immunological biochemicalrelationships among flagella...

INFECTION AND IMMUNITY, Aug. 1981, p. 602-6100019-9567/8 1/080602-09$02.00/0

Vol. 33, No. 2

Immunological and Biochemical Relationships Among FlagellaIsolated from Legionella pneumophila Serogroups 1, 2, and 3

J. A. ELLIOTT AND W. JOHNSON*Department of Microbiology, University of Iowa, Iowa City, Iowa 52242

Received 27 March 1981/Accepted 18 May 1981

Flagella were isolated from virulent Legionella pneumophila serogroups 1, 2,and 3. Antiserum made against purified serogroup 1 flagellin agglutinated live,flagellated serogroups 1, 2, and 3 but not heat-killed or nonflagellated bacteria. Asingle line of identity was seen in immunodiffusion slides between the flagellaisolated from the three serogroups and antibody to flagellin isolated from sero-groups 1, 2, and 3. Indirect immunoperoxidase staining showed that antibody toflagellin isolated from serogroup 1 organisms reacted with flagella on serogroup1, 2, and 3 bacteria. Indirect immunoperoxidase staining also showed that antibodyto flagellin isolated from serogroup 1 L. pneumophila did not react with theserogroup-specific cell surface antigen, thus demonstrating that the flagella- andthe serogroup-specific antigen are separate antigens. The amino acid content ofthe flagella from the three serogroups was essentially the same, with aspartate,glutamate, alanine, and threonine comprising 41% of the total. Thirty-five percentof the amino acids were hydrophobic, and there were no detectable amounts ofcysteine, tryptophan, or tyrosine.

There are currently six recognized serogroupsof Legionella pneumophila based on direct im-munofluorescent staining (17). Previous workfrom our laboratory has shown that the high-molecular-weight F-1 antigen is the major anti-gen detected by indirect immunofluorescencestaining and microagglutination assay tests andis responsible for the serogroup specificity of L.pneumophila (10, 11). Currently, little is knownabout the possible role of other antigens in theantigenic structure of L. pneumophila. Thoma-son et al. (22), Chandler et al. (2), and Rodgerset al. (20, 21) have shown that fresh isolates ofL. pneumophila have flagella. Chandler et al.(3) have also reported that flagellated bacteriaare present in tissue sections taken from thelungs of patients with confirmed Legionellosis.Since flagella are major antigens and have beenused to serotype other organisms, most notablySalmonella and Escherichia coli, we initiatedthis study to determine the immunological andbiochemical relationship of flagella isolated fromdifferent serogroups of L. pneumophila and todetermine the role of flagella in the antigenicstructure of L. pneumophila.

MAT'ERIALS AND METHODSOrganisms. L. pneumophila serogroups 1 (Phila-

delphia 2), 2 (Togus), and 3 (Bloomington 2) wereobtained from the Centers for Disease Control, At-lanta, Ga. Nonflagellated bacteria used for agglutina-tion studies were obtained by passage of cultures on

supplemented Mueller-Hinton agar (8). Flagellatedbacteria were obtained from infected guinea pigs andstored as frozen (-70°C) spleen suspensions. Flagel-lated bacteria were recovered from the frozen guineapig spleen suspensions by swabbing the suspensionsonto charcoal-yeast extract medium (CYE) (7). Theguinea pig 50% lethal dose of the nonflagellated strains,as determined by the method of Reed and Muench(19), was greater than 1011 colony-forming units. Theguinea pig 50% lethal dose for the flagellated strainswas 1.5 x 105 colony-forming units for the serogroup1 strain, 2.6 x 105 colony-forming units for the sero-group 2 strain, and 7.6 x 106 colony-forming units forthe serogroup 3 strain.

Animals. Guinea pigs weighing 250 to 350 g wereused to maintain the flagellated bacteria. New Zealandwhite rabbits weighing 3 to 4 kg were used for antibodyproduction. All animals were purchased from localsuppliers.

Flagella preparation. Frozen guinea pig spleensuspensions containing serogroup 1, 2, or 3 were platedonto CYE agar and incubated at 35°C in a humidifiedincubator. After 4 days of incubation, the organismswere transferred onto fresh CYE medium. Thirtyplates were inoculated with each serogroup and incu-bated until confluent growth was obtained. The bac-teria were then washed off the plates with sterile 0.85%saline. The bacterial suspension was then forcedthrough a 27-gauge needle. After passing the suspen-sion through the needle a second time, the bacteriawere removed by centrifugation at 7,000 x g for 20min. The supernatant fluid containing the flagella wascentrifuged at 180,000 x g for 20 min. The pelletcontaining the flagella was then washed three timeswith distilled water. To determine the amount of

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L. PNEUMOPHILA FLAGELLA 603

bacterial contamination, a sample of the final pelletwas then prepared for electron microscopy.

Preparation of serogroup 1 F-1 antigen. Sero-group 1 F-1 antigen was prepared as previously de-scribed (11).SDS-PAGE. Sodium dodecyl sulfate-polyacryl-

amide slab gel electrophoresis (SDS-PAGE) was per-formed by the method of Laemmli (12). Protein bandswere located by staining the gels with Coomassiebrilliant blue R250.

Preparation of antisera. Antiserum was preparedagainst the serogroup 1 F-1 antigen as previouslydescribed (11). The flagellin from serogroups 1, 2, and3 was purified by using a preparative SDS-polyacryl-amide slab gel. A total of approximately 500 ,ug ofprotein as determined by the method of Lowry et al.(14) was applied to the top of the gel. After the gelwas run, several tracks were removed and preparedfor staining. The remaining unstained gel was cut into2-mm sections and placed into stoppered test tubes.After the position of the flagellin band was determinedby staining, the unstained sections containing the fla-gellin band were minced and injected intramuscularlyinto 3.0-kg rabbits (23). The rabbits were boosted inthe same manner 3 weeks later and bled via a marginalear vein 2 weeks after the second injection.

Slide agglutination. Antigens for the slide agglu-tination tests were prepared by growing flagellatedand nonflagellated strains of serogroups 1, 2, and 3 onCYE agar as previously described. To insure that theflagella remained on the bacteria, the bacteria weregently removed by flooding to plates with sterile 0.85%saline. Two drops of live flagellated or nonflagellatedbacteria or heat-killed cells (101°C 1 h) was thenmixed with 1 drop of anti-serogroup 1 flagellin. Testswere scored as positive (agglutination observed within15 min) or negative (no agglutination).Immunodiffusion. Ouchterlony double immuno-

diffusion was performed in 1% agarose made up in 0.1M Veronal buffer (pH 8.6) containing 3% polyethyleneglycol 6,000 and 0.4% sodium azide. Five milliliters ofmelted agar mixture was poured into a plastic petridish (15 by 60 mm; Falcon Plastics, Oxnard, Calif.),and 3-mm wells were cut into the solidified agar witha gel punch. Antiserum and antigen were then addedto the appropriate wells with 50-,ul micropipettes.

Preparation of bacteria for electron micros-copy. Flagellated bacteria from each serogroup weregrown from stock frozen guinea pig spleen suspensionsby plating onto CYE agar. After initial growth wasobtained, the bacteria were transferred onto threeplates of CYE. The bacteria were incubated for 3 daysat 35°C. Initial experiments showed that the flagellawere sheared from the bacteria unless they were han-dled very gently. The plates were first flooded with 5ml of sterile 0.85% saline for 1 h, after which thebacteria were removed from the surface of the agar bygently rocking the plates from side to side. A 0.75-mlportion of the bacterial suspension was then added to0.25 ml of 4% glutaraldehyde to obtain a final concen-tration of 1% glutaraldehyde. The bacteria were fixedfor 1 h and then stained with 2% phosphotungstic acidfor 15 s.

Indirect immunoperoxidase staining. Indirectimmunoperoxidase staining was performed by a mod-

ification of the method outlined by Bohn (1). Thismodification consisted of directly absorbing the glu-taraldehyde-fixed bacteria onto 400-mesh carbon-coated grids before staining, to insure that the flagellawere not removed during the staining process. Thegrids were then washed by floating them on smalldrops of phosphate-buffered saline (0.15 M sodiumphosphate-0.85 M NaCl, pH 7.2). After they werewashed, the grids were floated on normal C3H mouseserum for 20 min and then washed, by floating, withthree changes of phosphate-buffered saline for a totalof 20 min. The grids were floated on normal mouseserum, to reduce nonspecific absorption of rabbit anti-bodies onto the bacterial flagella or cells. The gridswere then placed on either anti-serogroup 1 F-1 serumor anti-serogroup 1 flagellin serum for 20 min. Afterthey were washed again with phosphate-buffered sa-line, the grids were floated on horseradish-peroxidase-labeled goat anti-rabbit immunoglobulin G (IgG; MilesLaboratories, Inc., Elkhart, Ind.) for 20 min. The gridswere then washed with phosphate-buffered saline for20 min, and the stain was developed for 10 min with amixture of 0.03% diaminobenzidine tetrahydrochlorideand 0.01% H202 in 0.05 M tris(hydroxymethyl)amino-methane (Tris) buffer (pH 7.6). After they werestained, the grids were washed for 20 min with Trisbuffer.

Bacteria used for ultrathin sectioning were stainedin the same manner except that reactions and wash-ings were performed in culture tubes (12 by 75 mm).

Electron microscopy. Whole cell and flagellamounts were made on carbon-coated 400-mesh coppergrids and negatively stained with 2% phosphotungsticacid.

Ultrathin sections were placed on uncoated gridsand postsection stained with uranyl acetate and leadcitrate. All grids were viewed and photographed usinga JEOL 100B electron microscope.Amino acid analysis. Amino acids were analyzed

in a Beckman 121MB amino acid analyzer after hy-drolysis in 6 M HCI at 110°C for 24 and 72 h. Thevalues for serine and threonine were determined bylinear extrapolation to zero time. The analyses werenormalized for proline, glycine, and alanine. Trypto-phan was determined after hydrolysis in p-toluenesul-fonic acid by the procedure of Liu and Chang (13).

RESULTSFlagella. Figure 1 shows flagellated bacteria

of L. pneumophila serogroups 1 (Fig. 1A), 2 (Fig.1B), and 3 (Fig. 10). All serogroups have a singlepolar flagellum. However, these flagella are eas-ily sheared when the organisms are washed offthe surface of the agar medium, and many un-attached flagella can be seen, as shown in Fig.1D.SDS-PAGE. Flagella were dissociated into

flagellin subunits with 2-mercaptoethanol andSDS before being analyzed by SDS-PAGE. Ascan been seen in Fig. 2, the flagellin subunits ofserogroups 1, 2, and 3 show one major proteinband with a molecular weight of approximately47,000 when compared with the standards.

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FIG. 1. Electron micrographs showing flagella of L. pneumophila. (A) serogroup 1; (B) serogroup 2; (C)serogroup 3; (D) serogroup 1. Bar equals 1 umn.

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TABLE 1. Slide agglutination of L. pneumophilaserogroups 1, 2, and 3 with antiserum to flagellin

isolated from L. pneumophila serogroup 1

Agglutination with serogroup:Antigen used

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Flagellated cells + + +Heat-killed cellsa - - -Nonflagellated cells

aCells were heated at 101°C for 1 h.

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FIG. 2. SDS-PAGE of purified flagella isolatedfrom L. pneumophila. 1, Serogroup 1; 2, serogroup 2;3, serogroup 3. The standards were bovine serum

albumin (BSA), ovalbumin (OVA), ribonuclease(RNase).

Slide agglutination. The results of aggluti-nation studies (Table 1) show that antibody toflagellin from serogroup 1 reacts with flagellatedbacteria of serogroups 1, 2, and 3 but does notreact with nonflagellated organisms of the sameserogroups. When flagellated bacteria from thethree serogroups are heat killed by being heatedat 101°C for 1 h, they no longer agglutinate withanti-flagellin antibody. This suggests that theflagellar antigen is either destroyed or removedfrom the bacteria by heating.Immunodiffusion. Results obtained from

SDS-PAGE and agglutination studies suggested

that the flagella from L. pneumophila sero-groups 1, 2, and 3 were antigenically similar. Theimmunological relatedness of the flagellin fromserogroups 1, 2, and 3 was determined by im-munodiffusion. As can be seen in Fig. 3A, anti-bodies to flagellin isolated from serogroups 1, 2,and 3 react with a single line of identity whendiffused against flagella isolated from serogroup1 bacteria. Similar results were obtained whenthe three anti-flagellin antibodies were diffusedagainst serogroup 2 and 3 flagella (Fig. 3B andC).Indirect immunoperoxidase staining. In-

direct immunoperoxidase staining was used toinsure that antibody to the flagellin subunitswas specific for flagella. Figure 4A illustratescontrol cells incubated with normal rabbit se-rum. The serogroup 1 anti-flagellin antibodyreacted with the native flagella of serogroups 1,2, and 3 bacteria (Fig. 4B-D). Figure 5 showsthat the flagellin antibody reacted with the na-tive flagella (Fig. 5A) but not the cell surface(Fig. 5B). These results confirned that the an-tibody against flagellin prepared by SDS-PAGEreacted with native flagella but not with any cellsurface antigen. In contrast, antibody againstthe serogroup 1-specific F-1 antigen did not reactwith the flagella, as seen in Fig. 6A, but did reactwith F-1 antigen located on the cell surface ofserogroup 1 bacteria (Fig. 6B).Amino acid analysis. Amino acid analysis

of the isolated flagella from serogroups 1, 2, and3 is presented in Table 2. No cystine, tryptophan,or tyrosine was detected in any of the flagellinsubunits.

DISCUSSIONIsolates of L. pneumophila can be divided into

six serogroups based on the detection of sero-group-specific cell surface antigens by direct im-munofluorescent staining (17). The serogroup-specific antigen is also the major antigen de-tected in human convalescent serum by both theindirect immunofluorescent and microagglutin-ation assays (10). However, the exact chemicalcomposition of the serogroup-specific antigendetected by these serological tests has not yet

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FIG. 3. Immunodiffusion analysis of flagella iso-lated from L. pneumophila serogroups 1, 2, and 3. H-1, Serogroup 1 flagella, H-2, serogroup 2 flagella; H-3, serogroup 3 flagella. AH-1, Antibody to serogroup1 flagellin; AH-2, antibody to serogroup 2 flagellin;AH-3, antibody to serogroup 3 flagellin.

been elucidated. Wong et al. (25) have reportedthe isolation of a serospecific antigen composedof a lipid-protein-carbohydrate complex. In ad-dition, they reported the isolation of a proteinfraction which contained a common antigen.Johnson et al. (10) reported the isolation of ahigh-molecular-weight carbohydrate antigen.This antigen (F-1) was shown to be the sero-group-specific antigen (11). Recently it was re-ported that the F-1 serogroup-specific antigen isa cell surface component (5). This was confirmedin the present study by indirect immunoperoxi-dase staining, which showed that antibody tothe F-1 antigen reacted with a cell surface anti-gen but did not react with flagella.Thomason et al. (22), Chandler et al. (2), and

Rodgers et al. (20, 21) have shown that freshisolates of L. pneumophila have flagella. Sincemost flagella are good antigens, it appeared im-portant to determine the role of flagella in theantigenic structure of L. pneumophila. The fla-gella isolated from L. pneumophila serogroups1, 2, and 3 all had subunits with a molecularweight of approximately 47,000 as determinedby SDS-PAGE with approximately the sameamino acid content. The amino acid content ofthe flagellin subunits isolated from L. pneumo-phila is similar to that of flagellin subunits iso-lated from Salmonella and Bacillus (9, 15, 16).No cystine, tryptophan, or tyrosine was de-tected. Forty-one percent of the subunit wascomposed of aspartic acid, glutamic acid, ala-nine, and threonine, and approximately 35% ofthe molecule was composed of hydrophobicamino acids. Only small amounts of proline,histidine, and methionine were detected.Of major interest is the fmding that the fla-

gellin subunits of L. pneumophila serogroups 1,2, and 3 are antigenically identical. Indirect im-munoperoxidase labeling showed that antibodyto serogroup 1 flagellin reacted with the flagellaof serogroups 1, 2, and 3 (Fig. 4), and immuno-diffusion studies showed that antibody to sero-group 1, 2, and 3 flagellin reacted with a singleline of identity with flagella isolated from sero-group 1, 2, or 3 (Fig. 3). These studies are con-sistent with the amino acid analysis, whichshowed that the subunits from all three sero-groups had the same molecular weight and thesame amino acid content. Our laboratory is cur-rently sequencing the subunits to confirm thestructural identity of the flagellin subunits iso-lated from these three serogroups of L. pneu-mophila.The indirect immunoperoxidase labeling ex-

periments clearly established that the sero-group-specific F-1 antigen is distinct from thecommon flagella antigen. Since the serogroup-specific F-1 antigen is the major antigen detected

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TABLE 2. Amino acid composition offlagellinisolated from L. pneumophila serogroups 1, 2, and 3

No. of residues in flagellin isolated from

Amino acid serogroup:

1 2 3

Aspartic acid 35 34 36Threonine 17 17 19Serine 18 20 22Glutamic acid 35 32 34Proline 7 8 8Glycine 37 39 36Alanine 30 30 26Valine 20 21 22Methionine 4 6 4Isoleucine 17 18 21Leucine 26 28 24Phenylalanine 10 9 10Lysine 20 21 18Histidine 4 4 4Arginine 8 8 9

by the microagglutination and indirect and di-rect immunofluorescent assays (10, 18), thequestion arises as to why antibodies to the com-mon flagella antigen are not detected by thecurrent serological procedures. The data in Ta-ble 1 on the heat lability of the flagella antigenin slide agglutination studies suggest a possibleanswer. The cells used as antigen in the micro-agglutination and indirect immunofluorescenceassays are either heated at 1010C for 1 h (6) or

boiled for 15 min (24). These procedures destroythe flagella antigen but do not affect the heat-stable F-1 antigen. Preliminary studies in ourlaboratory have shown that the flagella of L.pneumophila can no longer be seen in electronmicrographs of cells heated at 1010C for 1 h(unpublished data). Therefore, any serologicaltest using heat-killed antigen suspensions willdetect only the heat-stable F-1 antigen.The failure to detect the common flagellar

antigens by direct immunofluorescent stainingappears also to relate to the heat lability of theflagella. Slides prepared for direct immunofluo-rescent staining are air dried, then heat fixed (4).Studies in our laboratory using anti-flagellin an-tibody and indirect immunofluorescence stain-ing show that the anti-flagellin antibody doesnot react with flagella on cells that have beenheat fixed (unpublished data). Therefore, theflagella would probably not be seen in directimmunofluorescence studies if the specimenwere heat fixed.The presence of a common flagellar antigen in

L. pneumophila serogroups 1, 2, and 3 suggeststhat similar antigens may be present on otherserogroups of L. pneumophila or possibly even

other species of Legionella. The presence of

such common antigens may be of value in theserological identification and classification ofLe-gionella. In addition, since the current serologi-cal tests do not detect antibodies to the flagellarantigens, it would be of interest to determinewhether antibodies to the common flagellar an-tigens could be detected in convalescent serum.Our laboratory is currently in the process ofdeveloping an enzyme-linked immunosorbentassay to determine the levels of anti-flagellaantibody in acute and convalescent serum andthe specificity of these antibodies for flagellaisolated from L. pneumophila. Further studieson the flagellar antigens and the serogroup-spe-cific F-1 antigens may be of value in establishinga serotyping system to study the epidemiologyof infections caused by Legionella.

ACKNOWLEDGMENTSWe thank Raymond Blakely of the Protein Structure Fa-

cility, University of Iowa, for his assistance in the amino acidanalysis, and Mark Urbanowski, Department of Microbiology,for assistance with the electron micrographs.

This study was supported by Public Health Service grantAI-15807.

LITERATURE CITED1. Bohn, W. 1976. A fixation method for improved penetra-

tion in electron microscopical immunoperoxidase stud-ies. J. Histochem. Cytochem. 26:293-297.

2. Chandler, F. W., I. L. Roth, C. S. Callaway, J. L.Bump, B. M. Thomason, and R. E. Weaver. 1980.Flagella on Legionnaires' disease bacteria. Ann. Int.Med. 93:711-714.

3. Chandler, F. W., B. M. Thomason, and G. A. Hebert.1980. Flagella on Legionnaires' disease bacteria in thehuman lung. Ann. Int. Med. 93:715-716.

4. Cherry, W. B., B. Pittman, P. P. Harris, G. A. Hebert,B. M. Thomason, L. Thacker, and R. E. Weaver.1978. Detection of Legionnaires disease bacteria bydirect inununofluorescent staining. J. Clin. Microbiol.8:329-338.

5. Elliott, J. A., W. Johnson, and C. M. Helms. 1981.Ultrastructural localization and protective activity of ahigh-molecular-weight antigen isolated from Legionellapneumophila. Infect. Immun. 31:822-824.

6. Farshy, C. E., G. C. Klein, and J. C. Feeley. 1978.Detection of antibodies to Legionnaires disease organ-ism by microagglutination and micro-enzyme-linked im-munoabsorbent assay tests. J. Clin. Microbiol. 7:327-331.

7. Feeley, J. C., R. J. Bigson, G. W. Gorman, N. C.Langford, J. K. Rasheed, D. C. Mackel, and W. B.Baine. 1979. Charcoal-yeast-extract agar: primary iso-lation medium for Legionella pneumophila. J. Clin.Microbiol. 4:437-441.

8. Feeley, J. C., G. W. Gorman, R. E. Weaver, D. C.Mackel, and H. Smith. 1978. Primary isolation mediafor Legionnaires disease bacterium. J. Clin. Microbiol.8:320-325.

9. lino, T. 1969. Genetics and chemistry of bacterial flagella.Bacteriol. Rev. 33:454-475.

10. Johnson, W., J. A. Elliott, C. M. Helms, and E. D.Renner. 1979. A high molecular weight antigen inLegionnaires disease bacterium: isolation and partialcharacterization. Ann. Int. Med. 90:638-641.

11. Johnson, W., E. Pesanti, and J. Elliott. 1980. Sero-specificity and opsonic activity of antisera to Legionella

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pneumophila. Infect. Immun. 26:698-704.12. Laemmli, U. K. 1970. Cleavage of structural proteins

during the assembly of the head of bacteriophage T4.Nature (London) 227:680-685.

13. Liu, T. Y., and Y. H. Chang. 1971. Hydrolysis of proteinswith p-toluenesulfonic acid: determination of trypto-phan. J. Biol. Chem. 246:2842-2848.

14. Lowry, 0. H., J. Rosebrough, A. L. Farr and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

15. Martinez, R. J., D. M. Brown, and A. N. Glazer. 1967.The formation of bacterial flagella. III. Characterizationof the subunits of the flagella of Bacillus subtilis andSpirillum serpens. J. Mol. Biol. 28:45-51.

16. McDonough, D. 1965. Amino acid composition of anti-genically distinct Salmonella flagellar proteins. J. Mol.Biol. 12:342-355.

17. McKinney, R. M., H. W. Wilkinson, H. M. Sommers,B. J. Fikes, K. R. Sasseville, M. M. Yongbluth, andJ. S. Wolf. 1980. Legionella pneumophila serogroup 6:isolation from cases of legionellosis, identification byimmunofluorescence staining, and immunological re-sponse to infection. J. Clin. Microbiol. 12:395-401.

18. Ormsbee, R. A., M. G. Peacock, G. L. Lattimer, L. A.Page, and P. Fiset. 1978. Legionnaires disease: anti-genic peculiarities, strain differences, and antibiotic sen-sitivities to the agent. J. Infect. Dis. 138:260-264.

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19. Reed, L. J., and H. Muench. 1938. A simple method ofestimating fifty per cent endpoints. Am. J. Hyg. 27:493-497.

20. Rodgers, F. G., P. W. Greaves, and A. D. Macrae.1979. Flagella and fimbriae on Legionella organisms.Lancet ii:753-754.

21. Rodgers, F. G., P. W. Greaves, A. D. Macrae, and M.J. Lewis. 1980. Electron microscopic evidence of fla-gella and pili on Legionella pneumophila. J. Clin. Pa-thol. 12:1184-1188.

22. Thomason, B. M., F. W. Chandler, and D. G. Hollis.1979. Flagella on Legionnaires disease bacteria: an in-terim report. Ann. Int. Med. 91:224-226.

23. Weintraub, M., and S. Raymond. 1963. Antiserumsprepared with acrylamide gel used as adjuvant. Science142:1677-1678.

24. Wilkinson, H. W., B. J. Fikes, and D. D. Cruce. 1979.Indirect immunofluorescence test for serodiagnosis ofLegionnaires disease: evidence for serogroup diversityof Legionnaires disease bacterial antigens and for mul-tiple specificity of human antibodies. J. Clin. Microbiol.9:379-383.

25. Wong, K. H., W. 0. Schalla, R. J. Arko, J. C. Bullard,and J. Feeley. 1979. Immunochemical, serologic andimmunologic properties of major antigens isolated fromthe Legionnaires disease bacterium. Ann. Int. Med. 90:634-638.


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