effectof bacterial proteases on the size of flagellin subunits wasdetermined by gel electrophoresis of flagellar filaments thatwere isolated and purified in the presence or absence ofphenylmethylsulfonyl fluoride.
Immunoblotting of purified filaments. Flagellin was de-tected by immunolabeling with H7-specific antisera. Antise-rum to purified H7 flagellar filaments was raised by threesubcutaneous injections of 100 ptg of antigen mixed inFreund complete adjuvant (Difco) in 2-kg male New ZealandWhite rabbits (P. Sherman and R. Soni, J. Med. Microbiol.,in press). Antiserum was collected by venipuncture 2 weeksafter the final injection ofantigen. A second rabbit antiserumto H7 flagella was purchased from a commercial source(Difco). Dot blot immunoassays were performed with 10 ,ugof purified H7 flagellar filaments and bovine serum albumin(BSA; Sigma) as antigens and a 1:50 dilution of each of thetwo rabbit antisera (26).Western blots (immunoblots) were performed to establish
the immunogenicity of filaments on intact flagella. For West-ern immunoblotting, H7 flagellin was transferred from poly-acrylamide gels to nitrocellulose paper as described previ-ously (27). Nitrocellulose paper was then incubated with a1:50 dilution of a rabbit antiserum previously raised by us tointact whole 0157:H7 organisms (24), commercial rabbitanti-H7 antiserum (Difco), and Vero toxin antibody-positivehuman serum obtained from a patient previously infectedwith E. coli 0157:H7 (serum kindly provided by M. Karmali,The Hospital for Sick Children). Binding of antibody toflagellin was detected by incubation of nitrocellulose blotswith a 1:5,000 dilution of protein A-horseradish peroxidaseconjugate (Bio-Rad).
Isoelectric point. The pIs of flagellins purified from E. coli0157:H7 and S. enteritidis were calculated after isoelectricfocusing on horizontal slab gels (Servalyt; Serva, Heidel-berg, Federal Republic of Germany).Amino acid analysis. Amino acid compositions of purified
flagellin were determined by the Biotechnology ServiceCentre, Research Institute, The Hospital for Sick Children,by the Pico-Tag system after samples had been hydrolyzedin 6.0 N hydrochloric acid-10.1% phenol for 24 h at 110°C.Results of the means of two experiments are expressed asmoles per 100 moles.Immunoelectron microscopy. To confirm that the final
flagellin preparation contained reconstituted flagellar fila-ments, we incubated 0.01 ml of the rabbit antiserum raisedby us at a 1:50 dilution for 2 h at 37°C with 1.0 ml of 0.1%glutaraldehyde-fixed E. coli 0157:H7 which had previouslybeen suspended in 1% BSA for 1 h at 37°C. After incubationwith antiserum, bacteria were washed three times in sterilesaline and placed onto 300-mesh Formvar-coated nickelgrids. To decrease nonspecific binding of gold, we incubatedthe grids with 10% normal goat serum for 1 h at roomtemperature. Grids were then incubated with 16-nm colloidalgold particles conjugated to goat anti-rabbit immunoglobulinG (Janssen Life Sciences Products, Olen, Belgium) over-night at 4°C followed by four washings of the grids withphosphate-buffered saline. After negative staining with 2%phosphotungstic acid, localization of gold particles that werebound to antibody adherent to surface constituents of E. coli0157:H7 was examined in a Philips 300 transmission elec-
le
0
FIG. 1. Transmission electron photomicrograph showing flagella(arrows) expressed on the surface of E. coli 0157:H7 strain CL-56.
tron microscope at 60 kV as described previously (20).Preimmune rabbit serum and antiserum raised to outermembranes of E. coli 0157:H7 (Sherman and Soni, in press)were used as negative controls.
RESULTS
Under the growth conditions used, E. coli CL-56 wasmotile when examined by phase-contrast microscopy andflagella were expressed on the cell surface of bacteria whenintact organisms were examined by transmission electronmicroscopy (Fig. 1). After disruption of flagella from bacte-ria and further purification by ammonium sulfate precipita-tion and dialysis, reconstituted flagellar filaments were dem-onstrated by transmission electron microscopy (Fig. 2).The flagellin protein subunits making up the reconstituted
flagellar filaments isolated from E. coli 0157:H7 differed inseveral respects from those purified from the S. enteritidisreference strain. The predominant flagellin band from E. coliCL-56 was larger in size than the flagellin subunits of S.enteritidis as determined by electrophoresis in the presenceof 0.1% sodium dodecyl sulfate and 0.5 M urea (Fig. 3). Theapproximate molecular weight (Mr) of H7 flagellin subunitswas 66,000 compared with an approximate Mr of 58,000 forflagellin subunits isolated from S. enteritidis; the latter isequivalent to the molecular weight determination reportedby Ibrahim et al. (8).Both the E. coli and S. enteritidis strains appeared to
elaborate proteases that degraded flagellin subunits since
FIG. 2. Transmission electron photomicrographs demonstrating reconstituted flagellar filaments from E. coli 0157:H7 (A) and S.enteritidis (B) after disruption from intact bacteria and purification as described in Materials and Methods.
electrophoresis of flagellin purified in the absence of theproteinase inhibitor phenylmethylsulfonyl fluoride resultedin a shift of the predominant protein band to a lowerapparent Mr of approximately 21,000 (Fig. 4). The majorprotein band for E. coli 0157:H7 flagellin was still larger insize than that of S. enteritidis flagellin extracted in theabsence of proteinase inhibitors. The presence of a singleprotein band rather than a series of proteins of various
1 2 3
92.5 ~ P
66.2 --
45.0 -à- -*
31.0 -
21.5 oe
14.4 -
molecular masses suggests that the action of proteinases isquite specific rather than cleaving flagellin protein randomlyat multiple sites.
After electroelution from polyacrylamide gels and isoelec-tric focusing, flagellin extracted from E. coli 0157:H7 in thepresence of proteinase inhibitor had a pI of 5.42 in compar-ison with a pI of 5.59 for the flagellin of the S. enteritidisreference strain. The lower isoelectric point for E. coli H7flagellin is likely due to the relatively greater molar concen-trations of acidic amino acids aspartic acid and glutamicacid. The amino acid composition of extracted flagellin isshown in Table 1. The abundance of the acidic amino acidresidues aspartic acid and glutamic acid in flagellin prepara-tions has been noted previously in analyses of other purifiedflagellin proteins (5, 14, 20). Similar to previous findings (14),
1 2 3
92.5 -66.2 -
45.0 -
31.0 -
21.5 -
14.4 -
FIG. 3. Flagellin subunits of flagella purified from E. coli sero-
type 0157:H7 (lane 2) and S. enteritidis (lane 3) after gel electro-phoresis in 10% polyacrylamide in the presence of 0.1% sodiumdodecyl sulfate and 0.5 M urea as described previously (6). Majorflagellin bands are indicated by the adjacent arrows. Lane 1 containsknown molecular weight standards (103).
- _-
FIG. 4. Sodium dodecyl sulfate-polyacrylamide gel electropho-resis of flagellins purified from E. coli 0157:H7 (lane 2) and S.enteritidis (lane 3) in the absence of the proteinase inhibitor phen-ylmethylsulfonyl fluoride. The major protein band (arrowhead) isapproximately 21,000 molecular weight. Lane 1 contains molecularweight standards (103).
a Mean of two separate experiments.b Results of one experiment.
neither E. coli nor S. enteritidis flagellin contained detect-able tryptophan. E. coli H7 flagellin did not contain histidineresidues, in contrast to a low level of histidine in flagellinfrom the S. enteritidis reference strain, findings that are
consistent with those reported previously (14).In addition to electron microscopic evidence of reconsti-
tuted flagella shown in Fig. 2A, the material purified from E.coli 0157:H7 strain CL-56 was confirmed as flagellar fila-ments by two other complementary methods. First, purifiedmaterial reacted in a dot blot immunoassay with a rabbitantiserum prepared to H7 flagella (Fig. 5, bottom half of lane1 and lane 2). The antiserum did not react nonspecificallywith BSA (Fig. 5, top half of lanes 1 and 2) or withheterologous antigens such as bacterial outer membraneextracts, 0157 lipopolysaccharide, and flagellar filaments ofS. enteritidis (data not shown). Second, immunogold label-ing of intact E. coli 0157:H7 showed that the H7 flagellar
1 2
FIG. 5. Dot blot immunoassay with BSA (top half of each lane)and purified H7 flagellar filaments (bottom half of each lane). Rabbitantisera, one raised by us to the H7 flagellin preparation (lane 1) anda second commercially available H7 antiserum (lane 2), were
incubated on nitrocellulose strips on which BSA and H7 flagellarfilaments had been placed as antigens. A positive reaction withprotein A-peroxidase conjugate is shown with flagellar preparationsin the bottom half of each lane. Nonspecific binding of antisera toBSA (top half of each lane) was not observed.
antiserum raised by us in rabbits localized to flagella pro-truding from the cell surface of intact organisms (Fig. 6A). Incontrast, localization of gold particles to flagella was notobserved when preimmune rabbit serum or heterologousantiserum was incubated with organisms placed onto grids(data not shown). Incubation of S. enteritidis with H7antiserum did not result in immunogold labeling of flagella(Fig. 6B).To determine the immunogenicity of flagellin constituents
of H7 flagella, we next performed Western immunoblots.Antiserum raised in rabbits to intact E. coli 0157:H7 (24)(Fig. 7, lane 3) bound to protein in the flagellin preparationthat was the same molecular size as the flagellin protein withwhich a commercial H7 flagellar antiserum reacted (Fig. 7,lane 2). Furthermore, serum obtained from a patient previ-ously ill with E. coli 0157:H7-associated hemorrhagic colitisalso bound to flagellin proteins on Western immunoblots(Fig. 7, lane 4).
DISCUSSIONWe demonstrated that flagella isolated and purified from
E. coli 0157:H7 can be dissociated and reconstituted. Re-constituted flagellar filaments were observed by electronmicroscopy after negative staining. These filaments wereantigenically recognized by a commercially available H7flagellar antiserum, and by using immunogold labeling, weshowed that rabbit antiserum raised to the purified materiallocalized to flagellar filaments on intact E. coli 0157:H7.
In this study, the flagella of E. coli 0157:H7 were charac-terized ultrastructurally, biochemically, and antigenically.H7 flagellar filaments were shown to differ in several re-spects from those isolated from a reference S. enteritidisstrain. Our results indicated that H7 flagella of E. coliserotype 0157:H7 are composed of flagellin subunits with anapparent molecular weight of 66,000. The subunits are,therefore, larger than the 58-kilodalton subunits that makeup flagellar filaments on S. enteritidis (8). Since flagellinssynthesized by gram-negative bacteria are nonglycosylatedproteins (10, 25), the differences in molecular weight must berelated to varying amino acid sequences.The apparent molecular weight of H7 flagellin subunits
determined by us is larger than that previously reported forflagellin purified from laboratory E. coli K-12, for which theMr was variously reported as 51,172 (14) and 60,000 (13).Lower-molecular-weight protein bands shown on the gel inFig. 4 may be fragments of flagellin rather than nonflagellarcontaminants since many of these bands were also observedafter gel electrophoresis of the electroeluted 66,000-Mr band(data not shown). Similar findings have also been observedby others (8). The small amount of cysteine found to bepresent in flagellin by amino acid analysis (Table 1) couldindicate the presence of a small amount of contaminatingproteins since purification of flagellin from E. coli and othergram-negative bacteria has not indicated the presence ofpotential sulfhydryl bonds (14, 21).Antiserum raised to flagellin was shown by immunoelec-
tron microscopy (Fig. 6) to react with multiple antigenic sitesalong the length of the flagellar filament on intact E. coli0157:H7. Lack of aggregation of gold particles at either thetip or the base of the flagellar filament indicates that theantiserum was raised to the repeating flagellin subunits thatform the filament rather than to copurified basal bodies orhook-associated proteins that attach the filament to thebacterial cell surface.The findings with immunoblots also indicated that flagellin
of H7 flagella is immunogenic. Flagellin subunits of H7
FIG. 6. Immunogold labeling of flagella after incubation of E. coli 0157:H7 (A) and S. enteritidis (B) with rabbit anti-H7 antiserumfollowed by immunogold conjugated to goat anti-rabbit immunoglobulin G. Gold particles are clustered along the length of the flagellarfilament on E. coli 0157:H7.
flagella were recognized by antibodies contained in antise-rum from a rabbit immunized with whole E. coli 0157:H7. Inaddition, serum obtained from a patient after an acute illnesscharacterized by hemorrhagic colitis and in whom E. coli0157:H7 was isolated in stool specimens contained antibod-ies to flagellin subunits of H7 flagella. Although this is thefirst report of the immunogenicity of H7 flagella, antigenicityof flagellin purified from other gram-negative enteric patho-gens has been reported previously (18).The functional role of H7 flagella in promoting virulence of
E. coli 0157:H7 infection in humans is not yet defined.However, expression of flagella by other enteric pathogenssuch as Campylobacter jejuni (19) and Vibrio cholerae (28)appears to be an important virulence factor. Increasedmotility allows organisms to both colonize and penetrate thesurface mucous layer which overlies the lining of the gastro-intestinal tract (16). Penetration of the mucous layer resultsin increased exposure of organisms to the surface ente-rocytes and renders the organisms less susceptible to clear-ing from the bowel by normal peristaltic activity (19, 28). Inaddition to virulence associated with motility properties ofbacteria, certain flagella may also function as bacterialattachment factors, or adhesins, that promote binding ofpathogenic organisms to receptors present on the surface ofenterocytes and colonocytes (1, 2, 21).
Using a tissue culture adherence assay we previouslydemonstrated that adherence of E. coli 0157:H7 strainCL-56 to human epithelial cells is inhibited by extracts ofbacterial outer membranes. In contrast, equal concentra-tions of H7 flagella and 0157 lipopolysaccharide did notinhibit adherence of organisms to human epithelial cells(Sherman and Soni, in press). Nevertheless, H7 antigensappear to be an important factor in the pathogenesis ofhuman disease because nonmotile, aflagellar E. coli strainsof the 0157 serogroup are much less frequently isolated instool specimens from symptomatic patients than are E. colistrains of the 0157:H7 serotype.
1 2 3 4
92.5-66.2 -
45.0 -
31 o--
21.5 -
1 4.4 -j
FIG. 7. Western immunoblotting of flagellar filaments extractedfrom E. coli 0157:H7 demonstrating antigenicity of intact flagella.Flagellar filaments that were transferred to nitrocellulose paper wereincubated with whole-cell 0157:H7 rabbit antiserum (lane 2), com-mercially available H7 rabbit antiserum (lane 3), and serum obtainedfrom a patient previously ill with hemorrhagic colitis and in whom E.coli 0157:H7 was isolated in the stools (lane 4). The arrowheadindicates a positive reaction of antiserum with flagellin proteins ineach lane. Molecular weight standards (103) transferred to nitrocel-lulose paper and stained with amido black are shown in lane 1.
Expression of flagella by bacteria is subject to phasevariation. For example, certain aflagellar strains can revertto expression of flagellar antigens under the appropriateenvironmental conditions (3). Therefore, generation of E.coli 0157 isogenic mutants that are deficient only in thechromosomal genes coding for H7 flagella is necessary toestablish more definitively the functional significance ofthese surface structures in the pathogenesis of disease asso-ciated with these newly recognized enterohemorrhagic E.coli strains.
ACKNOWLEDGMENTS
This work was supported by a grant from the Medical ResearchCouncil of Canada. P. Sherman is the recipient of a Career ScientistAward from the Ontario Ministry of Health Research PersonnelDevelopment Program.
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