INFECTION AND IMMUNITY, Jan. 1990, p. 189-196

Vol. 58, No. 1

0019-9567/90/010189-08$02.00/0 Copyright © 1990, American Society for Microbiology

The 75-Kilodalton Protein of Chlamydia trachomatis: a Member of the Heat Shock Protein 70 Family? SANDRA L. DANILITION,'t IAN W. MACLEAN,2 ROSANNA PEELING,' SCOTT WINSTON,2 AND ROBERT C. BRUNHAM1* Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba R3E 0W3,1 and Allelix Inc., Mississauga, Ontario L4V JPJ,2 Canada Received 30 May 1989/Accepted 2 October 1989

The gene encoding a 75-kilodalton (kDa) protein of Chlamydia trachomatis was cloned, expressed, and sequenced. Genomic libraries from C. trachomatis serovar D DNA were constructed in vectors pUC18 and Agtll and were screened with a panel of monoclonal antibodies against C. trachomatis antigens. The only recombinants identified were those that reacted with antibody UM-13, which has specificity for a genus-specific epitope on the 75-kDa protein. The gene was localized to a 2.9-kilobase DNA fragment and sequenced. The gene consists of a long open reading frame of 1,956 nucleotides, which translates into 652 amino acids totalling 70,558 daltons in mass. Putative promoter elements and a ribosome binding site were identified within 5'-flanking sequences, and a typical rho-independent terminator was identified within 3'-flanking sequences. Screening of the GenBank nucleic acid sequence data bank revealed extensive similarity between the chlamydial 75-kDa gene and the heat shock protein 70 (hsp70) family of proteins. In particular, 71 and 69% amino acid sequence similarities were identified with hsp70 of Escherichia coli and BaciUus megaterium, respectively. Polyclonal antibodies were produced to the recombinant antigen in rabbits and detected epitopes on elementary bodies in enzyme-linked immunosorbent and indirect microimmunofluoresence assays. Antibodies reacted with an antigen of identical molecular mass in L2 and C serovars in an immunoblot assay and neutralized these serovars in cell culture. The 75-kDa protein appears to be a chlamydial homolog of hsp70, is immunoaccessible on native elementary bodies, and is a target for neutralization.

Chlamydia trachomatis is an intracellular bacterial parasite that replicates via an intricate life cycle involving eucaryotic host cells. The infectious extracellular form of the organism, termed the elementary body (EB), is a sporelike structure which is metabolically inactive. Three cysteinerich outer membrane proteins of 57, 40, and 12.5 kilodaltons (kDa) in molecular mass are speculated to interact via disulfide bonds within the outer membrane of the EB, enabling it to remain structurally stable and impervious to entry of small molecules (1, 21, 22). The second form of the organism, termed the reticulate body, is found within the infected eucaryotic host cell within a membrane-lined vacuole. The reticulate body is approximately threefold larger in diameter than the EB, metabolically active, and osmotically unstable. The 40-kDa protein is found in the outer membrane of reticulate bodies, although the 57- and 12.5-kDa proteins are typically absent (11). Near the end of the growth cycle, newly synthesized 57- and 12.5-kDa cysteine-rich proteins are incorporated into the reticulate body outer membrane (12, 21). As the organism condenses into EBs, all three cysteine-rich proteins are thought to interact to confer the properties necessary for the extracellular survival of the EB. Proteins other than the 57-, 40-, and 12.5-kDa proteins are also found in the outer membrane of C. trachomatis (6). Using monospecific polyclonal antibodies, we noted that a highly conserved 75-kDa protein was immunoaccessible in the outer membrane and a target for neutralizing antibody (17). At this time, only a limited number of chlamydial genes have been cloned. To help gain an understanding of the

biology of chlamydiae, using a molecular approach, we chose to clone one of the outer membrane antigens. This work describes the cloning, expression, and sequencing of the gene which encodes the 75-kDa protein. MATERIALS AND METHODS Organisms: growth and purification. C. trachomatis serovar D (UW3/Cx) was propagated in HeLa 229 cells and purified as described previously (24). Escherichia coli Y1089 (r-), Y1090 (r-), JM101, JM109, and DH5-a were used as recipients of recombinant DNA (31, 33). The vectors utilized were Xgtll, pUC19, pUC18, M13mpl8, and M13mpl9. DNA isolation and manipulations. Chlamydial DNA was isolated from purified EBs as described previously (19). Plasmid and bacteriophage DNAs were isolated and purified by standard methods and treated by standard recombinant DNA techniques (18). All DNA-modifying enzymes were used as specified by the manufacturer. Construction of libraries. A genomic library of chlamydial DNA was constructed in vector Xgtll by the insertion of EcoRI fragments with packaging and screening in accord with the manufacturer's instructions (Promega Biotec, Madison, Wis.). A partial genomic library of chlamydial DNA was constructed in plasmid vector pUC18. Chlamydial DNA, partially digested with Sau3AI, was separated electrophoretically, and DNA within the size range of 5 to 7.5 kilobases (kb) was excised, purified, and subsequently ligated to the BamHI site of pUC18. Immunoblotting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Screening of the libraries involved immunoblotting either plaques or colonies, essentially as described previously (33). Screening was carried out with a series of monoclonal antibodies with specificities against chlamydial antigens. These monoclonal antibodies have

Corresponding author. t Present address: Division of Biological Research, The Ontario Cancer Institute, and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada M4X 1K9. *

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specificities to chlamydial antigens of molecular mass 75, 60, 57, 40, 32, and 29 kDa (17). Whole-cell lysates of transformants were screened by electrophoresis in 10 or 12.5% sodium dodecyl sulfate-polyacrylamide gels and subsequently analyzed by either silver staining or immunoblotting, as described previously (17). Deletions. For the purpose of sequencing the cloned gene, a variety of deletions were created, using the enzyme T4 DNA polymerase (Pharmacia, Inc., Piscataway, N.J.) or exonuclease III and Si nuclease (Bethesda Research Laboratories, Inc., Gaithersburg, Md.) (8, 10, 14). Sequencing. The various clones were sequenced by the Sanger dideoxy chain termination method (26). The sequencing kits used were purchased from Bio-Rad Laboratories, Richmond, Calif., and United States Biochemical Corp., Cleveland, Ohio, and contained Klenow or Sequenase enzymes, respectively. Computer analysis. Sequence analysis was carried out with the aid of the Pustell Sequence Analysis Programs (IBI) and the University of Wisconsin GCG programs. Immunologic characterization of the recombinant 75-kDa protein. Recombinant 75-kDa protein was purified from E. coli DH5-a containing plasmid pERU-S2 after lysis with 2% Sarkosyl. The cell supernatant was run over an affinity column containing the capture antibody UM-13 (17). Antigen was eluted with 0.1 M sodium acetate-0.15 M sodium chloride, pH 4, and the fractions containing the purified antigen were pooled. Two rabbits were injected intramuscularly five times over a 2-month period with 50 pig of purified antigen mixed with an equal volume of complete or incomplete Freund adjuvant. Immune serum was fractionated on a protein A-Sepharose column (24), and purified immunoglobulin G (IgG) was characterized in microimmunofluorescence, enzyme-linked immunosorbent, immunoblot, and neutralization assays (17). RESULTS Isolation of the gene encoding the 75-kDa protein. The chlamydial genomic library in Xgtll was screened with a pool of monoclonal antibodies to six different C. trachomatis antigens (17). Of the plaques screened, 75% contained inserts and approximately 10% reacted with the pool of monoclonal antibodies. Five of the positive recombinants were isolated, and all were immunoreactive to UM-13, a monoclonal antibody with specificity to a Chlamydia genusspecific epitope on a 75-kDa protein. An immunopositive recombinant that expressed a fusion protein of ,-galactosidase and the 75-kDa protein was identified. The recombinant expressed a 150-kDa fusion protein that was immunoreactive with monoclonal antibody UM-13 by immunoblot (data not shown). Restriction analysis of the recombinant revealed a chlamydial DNA insert of 2.2 kb. This recombinant was used in partial sequencing of the 75-kDa gene. A partial library of chlamydial genomic DNA was constructed in pUC18 by insertion of Sau3AI-partially-digested DNA of 5 to 7.5 kb into the BamHI site of pUC18. Immunoblot screening of the library revealed a recombinant, pERU-Si, that expressed the 75-kDa protein. Whole-cell extracts of E. coli harboring the recombinant plasmid contained a protein which comigrated with the 75-kDa protein of C. trachomatis (Fig. 1A). The recombinant protein reacted with monoclonal antibody UM-13 in an immunoblot of the same gel (Fig. 1B). A 2.9-kb restriction fragment containing the entire gene encoding the 75-kDa protein was subcloned into pERU-S2.

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identified. The terminator consists of a 13-base dyad (arFig. 3) beginning at base 1983 and ending at base 2012. The 3' end of the dyad is followed by eight thymidine residues in the DNA sequence. Transcription of this region would result in folding of the mRNA into a stem-and-loop structure with a calculated least free energy of -24.4 kcal (ca. -102.1 kJ) (34). Homology to heat shock proteins. Comparison of the nucleotide sequence of the 75-kDa gene to sequences of the GenBank nucleic acid sequence data base (release 55.0, March 1988) revealed a large degree of similarity to members of the heat shock protein 70 (hsp70) family (Fig. 4). The hsp70 amino acid sequences of E. coli dnaK and Bacillus megaterium dnaK showed the greatest sequence similarities to the 75-kDa gene, at 71.3 and 69.7%, respectively. Figure 4 shows the similarity between the inferred amino acid sequence of the 75-kDa protein and four heat shock proteins of procaryotic and eucaryotic origin (2, 4, 20, 28). There appear to be two general regions of strong similarity among the 75-kDa gene and the other sequences; the first and third quarters of the sequences are very similar and separated by a segment of divergent residues. Immunologic characterization. From immune serum, purified IgG reacted with an antigen of identical molecular mass (75 kDa) in both serovar L2 and C EBs in immunoblot assay and reacted with no other chlamydial antigen (data not shown). These antibodies reacted with whole chlamydial EBs in the microimmunofluorescence assay in a genus pattern and detected a surface-accessible antigen in the enzyme-linked immunosorbent assay format, using native EBs from both serovars L2 and C. Both L2 and C EBs were neutralized in cell culture with immune IgG in a concentration-dependent manner (Fig. 5). rows,

DISCUSSION The gene encoding the 75-kDa protein of C. trachomatis cloned, expressed, and sequenced. Prior studies with monospecific polyclonal antibodies (17) established that this protein can be detected on native EBs, although 2% Sarko-

was

syl extraction of whole EBs resulted in the solubilization of the protein separate from the outer membrane complex. In vitro and clinical studies suggest that the 75-kDa protein has immunobiologic significance. The protein is immunogenic in infected persons, with about 50% of individuals producing antibody to this antigen (5). Monospecific antibodies to the antigen neutralized C. trachomatis in cell culture (17), and serum antibody to the 75-kDa antigen has been correlated with protection against ascending fallopian tube C. trachomatis infection (5). Thus, we were interested in molecularly characterizing this chlamydial protein. The gene encoding the 75-kDa protein was isolated on a fragment of Sau3AI-digested C. trachomatis DNA. The recombinant molecule pERU-Sl was identified by expression of a protein immunoreactive with monoclonal antibody UM-13. Nucleotide sequence analysis identified typical procaryotic promoter elements and a rho-independent transcriptional terminator. Similar elements have been identified for other chlamydial genes (27); however, no chlamydial consensus sequences have been defined. Other investigators have cloned a gene which expresses a protein of similar molecular mass (3, 15, 23; R. S. Stephens, C. C. Kuo, and N. Agabian, Abstr. Annu. Meet. Am. Soc. Microbiol. 1982, B29, p. 35). Of considerable interest, Palmer and Falkow (23) observed that a gene encoding a chlamydial protein of 75 kDa was transcribed early during the developmental cycle, suggesting that this protein may be necessary for early biologic events in the developmental cycle and may be temporally regulated. Birkelund et al. (3) recently cloned a gene for a chlamydial 75-kDa immunogen. The restriction map is similar to the map described here. They (3) also showed that this gene was expressed early during infection. In this study, polyclonal antibodies to purified recombinant 75-kDa protein were capable of binding to EBs in microimmunofluorescence and enzyme-linked immunosorbent assays, suggesting that epitopes on this antigen are immunoaccessible on the surface of native EBs. Antibodies were also able to neutralize two antigenically distinct C. trachomatis serovars in cell culture. These results confirm

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A HEAT SHOCK PROTEIN OF C. TRACHOMATIS

VOL. 58, 1990

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previous results with monospecific antibodies produced to immunoaffinity-purified native antigen (17). Comparative nucleotide and amino acid analyses of the 75-kDa gene revealed marked sequence similarity to the hsp70 family of proteins (16). The high degree of sequence homology suggests that the chlamydial 75-kDa protein may have functions analogous to those of other hsp70s. Evolutionarily, heat shock proteins are highly conserved, and various activities have been attributed to them. Pelham (25) proposed that hsp70s bind to hydrophobic regions of pro-

teins and prevent or disrupt inappropriate protein-protein interactions. Two recently reported experiments strongly support this model. An hsp70 was identified in yeasts and shown to assist in two activities: (i) transmembrane protein export (9) and (ii) ATP-dependent unfolding of preproa-factor prior to membrane translocation (7). It may be that the 75-kDa protein functions as a stress protein important for EB protein reorganization as it enters an intracellular environment.

The 75-kDa protein is also highly homologous to a portion

194

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FIG. 4. Amino acid comparison of the 75-kDa protein to heat shock proteins. Chlamydia (the 75-kDa protein), E. coli (dnaK), B. megaterium (dnaK), Xenopus laevis (hsp7OB), rat (immunoglobulin heavy chain binding protein). A dot indicates the same amino acid as in the 75-kDa protein.

A HEAT SHOCK PROTEIN OF C. TRACHOMATIS

VOL. 58, 1990

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('g gOG) FIG. 5. Neutralization of C. trachomatis serovars L2 (A) and C (B) in cell culture. Results are expressed as mean + standard deviation of number of inclusions per cover slip in triplicate culture for EBs treated with nonimmune IgG at 70 F±g or immune IgG at 70, 7, 0.7, or 0.07 ALgANTIBODY CONCENTRATION (ug IGG)

of a 71-kDa mycobacterial antigen which bears very. close amino acid sequence similarity to other hsp70s (32). The extensive similarity between the 75-kDa protein and the mycobacterial protein was identified from amino acids 440 to 500 of the 75-kDa protein. Figure 4 shows that this region of the heat shock proteins spans an area of high homology; this region may therefore constitute an important functional domain common to stress proteins of the hsp70 family. ACKNOWLEDGMENTS We thank Bruce Collier for technical advice, Gerry Bilan for preparation of the manuscript, Neil Miyamoto for critical reading of the manuscript, and Lorne Reid for assistance in computer analysis. This work was supported by a grant from the Medical Research Council of Canada. LITERATURE CITED 1. Bavoil, P., A. Ohlin, and J. Schachter. 1984. Role of disulfide bonding in outer membrane structure and permeability in Chlamydia trachomatis. Infect. Immun. 44:479-485. 2. Bienz, M. 1984. Xenopus HSP70 genes are constitutively expressed in injected oocytes. EMBO J. 3:2477-2483. 3. Birkelund, S., A. G. Lundenose, and G. Christiansen. 1989. Characterization of native and recombinant 75-kilodalton immunogens from Chlamydia trachomatis serovar L2. Infect. Immun. 57:2683-2690. 4. Bordwell, J. C. A., and E. A. Craig. 1984. Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnak gene are homologous. Proc. Natl. Acad. Sci. USA 81:848-852. 5. Brunham, R. C., R. Peeling, I. Maclean, J. McDowell, K. Persson, and S. Osser. 1987. Post abortal Chlamydia trachomatis salpingitis: correlating risk with antigen-specific serological responses and with neutralization. J. Infect. Dis. 155:749-755. 6. Caldwell, H. D., J. Kromhout, and J. Schachter. 1981. Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis. Infect. Immun. 31:11611176. 7. Chirico, W. J., M. G. Waters, and G. Blobel. 1988. 70K heat shock related proteins stimulate protein translocation into microsomes. Nature (London) 332:805-809. 8. Dale, R. M., B. A. McClure, and J. P. Houchins. 1985. A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the corn mitochondrial 18S rDNA. Plasmid 13:31-40. 9. Deshaies, R. J., B. D. Koch, M. Werner-Washburne, E. A. Craig, and R. Schekman. 1988. A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature (London) 332:800-805.

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