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Comparison of isolates of Chlamydia psittaci of ovine, avian and feline origin by analysis of polypeptide profiles form purified elementary bodies
Ii, terinary Microbiology, 26 ( 1991 ) 269-278 Elsevier Science Publishers B.V., Amsterdam
269
Comparison of isolates of Chlarnydia psittaci of ovine, avian and feline origin by analysis of polypeptide profiles from purified elementary bodies Margaret McClenaghan ~, Neil F. Inglis and Alan J. Herring Moredun Research Institute, 408 Gihnerton Road, Edinburgh EH17 7JH, UK (Accepted 9 August 1990) ABSTRACT McClenaghan, M., Inglis, N.F. and Herring, A.J., 1991. Comparison of isolates of Chlamydia psittaci ofovine, avian and feline origin by analysis of polypeptide profiles from purified elementary bodies. lJk,t. Microbiol., 26: 269-278. The single species Chlamydia psittaci is a diverse grouping which contains several different types of chlamydial strain for which there is no generally accepted typing method. The results obtained when profiles of polypeptides from purified elementary bodies are compared are consistent with type designations obtained using other criteria. However, the method still requires large scale culture and extensive purification of the chlamydial cells.
INTRODUCTION
Within the genus Chlamydia a diverse group of isolates comprises the species C. psittaci. Two recent D N A reassociation studies (Cox et al., 1988; Fukushi and Hirai, 1989 ) have shown sufficient divergence between the genomes of various types of C. psittaci to divide the group into at least four separate species. At present there is no generally accepted and widely accessible method available for typing isolates. However, clear differences between types have been demonstrated by restriction endonuclease (RE) analysis (McClenaghan et al., 1984; Timms et al., 1988; Fukushi and Hirai, 1989; Andersen and Tappe, 1989), the micro-immunofluorescence ( M I F ) t e s t (PerezMartinez and Storz, 1985), monoclonal antibody reactivities (Delong and Magee, 1986; Fukushi et al., 1987), immunoblotting (Fukushi and Hirai, 1988; Winsor and Grimes, 1988) and a variety of biological parameters (Schachter et al., 1974; Spears and Storz, 1979; Buzoni-Gatel and Rodolakis, 1983; Anderson, 1986; Rodolakis et al., 1989). In this communication we compare the polypeptide profiles of purified el~Present address: Institute of Animal Physiology and Genetics, Edinburgh Research Station, Roslin, Midlothian, UK. 0378-1135/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
270
M. McCLENAGHAN ET AL.
ementary bodies (EBs) from nine isolates of C. psittaci and one of C. trachomatis. The results support those of previous comparisons by RE analysis and MIF and give further data for strain discrimination. The use of polypeptide profiling as a typing method is discussed. MATERIALS AND METHODS
Chlamydiae Of the C. psittaci isolates used in this study, Cal 10, avian 725 (cockatiel), avian 741 (wood pigeon), ovine abortion (OA) strains $26/3 and A22 and ovine arthritis isolate P787 have all been described previously (McClenaghan et al., 1984). Strain $57/3 is a more recent OA strain from the same farm as $26/3. Both A22 and $26/3 are high passage level strains (more than 40 passes for A22) $57/3 was used at the third pass. Strain CH1287 is from a case of ovine conjunctivitis. The feline pneumonitis isolate was received as purified elementary bodies and was the kind gift of Dr. S.J. Richmond, University of Manchester. The L2-serovar of C. trachomatis was supplied by Dr. I. Smith, Edinburgh University.
Culture and purification Both growth in cell culture and purification of elementary bodies (EBs) on gradients of "Renografin" (Squibb) were performed as described previously (McClenaghan et al., 1984). The protein content of purified EB preparations was estimated using the dye-binding method of Bradford (1976). Prior to electrophoresis, EB suspensions were diluted with an equal volume of × 2 concentration denaturing buffer to yield a final concentration of 63 m M TrisHC1, pH 6.8, 5% (v/v) B-mercaptoethanol, 2% w/v sodium docecyl sulphate ( SDS ), 10% (w/v) sucrose and 0.001% bromophenol blue. Denaturation was for 90 s at 100 ° C.
Polyacrylarnide gel electrophoresis (PAGE) Chlamydial proteins were separated using 0.75 m m thick, 10% (w/v) gels, with a 3% (w/v) stacking gel and the discontinuous buffer system of Laemmli (1970). Gels were run for 2 h using constant current conditions and a current density of 18 m A / c m z. Approximately 10/~g of protein were loaded per track. Staining with silver was performed using either the method of Morrissey ( 1981 ) or that of Sammons et al. ( 1981 ). Molecular weights were estimated using a sonic digitizer to input band positions into a BBC-B microcomputer running a MAP-GEL-2 programme which was kindly communicated by J. Coadwell.
271
C O M P A R I S O N O F ISOLATES O F CHLAMYDIA PSITTACI
1
2
3
4
5
6
7
8
9
10
a b C
e ~
f~
~
~,r ~
~.,~
g~
h
m J
Fig. 1. Polyacrylamide gel electrophoresis of total chlamydial elementary body polypeptides. Track ( 1 ) molecular weight standards - a: ~-galactosidase, 116 000; b: phosphorylase-A, 94 000; c: ovotransferrin, 77 000; d: bovine serum albumin, 67 000; e: ovalbumin, 45 000; f: chymotrypsinogen A, 26 000; g: myoglobin, 17 000; h: cytochrome C, 12 000. Track (2) C. trachomatis serovar L2. Track (3) Cal 10. Track (4) avian isolate 725. Track (5) ovine abortion A22. Track (6) ovine abortion $26/3. Track (7) ovine abortion $57/3. Track (8) ovine arthritis P787. Track (9) ovine conjunctivitis CH1287. Track (10) feline pneumonitis. The gel was stained by the method of Morrissey ( 1981 ).
272
M. McCLENAGHANET AL.
12345678910
a, ill
d
f, 9i
~
Fig. 2. Polyacrylamide gel analysis of Eco R 1 digests of chlamydial genomic DNA. Tracks ( 1 and 10) molecular weight standards - sizes in kilobase pairs are a: 6.7; b: 4.4; c: 2.3: d: 2.0: c: 1.35; f: 1.08 and g: 0.87. Track (2) avian 725. Track (3) avian 741. Track (4) Cal 10. Track ( 5 ) feline pneumonitis. Track (6) ovine abortion A22. Track ( 7 ) ovine conjunctivitis CH 1287. Track (8) ovine arthritis P787. Track (9) ('. trachomatis L2.
COMPARISONOF ISOLATESOF CHLAMYDL4 PSITTACI
273
TABLE 1 Molecular weight estimates for the major outer membrane proteins Strain
Type/origin
Size of MOMP in kDa
L2 Ca110
C. trachomatis L2 strain
40 38
725 A22 S/26/3 $57/3 P787 CH 1287 Fel. Pn.
Laboratory strain (origin: case of ornithosis ) Avian strain (cockateil) Ovine abortion Ovine abortion Ovine abortion Ovine arthritis Ovine conjunctivitis Feline pneumonitis
38 36.5 36.5 36.5 37.5 37.5 37
Restriction endonuclease analysis Analysis of chlamydial DNA by PAGE following digestion with the restriction enzyme Eco RI was performed exactly as described in McClenaghan et al. (1984). RESULTS
The polypeptide profiles obtained by SDS-PAGE analysis of purified EBs from eight isolates of C. psittaci and one isolate of C. trachomatis are shown in Fig. 1. The profiles display similar numbers and size distribution of peptides with a single dominant band, the major outer membrane protein (MOMP, see below), which migrates with an apparent size of about 40 kilodaltons (kDa). Within the eight C. psittaci isolates four profile types were evident. The three OA isolates showed very similar profiles although a minor quantitative difference was found in a band migrating at about 90 kDa in isolate $57/3 (arrowed in Fig. 1 ). Likewise, the other two ovine isolates from a case of arthritis (P787) and of conjunctivitis (CH 1287 ) showed profiles which were closely related to each other but clearly distinct from the OA and non-ovine isolates. The Cal 10 and psittacine 725 isolate displayed a third profile type, the between-isolate variation was more marked than that seen within the two ovine types but the general distribution of bands and sizes of the MOMPs were similar. One clear difference between the avian isolates was a prominent band in the Cal 10 profile of about 80 kDa in size, the significance of this band is unknown but it has appeared in analyses of subsequent preparations (T.W. Tan and A.J. Herring, unpublished results). The single feline isolate gave a profile unrelated to the others as did C. trachornatis L2 which was included as a control.
274
M. McCLENAGHAN ET AL.
1
2
3
4 ,~a
~b ,IC
"d
,~f
~
"~g ~h
Fig. 3. Comparison ofchlamydial polypeptide profiles after staining by the method of Sammons et al. (1981 ) (tracks 1 and 2) and the method of Morrissey (1981 ) (tracks 3 and 4). Tracks (1 and 3) ovine arthritis P787. Tracks (2 and 4) ovine abortion A22. The positions of the marker bands a-h described in Fig. 1 are indicated. The arrows indicate a blue staining band. T h e a p p a r e n t m o l e c u l a r weights o f the M O M P s for each isolate are s h o w n in T a b l e 1. T h e y were e s t i m a t e d f r o m a gel in which the same p o l y p e p t i d e p r e p a r a t i o n s were r u n beside size m a r k e r p r o t e i n s in c e n t r e tracks to m i n i m ise distortion. Each t y p e a p p e a r s to h a v e a characteristic M O M P m o l e c u l a r weight a n d in all analyses the relative sizes o f the M O M P s were the same. Fig. 2 shows d a t a which e x t e n d o u r p r e v i o u s g e n o m i c D N A R E analysis to include the o v i n e c o n j u n c t i v i t i s isolate ( C H 1287 ). This y i e l d e d a profile similar but not identical to the o v i n e arthritis isolate ( P 7 8 7 ) . T h u s the groupings based on R E a n d p o l y p e p t i d e profiling are entirely consistent.
COMPARISON OF ISOLATES OF C H L A M Y D I A PSITTACI
275
Fig. 3 shows a comparison of the abortion and arthritis isolate polypeptide profiles stained using two different silver staining protocols. The method of Sammons et al. ( 1981 ) stains different polypeptide species different colours and was used to attempt to deduce relationships between individual peptide bands in the profiles of different chlamydial types. It was found to stain clearly only a sub-set of the bands seen with the Morrissey protocol. Many major polypeptide species, and especially the MOMP, were only barely detectable by this method. In contrast, the Morrissey protocol yielded a profile essentially identical with that found for Commassie blue staining but with far better detection of minor components (results not shown). Colour differentiation of bands was not an expected feature of the Morrissey protocol but was observed to a variable extent. One low molecular weight peptide in both the OA and P787 isolates stained a distinct blue colour (arrowed in Fig. 3 ). DISCUSSION
The polypeptide constituents of C. trachornatis EBs have been described by several authors (reviewed by Newhall, 1988). There is a large measure of agreement in the published profiles and the M O M P is always the dominant feature. The profile shown above for purified EBs of C. trachornatis serovar L2 is very similar to that obtained by Coomassie blue staining by Newhall ( 1988 ). This confirms that the Morrissey silver staining protocol stains proteins quantitatively as claimed (Morrisey, 1981 ). Data showing comparisons of polypeptide profiles of C. psittaci EBs from a variety of types have not been published. However, Vitu and Russo (1984) report finding no substantial difference between isolates of ovine, caprine, bovine and canine origin. They mention one minor quantitative change in the caprine strain at 88 kDa possibly similar to the one observed above in the OA isolate $57/3. They also found clear differences between the one avian strain examined and their m a m m a l i a n strain panel. It is possible that the variation in the level of the 88 kDa band is associated with pass level, we hope to investigate this possibility further. More recently, Fukushi and Hirai ( 1988 ) published data for detergent extracted C. psittaci EBs from 16 isolates, mostly of avian origin. They analysed the insoluble material left after extraction and their gels showed the MOMP and small amounts of other polypeptides. The panel of strains compared included an ovine arthritis isolate, two feline isolates with Cal 10 and C. trachomatis L2 as standards. The relative sizes allocated to the MOMPs were as shown in Table 1 but their molecular weight estimates were all slightly higher. This was probably due to the relatively heavy gel loadings used in this study in order to reveal total polypeptide profiles. Their analysis was complemented by immunoblotting using anti-MOMP sera; this test grouped Cal 10 with the avian isolates whilst the feline pneumonitis and ovine arthritis iso-
276
M. McCLENAGHAN E T A [ .
lates were in separate groups. These results, taken with the RE analyses, DNA reassociation studies, MIF data and blotting studies referred to above, give groupings consistent with those deduced from the profiles presented in this paper. Recently Buzoni-Gatel et al. (1989) published a comparison of polypeptide profiles of soluble SDS extracts of purified EBs from ovine chlamydial strains that were classified as either "invasive" or "non-invasive" in a mouse infection test (Rodolakis et al., 1989). The two strain types clearly showed different profiles but the distribution of bands was completely different from that shown in this paper and from other published profiles of chlamydial EB proteins. In particular, the MOMP band was absent from the gels. These results may be due either to differential staining with silver, as reported above for the Sammons et al. ( 1981 ) protocol, or to the failure of SDS, used at low temperature and in the absence of a disulphide bond reducing agent, to solubilise the structural components of the chlamydial outer membrane (Baviol et al., 1984). Since protein gel electrophoresis is a widely used and familiar technique it has some advantages for the characterisation of chlamydial isolates. However, it suffers from the same disadvantages as RE analysis in that it requires extensive growth in culture and purification of the EBs for both test isolate and standard strains. The need for purification is reduced for both methods ifa blotting technique is used with either cloned DNA or antibody probes but once again culture is necessary and inter-laboratory comparisons require wide access to the same probes, which is often difficult to achieve. C. trachomatis typing has been successfully achieved by exploiting the antigenic variability in the MOMP (Wang and Grayston, 1982). That similar variability exists between types of C. psittaci has been demonstrated by immunoblotting studies (Fukushi and Hirai, 1988; Winsor and Grimes, 1988) and by direct sequencing of the MOMP gene (Pickett et al., 1988; Zhang et al., 1989; Herring et al., 1989). As for C. trachornatis inter-type MOMP sequence variability is confined to four internal "variable domains". There is considerable sequence conservation at the 5' and 3' termini of the MOMP gene that may be exploited to amplify the MOMP sequence using the polymerase chain reaction (PCR; Saiki et al., 1988 ). Our preliminary results suggest that PCR can be used to both detect and type C. psittaci isolates in a test which eliminates or minimises the need for cell culture (Herring et al., 1990 ). We are currently investigating this approach for the combined detection and typing of C. psittaci infection in the context of ovine and human infections. ACKNOWLEDGEMENTS
We thank Dr. S. Richmond, of the Department of Medical Microbiology, University of Manchester and Dr. I. Smith of the Department of Bacteriol-
COMPARISON OF ISOLATES OF CHL,,tMYDIA PSITTI-I(7
277
ogy, University of Edinburgh for supplying strains and Dr. J. Coadwell of the Institute of Animal Physiology and Genetics Research, Babraham for the "Mapgel 2" programme.
REFERENCES Andersen, A.A. and Tappe, J.P., 1989. Genetic, immunologic and pathologic characterization of avian chlamydial strains. JAVMA, 195: 1512-1516. Anderson, I.E., 1986. Comparison of the virulence in mice of some ovine isolates of Chlamydia psittaci. Vet. Microbiol., 12:213-220. Bradford. M,M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bonding. Anal. Biochem., 72: 248-254. Bavoil, P., Ohlin, A. and Schachter, J., 1984. Role of disulfide bonding in outer membrane structure and permeability in Chlamydia trachomatis. Infect, Immun., 44: 479-485. Buzoni-Gatel, D. and Rodolakis, A., 1983. A mouse model to compare virulence of abortive and intestinal ovine strains Chlamydial psiltaci: Influence of the route of inoculation. Ann. Microbiol. (Inst. Pasteur), 134A: 91-99. Buzoni-Gatel, D., Layachi, K., Dubray, G. and Rodolakis, A., 1989. Comparison of protein patterns between invasive and non-invasive ovine strains of Chlamydia psittaci. Res. Vet. Sci., 46: 40-42. Cox, R.L., Kuo, C-C., Grayston, J.T. and Campbell, L.A., 1988. Deoxyribonucleic acid relatedness of Chlamydia sp. strain TWAR to Chlamydia trachomatis and Chlamydia psittaci. Int. J. Syst. Bacteriol., 38: 265-268. Delong, W.J. and Magee, W.E., 1986. Distinguishing between ovine abortion and ovine arthritis Chlamydia psittaci isolates with specific monoclonal antibodies. Am. J. Vet. Res., 47:15201523. Fukushi, H. and Hirai, K., 1988. lmmunochemical diversity of the major outer membrane protein of avian and mammalian Chlamydia psittaci. J. Clin. Microbiol., 26: 675-680. Fukushi, H. and Hirai, K., 1989. Genetic diversity of avian and mammalian Chlamydia psittaci strains and relation to host origin. J. Bacteriol., 171: 2850-2855. Fukushi, H., Nojiri, K. and Hirai, K., 1987. Monoclonal antibody typing of Chlamydia psittaci strains derived from avian and mammalian species. J. Clin. Microbiol., 25:1978-1981. Herring, A.J., Tan, T.W., Baxter, S., Inglis, N.F. and Dunbar, S., 1989. Sequence analysis of the major outer membrane protein of an ovine abortion strain of Chlamydia psittaci. FEMS Microbiol. Lett., 65: 153-158. Herring, A.J., Tan, T.W. and Baxter, S., 1990. Chlamydial abortion in sheep: molecular approaches to vaccination, pathogen detection and strain typing. In: W.R. Bowie, H.D. Caldwell, R.P. Jones, P.A. Mardh, G.L. Ridgeway, J. Schachter, W,E. Stature and M.E. Ward (Editors), Chlamydial Infections. Cambridge University Press, Cambridge, UK, pp. 378382. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 277: 680-685. McClenaghan, M., Herring, A.J. and Aitken, I.D., 1984. Comparison of Chlamydia psittaci isolates by DNA restriction endonuclease analysis. Infect. lmmun., 45: 384-389. Morrisscy, J.H., 1981. Silver stain for proteins in polyacrylamide gels: A modified procedure with enhanced uniform senstivity. Anal. Biochem., 117: 307-310. Newhall, W.J., 1988. Macromolecular and antigenic composition of Chlamydiae. ln: A.L. Barton (Editor), Microbiology of Chlamydia. CRC Press, Florida, pp. 47-70.
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Perez-Martinez, J.A. and Storz, J., 1985. Antigenic diversity ofChlam.vdia psittaci of mammalian origin determined by microimmunofluorescence. Infect. Immun., 50: 905-910. Pickett, M.A., Everson, J,S. and Clarke, I.N., 1988. Chlamydia psittaci ewe abortion agent: complete nucleotide sequence of the major outer membrane protein gene. FEMS Microbio]. Letters, 55: 229-234. Rodolakis, A., Bernard, F. and Lantier, F., 1989. Mouse model for evaluation of virulence of Chlamydia psittaci isolated from ruminants. Res. Vet. Sci., 46: 34-39. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A., 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239: 487-491. Sammons, D.W., Adams, L.D. and Nishizawa, E.E., 1981. Ultrasensitive silver-based color staining of polypeptides in polyacrylamide gels. Electrophoresis, 2:135-141. Schachter, J., Banks, J., Sugg, N., Sung, M., Storz, J. and Meyer, K.F.. 1974. Serotyping of Chlamydia. Infect. Immun., 9: 92-94. Spears, P. and Storz, J., 1979. Biotyping of Chlamydia psittaci based on inclusion morphology and response to deithylaminoethyl-dextran and cycloheximide. Infect. Immun., 24: 224232. Timms, P., Eaves, F.W., Girjes, A.A. and Lavin, M.F., 1988. Comparison of Chlamydia psiltaci isolates by restriction endonuclease and DNA probe analyses. Infect. Immun., 56: 287-290. Vitu, C. and Russo, P., 1984. Compairison de differentes souches de Chlamydia psittaci par electrophorese en gel de polyacrylamide. Les Colloques de I'INRA, number 28. Wang, S.P. and Grayston, J.T., 1982. Microimmunofluorescence antibody responses in Chlamydia trachomatis infection, a review, in Chlamydial infections. In: P.A. Mardh, K.K. Holmes, J.D. Oriel, P. Piot and J. Schachter (Editors). Microbiology of Chlamydia. Elsevier, Amsterdam, pp. 301-316. Winsor, D.K., Jr. and Grimes, J.E., 1988. Relationship between infectivity and cytopathology for L-929 cells, membrane proteins and antigenicity of avian isolates of Chlamydia psittaci. Avian Dis., 32:421-431. Zhang, Y.-X., Morrison, S.G., Caldwell, H.D. and Baehr, W,, 1989. Cloning and sequence analysis of the major outer membrane protein genes of two Chlamydia psittaci strains. Infect. Immun., 57: 1621-1625.
269
Comparison of isolates of Chlarnydia psittaci of ovine, avian and feline origin by analysis of polypeptide profiles from purified elementary bodies Margaret McClenaghan ~, Neil F. Inglis and Alan J. Herring Moredun Research Institute, 408 Gihnerton Road, Edinburgh EH17 7JH, UK (Accepted 9 August 1990) ABSTRACT McClenaghan, M., Inglis, N.F. and Herring, A.J., 1991. Comparison of isolates of Chlamydia psittaci ofovine, avian and feline origin by analysis of polypeptide profiles from purified elementary bodies. lJk,t. Microbiol., 26: 269-278. The single species Chlamydia psittaci is a diverse grouping which contains several different types of chlamydial strain for which there is no generally accepted typing method. The results obtained when profiles of polypeptides from purified elementary bodies are compared are consistent with type designations obtained using other criteria. However, the method still requires large scale culture and extensive purification of the chlamydial cells.
INTRODUCTION
Within the genus Chlamydia a diverse group of isolates comprises the species C. psittaci. Two recent D N A reassociation studies (Cox et al., 1988; Fukushi and Hirai, 1989 ) have shown sufficient divergence between the genomes of various types of C. psittaci to divide the group into at least four separate species. At present there is no generally accepted and widely accessible method available for typing isolates. However, clear differences between types have been demonstrated by restriction endonuclease (RE) analysis (McClenaghan et al., 1984; Timms et al., 1988; Fukushi and Hirai, 1989; Andersen and Tappe, 1989), the micro-immunofluorescence ( M I F ) t e s t (PerezMartinez and Storz, 1985), monoclonal antibody reactivities (Delong and Magee, 1986; Fukushi et al., 1987), immunoblotting (Fukushi and Hirai, 1988; Winsor and Grimes, 1988) and a variety of biological parameters (Schachter et al., 1974; Spears and Storz, 1979; Buzoni-Gatel and Rodolakis, 1983; Anderson, 1986; Rodolakis et al., 1989). In this communication we compare the polypeptide profiles of purified el~Present address: Institute of Animal Physiology and Genetics, Edinburgh Research Station, Roslin, Midlothian, UK. 0378-1135/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
270
M. McCLENAGHAN ET AL.
ementary bodies (EBs) from nine isolates of C. psittaci and one of C. trachomatis. The results support those of previous comparisons by RE analysis and MIF and give further data for strain discrimination. The use of polypeptide profiling as a typing method is discussed. MATERIALS AND METHODS
Chlamydiae Of the C. psittaci isolates used in this study, Cal 10, avian 725 (cockatiel), avian 741 (wood pigeon), ovine abortion (OA) strains $26/3 and A22 and ovine arthritis isolate P787 have all been described previously (McClenaghan et al., 1984). Strain $57/3 is a more recent OA strain from the same farm as $26/3. Both A22 and $26/3 are high passage level strains (more than 40 passes for A22) $57/3 was used at the third pass. Strain CH1287 is from a case of ovine conjunctivitis. The feline pneumonitis isolate was received as purified elementary bodies and was the kind gift of Dr. S.J. Richmond, University of Manchester. The L2-serovar of C. trachomatis was supplied by Dr. I. Smith, Edinburgh University.
Culture and purification Both growth in cell culture and purification of elementary bodies (EBs) on gradients of "Renografin" (Squibb) were performed as described previously (McClenaghan et al., 1984). The protein content of purified EB preparations was estimated using the dye-binding method of Bradford (1976). Prior to electrophoresis, EB suspensions were diluted with an equal volume of × 2 concentration denaturing buffer to yield a final concentration of 63 m M TrisHC1, pH 6.8, 5% (v/v) B-mercaptoethanol, 2% w/v sodium docecyl sulphate ( SDS ), 10% (w/v) sucrose and 0.001% bromophenol blue. Denaturation was for 90 s at 100 ° C.
Polyacrylarnide gel electrophoresis (PAGE) Chlamydial proteins were separated using 0.75 m m thick, 10% (w/v) gels, with a 3% (w/v) stacking gel and the discontinuous buffer system of Laemmli (1970). Gels were run for 2 h using constant current conditions and a current density of 18 m A / c m z. Approximately 10/~g of protein were loaded per track. Staining with silver was performed using either the method of Morrissey ( 1981 ) or that of Sammons et al. ( 1981 ). Molecular weights were estimated using a sonic digitizer to input band positions into a BBC-B microcomputer running a MAP-GEL-2 programme which was kindly communicated by J. Coadwell.
271
C O M P A R I S O N O F ISOLATES O F CHLAMYDIA PSITTACI
1
2
3
4
5
6
7
8
9
10
a b C
e ~
f~
~
~,r ~
~.,~
g~
h
m J
Fig. 1. Polyacrylamide gel electrophoresis of total chlamydial elementary body polypeptides. Track ( 1 ) molecular weight standards - a: ~-galactosidase, 116 000; b: phosphorylase-A, 94 000; c: ovotransferrin, 77 000; d: bovine serum albumin, 67 000; e: ovalbumin, 45 000; f: chymotrypsinogen A, 26 000; g: myoglobin, 17 000; h: cytochrome C, 12 000. Track (2) C. trachomatis serovar L2. Track (3) Cal 10. Track (4) avian isolate 725. Track (5) ovine abortion A22. Track (6) ovine abortion $26/3. Track (7) ovine abortion $57/3. Track (8) ovine arthritis P787. Track (9) ovine conjunctivitis CH1287. Track (10) feline pneumonitis. The gel was stained by the method of Morrissey ( 1981 ).
272
M. McCLENAGHANET AL.
12345678910
a, ill
d
f, 9i
~
Fig. 2. Polyacrylamide gel analysis of Eco R 1 digests of chlamydial genomic DNA. Tracks ( 1 and 10) molecular weight standards - sizes in kilobase pairs are a: 6.7; b: 4.4; c: 2.3: d: 2.0: c: 1.35; f: 1.08 and g: 0.87. Track (2) avian 725. Track (3) avian 741. Track (4) Cal 10. Track ( 5 ) feline pneumonitis. Track (6) ovine abortion A22. Track ( 7 ) ovine conjunctivitis CH 1287. Track (8) ovine arthritis P787. Track (9) ('. trachomatis L2.
COMPARISONOF ISOLATESOF CHLAMYDL4 PSITTACI
273
TABLE 1 Molecular weight estimates for the major outer membrane proteins Strain
Type/origin
Size of MOMP in kDa
L2 Ca110
C. trachomatis L2 strain
40 38
725 A22 S/26/3 $57/3 P787 CH 1287 Fel. Pn.
Laboratory strain (origin: case of ornithosis ) Avian strain (cockateil) Ovine abortion Ovine abortion Ovine abortion Ovine arthritis Ovine conjunctivitis Feline pneumonitis
38 36.5 36.5 36.5 37.5 37.5 37
Restriction endonuclease analysis Analysis of chlamydial DNA by PAGE following digestion with the restriction enzyme Eco RI was performed exactly as described in McClenaghan et al. (1984). RESULTS
The polypeptide profiles obtained by SDS-PAGE analysis of purified EBs from eight isolates of C. psittaci and one isolate of C. trachomatis are shown in Fig. 1. The profiles display similar numbers and size distribution of peptides with a single dominant band, the major outer membrane protein (MOMP, see below), which migrates with an apparent size of about 40 kilodaltons (kDa). Within the eight C. psittaci isolates four profile types were evident. The three OA isolates showed very similar profiles although a minor quantitative difference was found in a band migrating at about 90 kDa in isolate $57/3 (arrowed in Fig. 1 ). Likewise, the other two ovine isolates from a case of arthritis (P787) and of conjunctivitis (CH 1287 ) showed profiles which were closely related to each other but clearly distinct from the OA and non-ovine isolates. The Cal 10 and psittacine 725 isolate displayed a third profile type, the between-isolate variation was more marked than that seen within the two ovine types but the general distribution of bands and sizes of the MOMPs were similar. One clear difference between the avian isolates was a prominent band in the Cal 10 profile of about 80 kDa in size, the significance of this band is unknown but it has appeared in analyses of subsequent preparations (T.W. Tan and A.J. Herring, unpublished results). The single feline isolate gave a profile unrelated to the others as did C. trachornatis L2 which was included as a control.
274
M. McCLENAGHAN ET AL.
1
2
3
4 ,~a
~b ,IC
"d
,~f
~
"~g ~h
Fig. 3. Comparison ofchlamydial polypeptide profiles after staining by the method of Sammons et al. (1981 ) (tracks 1 and 2) and the method of Morrissey (1981 ) (tracks 3 and 4). Tracks (1 and 3) ovine arthritis P787. Tracks (2 and 4) ovine abortion A22. The positions of the marker bands a-h described in Fig. 1 are indicated. The arrows indicate a blue staining band. T h e a p p a r e n t m o l e c u l a r weights o f the M O M P s for each isolate are s h o w n in T a b l e 1. T h e y were e s t i m a t e d f r o m a gel in which the same p o l y p e p t i d e p r e p a r a t i o n s were r u n beside size m a r k e r p r o t e i n s in c e n t r e tracks to m i n i m ise distortion. Each t y p e a p p e a r s to h a v e a characteristic M O M P m o l e c u l a r weight a n d in all analyses the relative sizes o f the M O M P s were the same. Fig. 2 shows d a t a which e x t e n d o u r p r e v i o u s g e n o m i c D N A R E analysis to include the o v i n e c o n j u n c t i v i t i s isolate ( C H 1287 ). This y i e l d e d a profile similar but not identical to the o v i n e arthritis isolate ( P 7 8 7 ) . T h u s the groupings based on R E a n d p o l y p e p t i d e profiling are entirely consistent.
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Fig. 3 shows a comparison of the abortion and arthritis isolate polypeptide profiles stained using two different silver staining protocols. The method of Sammons et al. ( 1981 ) stains different polypeptide species different colours and was used to attempt to deduce relationships between individual peptide bands in the profiles of different chlamydial types. It was found to stain clearly only a sub-set of the bands seen with the Morrissey protocol. Many major polypeptide species, and especially the MOMP, were only barely detectable by this method. In contrast, the Morrissey protocol yielded a profile essentially identical with that found for Commassie blue staining but with far better detection of minor components (results not shown). Colour differentiation of bands was not an expected feature of the Morrissey protocol but was observed to a variable extent. One low molecular weight peptide in both the OA and P787 isolates stained a distinct blue colour (arrowed in Fig. 3 ). DISCUSSION
The polypeptide constituents of C. trachornatis EBs have been described by several authors (reviewed by Newhall, 1988). There is a large measure of agreement in the published profiles and the M O M P is always the dominant feature. The profile shown above for purified EBs of C. trachornatis serovar L2 is very similar to that obtained by Coomassie blue staining by Newhall ( 1988 ). This confirms that the Morrissey silver staining protocol stains proteins quantitatively as claimed (Morrisey, 1981 ). Data showing comparisons of polypeptide profiles of C. psittaci EBs from a variety of types have not been published. However, Vitu and Russo (1984) report finding no substantial difference between isolates of ovine, caprine, bovine and canine origin. They mention one minor quantitative change in the caprine strain at 88 kDa possibly similar to the one observed above in the OA isolate $57/3. They also found clear differences between the one avian strain examined and their m a m m a l i a n strain panel. It is possible that the variation in the level of the 88 kDa band is associated with pass level, we hope to investigate this possibility further. More recently, Fukushi and Hirai ( 1988 ) published data for detergent extracted C. psittaci EBs from 16 isolates, mostly of avian origin. They analysed the insoluble material left after extraction and their gels showed the MOMP and small amounts of other polypeptides. The panel of strains compared included an ovine arthritis isolate, two feline isolates with Cal 10 and C. trachomatis L2 as standards. The relative sizes allocated to the MOMPs were as shown in Table 1 but their molecular weight estimates were all slightly higher. This was probably due to the relatively heavy gel loadings used in this study in order to reveal total polypeptide profiles. Their analysis was complemented by immunoblotting using anti-MOMP sera; this test grouped Cal 10 with the avian isolates whilst the feline pneumonitis and ovine arthritis iso-
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lates were in separate groups. These results, taken with the RE analyses, DNA reassociation studies, MIF data and blotting studies referred to above, give groupings consistent with those deduced from the profiles presented in this paper. Recently Buzoni-Gatel et al. (1989) published a comparison of polypeptide profiles of soluble SDS extracts of purified EBs from ovine chlamydial strains that were classified as either "invasive" or "non-invasive" in a mouse infection test (Rodolakis et al., 1989). The two strain types clearly showed different profiles but the distribution of bands was completely different from that shown in this paper and from other published profiles of chlamydial EB proteins. In particular, the MOMP band was absent from the gels. These results may be due either to differential staining with silver, as reported above for the Sammons et al. ( 1981 ) protocol, or to the failure of SDS, used at low temperature and in the absence of a disulphide bond reducing agent, to solubilise the structural components of the chlamydial outer membrane (Baviol et al., 1984). Since protein gel electrophoresis is a widely used and familiar technique it has some advantages for the characterisation of chlamydial isolates. However, it suffers from the same disadvantages as RE analysis in that it requires extensive growth in culture and purification of the EBs for both test isolate and standard strains. The need for purification is reduced for both methods ifa blotting technique is used with either cloned DNA or antibody probes but once again culture is necessary and inter-laboratory comparisons require wide access to the same probes, which is often difficult to achieve. C. trachomatis typing has been successfully achieved by exploiting the antigenic variability in the MOMP (Wang and Grayston, 1982). That similar variability exists between types of C. psittaci has been demonstrated by immunoblotting studies (Fukushi and Hirai, 1988; Winsor and Grimes, 1988) and by direct sequencing of the MOMP gene (Pickett et al., 1988; Zhang et al., 1989; Herring et al., 1989). As for C. trachornatis inter-type MOMP sequence variability is confined to four internal "variable domains". There is considerable sequence conservation at the 5' and 3' termini of the MOMP gene that may be exploited to amplify the MOMP sequence using the polymerase chain reaction (PCR; Saiki et al., 1988 ). Our preliminary results suggest that PCR can be used to both detect and type C. psittaci isolates in a test which eliminates or minimises the need for cell culture (Herring et al., 1990 ). We are currently investigating this approach for the combined detection and typing of C. psittaci infection in the context of ovine and human infections. ACKNOWLEDGEMENTS
We thank Dr. S. Richmond, of the Department of Medical Microbiology, University of Manchester and Dr. I. Smith of the Department of Bacteriol-
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ogy, University of Edinburgh for supplying strains and Dr. J. Coadwell of the Institute of Animal Physiology and Genetics Research, Babraham for the "Mapgel 2" programme.
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