Analysis of the variability in expression of Mycoplasma gallisepticum surface antigens

veterinary microbiology ELSEVIER

Veterinary Microbiology42 (1994) 147-158

Analysis of the variability in expression of Mycoplasma gallisepticum surface antigens Maricarmen Garcia

a Mohamed

G. Elfaki

b, Stanley H.

Kleven a,.

a Department of Avian Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602 USA b Department oflmmunology and Microbiology, Wayne State University, School of Medicine, Detroit, M148201 USA

Received 12 August 1993; accepted 23 March 1994

Abstract The in vitro expression of surface epitopes for different strains of Mycoplasma gallisepticum (MG) was studied with a panel of monoclonal antibodies (mAbs) using indirect colony immunostaining and Western blot (WB) analyses. Immunostaining of colonies with mAbs showed that five epitopes had different degrees of variable expression, while one epitope was permanently expressed in vitro. Colonies that failed to express the studied epitopes had the potential of phenotypically switching the expression of these epitopes in vitro. Variable and permanently expressed epitopes were associated with more than one protein and not all mAb-defined proteins were responsible for the immunostaining of intact MG colonies. The ability of MG to variably express their surface epitopes maybe the mechanism utilized by the microorganism to avoid the host immune response. Keywords: Mycoplasma gallisepticum; Surface antigen, bacteria; Antigen

1. Introduction Differences in virulence among M G strains had been reported by many researchers (Rodriguez and Kleven, 1980; Lin and Kleven, 1984; Levisohn et al., 1986; Yoder, 1986). Recently stronger evidence for heterogeneity among M G strains has evolved from studies comparing protein profiles by S D S - P A G E (Khan et al., 1987), Western blot analyses with polyclonal strain-specific antisera (Thomas and Sharp, 1988), and M G strain glycoprotein diversity (Thomas and Sharp, 1990). Genetic heterogeneity among M G strains has also * Correspondingauthor. Departmentof Avian Medicine,Collegeof VeterinaryMedicine,Universityof Georgia, 953 College Station Road, Athens, GA. 30602, USA. 0378-1135 / 94 / $07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI0378- 1135(94) 00056-3

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been reported from analyses of restriction endonuclease cleavage patterns (Khan and Yamamoto, 1989; Kleven et al., 1988) and by fingerprinting with a ribosomal RNA gene probe (Yogev et al., 1988, 1989). Studies with other mycoplasma species have demonstrated that monoclonal antibodies (mAbs) are a powerful tool to identify and characterize the expression of mycoplasma membrane antigens (Wise and Kim, 1987; Boyer and Wise, 1988; Watson et al., 1988). Although mAbs against several MG strains have been produced with the purpose of developing a specific probe (Hwang et al., 1989; Morsy et al., 1992), the restricted recognition of some strains or isolates suggests antigenic variation ( Panangala et al., 1992). The purpose of this study was to define and characterize the in vitro expression and distribution of several surface epitopes among MG strains and within populations of the same strain by using a panel of mAbs in order to determine if variable expression of surface epitopes is occurring in MG.

2. Materials and methods 2.1. Strains

The following Mycoplasma gallisepticum strains were utilized for production, screening, and analysis of mAbs: A5969 (Jungherr and Luginbuhl, 1952), R (Yoder et al., 1984), F (Rodriguez and Kleven, 1980; Carpenter et al., 1981; Glisson and Kleven, 1984), variant MG strains (K503, K703, K730) (Yoder, 1986), 6/85 (Evans and Hafez, 1992), ts-ll (Whithear et al., 1990) and $6 (208) (Zander, 1961). All MG strains were propagated in Frey's medium (Frey et al., 1968) with 12% swine serum (FMS), then filter cloned three times through a 0.45 ~m filter (Tully, 1983). All strains were also tested for purity by immunofluorescence (Talkington and Kleven, 1983 ). Filter-cloned MG strains grown in FMS were divided into 1 ml aliquots and stored at - 70°C in an equal volume of glycerol. Other avian mycoplasma species utilized were M. synoviae type strain WVU 1853 (Olson et al., 1963), M. iowae type strain 1-695 (Yoder and Hofstad, 1962), M. gallinarium PG16, and M. gallinaceum DD. 2.2. Monoclonal antibodies

A panel of seven established cell lines producing mAbs against M. gallisepticum were developed by Elfaki et al. ( 1990, 1993) and maintained by the Monoclonal Facility at the University of Georgia. These cell lines were identified as: 8F7 and 4G1, raised against whole cell preparations of F strain; 12D10, 9D4 and 6A10, raised against whole cell preparations of the $6 (208) strain (Elfaki et al., 1990); cell line 12D8 raised, against solubilized 6/85 membranes prepared as described elsewhere (Elfaki et al., 1991 ); and cell line 3D6, raised against a purified 98 kDa membrane protein of the $6 (208) strain (Elfaki et al., 1993). After thawing the cell lines were recloned by limiting dilution in order to screen clones against a wider number of MG strains. The mAbs produced by these cell lines were isotyped by an Enzyme-linked-immunosorbent assay (ELISA) using a complete set

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Table 1 mAbs and isotypes mAb

Isotypes

8F7/1~ 4G1/F~ 9D4/ $6" 12D10/$6a 6A10/$6a 3D6/S& 12D8/6/85a

IgG~ IgGi IgG2. IgGi IgGl IgG2b IgG1

"MG strains used to produceMAbs. of alkaline phosphatase conjugated isotype specific antibodies (Southern Biotechnology Associates, Inc., Birmingham, AL). The immunoglobulin class and subclass for each mAb is listed in Table 1. 2.3. In vitro passage procedure

The MG strains had the following initial passage levels: A5969 ( p > 500), F ( p l 9 ) , $6(p23), t s - l l ( p l 0 ) , 6/85(p7), R(p20), and variant strain K503 ( p > 3 0 0 ) , K703 (p > 300), K730 (p > 300). After 16 hr incubation or after cultures reached the log phase, using a 2% ( v / v ) inoculum, these were passed an additional 20 times in FMS medium. Aliquots from the initial, second, and the 20th passage ( + 20) were frozen at - 70°C for immunoblot analysis. In addition, colonies from these passages were grown on FMS agar for immunoperoxidase staining. 2.4. lmmunostaining o f colonies

The indirect immunoperoxidase assay (IIPA) performed as previously described by Bencina and Bradbury (1991) was used to determined if the epitopes recognized by the mAbs were surface-exposed and to characterize the in vitro expression of these epitopes. Colonies from the initial and the + 20 passage culture of MG strains were reacted against a 1:500 dilution of each of the mAbs. Reactions on colonies were observed under a microscope at 100 × power; positive immunostained colonies showed a brown stain on the surface. A sample of 100 colonies were examined for the initial and + 20 passage culture of each MG strain to determined the percentage of stained colonies. 2.5. Antigen preparation f o r western blot

Frozen aliquots of cultures from the initial and + 20 passage of MG strains A5969, F, $6, R, 6/85, ts-11, and K503 were thawed and centrifuged at 13 000 g for 10 min. Mycoplasma pellets were washed three times with 50/xl of PBS (pH 7.2) and carefully resuspended in lysis buffer with 2% SDS. The lysis buffer also contained 125 mM Tris pH 6.8, 10.0% ( v / v ) 2-beta mercaptoethanol, 20.0% glycerol, and 0.02% bromophenol blue. The proteins were then heated to 100°C for 5 min.

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2.6. Sds-page and immunoblotting

Six microliters of each sample was applied to a SDS-PAGE gel. The amount of protein per pellet was not determined, but the procedure was standardized to apply 10 6 cells per well on a slab gel. Samples were subjected to electrophoresis in a Bio-Rad Miniprotein II cell (Bio-Rad Laboratories, Inc. Hercules CA.) using the discontinuous buffer system described by Laemmli (Laemmli, 1970). The MG strain proteins were transferred to 0.20 p~m nitrocellulose (NC) membrane (Bio-Rad Laboratories, Inc. Hercules CA.), (Towbin et al., 1979) and blocked overnight in TBS with 5% bovine serum albumin (BSA) at room temperature. The mAbs were diluted to a 1:1000 (protein concentration about 10/xg/ml) solution of TBS with 2% normal rabbit serum (NRS) and BSA. The NC membranes were probed for 3 hr at room temperature with the appropriated mAbs. After washes in TBS with 0.05% Tween 20 the NC membranes were incubated with an affinity-purified alkaline phosphatase conjugated rabbit anti-mouse IgG (h + 1chain) (Zymed Laboratories, So. San Francisco, CA) diluted 1:1000 in TBS with 3% BSA for 1 hr at room temperature. This was followed by several washes. Immunoblots were developed with 0.06% (w/v) BCIPP-Toluidine and 0.4% ( v / v) nitroblue tetrazolium chloride (Boehringer Mannheim Biochemicals, Indianapolis, IN) diluted in 1 M Tris pH 10. 2. 7. Selective cloning

Colony impressions from the F-strain- + 20 passage culture were made on polyvinylidene difluoride (PVDF) membranes (0.1/z pore size Millipore, Bedford, MA) and immunostained with mAb 9D4/$6 following the immunobinding assay described by Kotani and Mc Garrity (1986). Positive and negatives colonies were localized by positioning the stained membrane above the agar plate. The selected colonies were expanded in 5 ml of FMS and plated on agar were selections were made. Six consecutive generations of negative clones were derived from a positive expanded-clone culture, and six consecutive generations of positive clones were derived from a negative expanded-clone culture. All the expanded clone cultures were analyzed by Westem blot with mAb 9D4/$6. 2.8. Physicochemical characterization

Partial physicochemical characterization of the epitopes recognized by mAbs 9D4/$6, 12D 10 / $6, and 6A 10/$6 was performed by mild trypsin and periodate oxidation treatment. A modification of the trypsin treatment of Rosengarten and Wise (1991) was utilized. Approximately 105 cells from aliquots of R-strain log phase culture (p22) were harvested and washed twice in PBS (pH 7.2) and once in PBS (pH 8.0), then incubated for l hr at 37°C in 25 ~1 of PBS (pH 8.0) containing either no trypsin or 0.08 ~g of trypsin (Sigma CO. St. Louis, MO. catalog # T1005). After this treatment, samples were immediately prepared and applied to SDS-PAGE and subsequently immunostained. Nitrocellulose membrane strips with transfer R strain proteins were treated with 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 20 mM or 50 mM periodic acid solution in sodium acetate buffer (pH 4.5) as described by Woodward et al. (1985). After this treatment membranes were probed with mAbs 9D4/$6, 12D10/$6 and 6A10/S6 as described above.

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3. Results 3.1. Immunostaining o f colonies N o n e o f the variant M G strains colonies (K503, K703, K 7 3 0 ) stained with any o f the seven m A b s utilized in this study. The m A b 1 2 D 8 / 6 / 8 5 only stained colonies f r o m M G strains 6 / 8 5 and A5969. Out o f the seven m A b s only 4 G 1 / F and 8 F 7 / F stained u n i f o r m l y 100% o f the M G strains A5969, F, t s - 1 1 , 6 / 8 5 , R, and $6 colonies. H o w e v e r , the percentage o f c o m p l e t e stained colonies for the a b o v e m e n t i o n e d M G strains ranged f r o m 32 to 98% for the initial and + 20 passage level w h e n stained with m A b s 9 D 4 / $ 6 , 1 2 D 1 0 / $ 6 , 6 A 1 0 / $6, 3 D 6 / $ 6 and 1 2 D 8 / 6 / 8 5 ( T a b l e 2). W h e n we e x a m i n e d these colonies under the m i c r o s c o p e they appear as a m i x population o f stained and unstained colonies in the same culture (Fig. l a ) . A particular partial staining was o b s e r v e d in a 5 - 1 2 % o f the colonies f r o m F-strain initial and + 20 passages respectively w h e n stained with m A b 6 A 1 0 / F (Fig. lb).

3.2. Western blot analysis B o t h a primary and a secondary antibody deletion controls, as w e l l as a swine serum control w e r e utilized in order to test the specificity o f the Western blot procedure. As expected, no reactions w e r e observed. W e s t e r n blot analysis results are s u m m a r i z e d in T a b l e 3. T w o proteins w e r e r e c o g n i z e d by three m A b s for all M G strains tested including the variant strain K503. A m o l e c u l a r weight of a p p r o x i m a t e l y 44 k D a was o b s e r v e d for the Table 2 Percent of immunostained MG strain colonies b at initial and initial + 20 in vitro passages level using monoclonal antibodies MG strain

A5969 F 6/85 ts-11 R $6

Passage

Initial Initial + 20 Initial Initial + 20 Initial Initial + 20 Initial Initial + 20 Initial Initial + 20 Initial Initial + 20

mAba 8F7/F

4G1/F

9D4/$6

12D10/$6

6A10/$6

3D6/$6

12D8/6/85

100 100 100d 100d 100 100 100 100 100 100 100 100

100 100 100d 100d 100 100 100 100 100 100 100 100

62 98 42 60 40 85 32 85 98 96 72d 98d

100 100 55 89 89 84 80 92 96 98 92d 90~

100 100 5c 12c 90 94 92 96 100 100 98d 96d

94 98 70 42 0 0 95 98 90 94 100d 98~

93 93 0 0 98d 98d 0 0 0 0 0 0

aMG variant strains (K503, K703, K730) colonies did not immunostain with any of the mAbs. bPercent of complete stained colonies. °Percent of partial stained colonies. dMG strain utilized to produce mAb

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Fig. 1. (a). The $6 (208)-strain p23 (int) immunostained with mAb 9D4/$6; showing unstained and stained colonies. (b). F-strain p19 (int) immunostained with mAb 6AI0/$6, showing partial stained colonies.

Table 3 Molecular weights (kDa) approximations of MG proteins recognized by monoclonal antibodies after 20 in vitro passages MG Strain

8F7/F

4G1/F

9D4/$6

12D10/S 6

6A10/S 6

3D6/$6

12D8/6/85

A5969 F 6/85 ts-11 R $6 K503 K703 K730

58 a 58, 79 79 58, 79 58, 79 58 Nt x NT b NT

58 58, 79 79 58, 79 58, 79 58 NP NT NT

44 44 44, 44, 44, 44, 44 44 44

44 44, 69 d 44, 69 d 44, 69 d 44, 69 44, 69 ° 44 44 44

75 NP 69 69 69 69 NP NT NT

119, 82-113 119, 82-113 NP 119, 82-113 119, 82-113 119, 82-113 119 i 19 119

79 79 69, 79 79 79 NP NP NT NT

69 d 69 d 69 69 d

aSurface proteins shown in bold. bNT, not tested. cNP, no proteins detected. d69 kDa protein detected after 20 in vitro passages.

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--98.0 --80.0 --64.4 --44.6

1

2

3

4

5

6

Fig. 2. Multiple and size variant proteins (approximadon in kDa) among MG strains initial passages in lanes: 1. R-strain probed with 8F7/F detected a 58 and 79 kDa. 2.6/85 strain probed with 8F7/F detected a 79 kDa. 3. Rstrain probed with 6A10/$6 detected a 69 kDa. 4. A5969-strain probed with 6A10/$6 detected a 75 kDa. 5. Rstrain probed with 12D8/6/85 detected a 79 kDa. 6. 6/85-strain probed with 12D8/6/85 detected a 69 and 79 kDa.

protein recognized by mAbs 9D4/$6 and 12D10/$6, and about 119 kDa for the protein recognized by mAb 3D6/$6. The mAbs 8F7/F, 6A10/$6, and 12D8/6/85 recognized four proteins among MG strains A5969, R, ts-11, 6/85, $6, and F-strain independently of the MG strain passage level. However, we observed that these mAbs recognized different size proteins in R, 6/85, and A5969 strains (Fig. 2). mAb 8F7/F recognized two proteins of about 58 and 79 kDa in R-strain, while it only recognized a 79 kDa in 6/85 strain, mAb 6A10/$6 recognized a protein of about 69 kDa in R-strain, while it recognized a protein of about 75 kDa in A5969 strain, mAb 12D8/6/85 recognized a 79 kDa protein in R-strain, while it recognized two proteins of about 69 and 79 kDa in 6/85 strain. In addition to the 119 kDa protein recognized by mAb 3D6/$6 a group of approximately 7 proteins with molecular weights ranging from about 113 to 82 kDa were recognized by this mAb independently of the passage level of A5969, F, 6/85, ts-! 1, R and $6 (Fig. 3).

211.0 119.0 98.0 80.6 64.4 44.6 1

2

3

4

5

6

Fig. 3. Western blot of MG strains initial passage probed with mAb 3D6/$6 arranged in lanes. 1. R. 2. $6 (208). 3. ts- 11.4. 6/85.5. F. 6. A5969. The epitope was recognized in proteins with a molecular masses of approximately 119, 113, 107, 98, 85, and 82 kDa.

154

M. Garcfa et al. / Veterinary Microbiology 42 (1994) 147-158 -98.0 - 80.0 -64.4 --44.6

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

Fig. 4. Western blot of initial (int) and + 20 passages ( + 20) of MG strains probed with mAb 9D4/$6 arranged in lanes: 1. M. iowae, 1695; 2. M. synoviae 1853 WV; 3. K503, int ; 4. K503, + 20 ; 5. R, int ; 6. R, + 20 ; 7. $6 (208), int ; 8. $6 (208), +20 ; 9. ts-ll, int ; 10. ts-ll, +20; 11.6/85, int ; 12. 6/85, +20 ; 13. F, int ; 14. F, +20 ; 15. A5969, int ; 16. A5969, +20. The epitope was recognized in proteins with molecular masses of approximately 44 and 69 kDa. A protein of a p p r o x i m a t e l y 69 k D a was r e c o g n i z e d f r o m the initial passage culture o f Rstrain by m A b s 9 D 4 / $ 6 and 1 2 D 1 0 / $ 6 . A f t e r 20 in vitro passage the 69 k D a protein was r e c o g n i z e d by 9 D 4 / $ 6 in $6, ts-11, and 6 / 8 5 but not in K503, A5969, or F-strain (Fig. 4). The m A b 1 2 D 1 0 / $ 6 r e c o g n i z e d the 69 k D a protein in $6, t s - l l , 6 / 8 5 and F strains after 20 in vitro passage but not in K503 or A 5 9 6 9 cultures.

a

- 98.0 - 80.0 - 64.4

-44.6

1

2

3

4

5

6

b 98.0 80.0

64.4

44.6

1

2

3

4

5

6

Fig. 5. Western blot analysis of expanded clones probed with mAb 9D4/$6. (a). Six generations of negative clones derived from a positive expanded-clone culture. (b). Six generations of positive clones derived from a negative expanded-clone culture.

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u

t

pa

u

t

pa

u

t

pa 98.0

80.0 64.4

44.6

1

2

3

4

5

6

7

8

9

Fig. 6. Proteinimmunoblotof R strain,initialpassage- untreated (u), trypsintreated (t), and periodicacidtreated (pa). Lanes 1 to 3 probedwith mAb 9D4/$6; lanes 4 to 6 probedwith mAb 12D10/$6; lanes 7 to 9 probedwith mAb 6A10/$6. 3.3. Selective cloning

Western blot analysis of selected clones showed that after three generations of selecting negative clones from a positive-clone culture of F-strain the mAb 9D4/S6-recognized epitope was no longer detected in association with the 69 kDa protein (Fig. 5a). However, the 69 kDa protein was detected in SDS-PAGE analysis of F-strain negative clones (data not shown). Additionally, after three generations of selecting negative clones from a positive-clone culture the mAb 9D4/S6-recognized epitope was detected in association with the 69 kDa surface protein of F-strain (Fig. 5b). 3.4. Physicochemical characterization

Trypsin treatment of R-strain cells resulted in the abolition of binding of mAbs 9D4/$6, 12D10/$6, and 6A10/$6. However, neither the low (0.1 mM) nor the high (100 mM) concentration solutions of periodic acid treatment abolished the binding of these mAbs to their respective epitopes. This indicates that the recognized epitopes on MG proteins are not carbohydrate but rather protein determinants (Fig. 6).

4. Conclusion

The determination of mAbs immunostaining patterns on intact MG colonies and the identification of the corresponding proteins by Western blot (WB) analysis permitted us to categorized the MG proteins as surface and non-surface proteins. With the exception of strain A5969, we found that intact colonies of MG strains which did not immunostain had only one protein bearing the recognized epitope of about 44, 79, or 119 kDa in size. Therefore, most likely these proteins are not exposed to the surface. The panel of mAbs recognized six different surface epitopes associated to six surface proteins, but not all MG strains tested reacted with all the mAbs. In addition to the lack of reactivity by atypical MG strains, few typical MG strain colonies did not react with mAbs

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3D6/$6, and 12D8/6/85 (Table 2). In Western blot analysis we found that different size proteins were recognized in strains R, 6/85, and A5969 by the same mAb. These two observations are evidence of the antigenic heterogeneity present among MG strains that had been reported by others (Panangala, et al). The immunostaining of MG colonies allowed us to observe the expression of the mAbsrecognized epitopes at a single colony level. We characterized the expression of the epitopes recognized by mAbs 8F7/F and 4G1/F as permanent, among MG strains, and among colonies from the same culture. On the other hand, the expression of surfaces epitopes recognized by mAbs 9D4/$6, 12D10/$6, and 6A10/S6, was characterized as variable among colonies of the same passage, between passages of the same strain, and among different MG strains. Among MG strains we found that F-strain seems to express the 9D4/ $6 and 6A10/S6-recognized epitope less frequently than R, $6, A5969, ts-11, and 6/85. Further analysis of clonal populations of F-strain 9D4/S6-recognized epitope showed that variable expression of this surface epitope seems to be a heritable trait passed from one clone to its progeny. The molecular basis for the variable expression of epitopes in MG is not known. The variable expressed epitopes recognized by mAbs 9D4/$6, 6A10/S6, and 12D10/$6 were found to be amino acid determinants associated with a surface protein of about 69 kDa. The presence of more than one epitope associated with a 67 kDa MG surface protein has been reported by Markham et al. (1990), and Bencina et al. (1993). The proteic nature of these epitopes indicates that post-translational modifications may not be the mechanism causing the variable expression of the surface epitopes associated with the 69 kDa protein. In addition, the fact that a 44 and 69 kDa, 58 and 79 kDa, 69 and 79 kDa, and 82-113 kDa share at least one epitope indicates a close relationship among all the mAbsdefined proteins reported here. Concurrent post-translation modifications are unlikely to occur, as explained for M. hyorhinis (Boyer and Wise, 1988), (Rosengarten and Wise, 1990). Recently, a report suggested that recombination events involving about 20 variant genes generates a repertoire of different size variant proteins of the 67 kDa polypeptide of MG (Browning et al., 1992). Our findings indicate that the variable expression of MG surface epitopes is a fact and in our opinion it should be taken into account during vaccine production and antigen production for serological tests.

Acknowledgements This work was supported by the Veterinary Medical Experiment Station, University of Georgia. We thank Dr. Dusan Bencina for his helpful comments in the preparation of this manuscript.

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