Antigenic Structure of Surface-Exposed Regions of the Major Outer-Membrane Protein of Chlamydia trachomatis

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1 REVIEWS OF INFECTIOUS DISEASES VOL. 10, SUPPLEMENT 2 JULY-AUGUST 1988 C, 1988 by The University of Chicago. All rights reserved /88/ $02.00 Antigenic Structure of Surface-Exposed Regions of the Major Outer-Membrane Protein of Chlamydia trachomatis Wilbert J. Newhall V From the Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana The major outer-membrane protein (MOMP) of Chlamydia trachomatis displays a number of surface-exposed epitopes. Some of these are shared by all or some of the 15 known serovars, whereas others are serovar-specific. With use of epitope-mapping analysis of limited proteolytic digests of purified MOMPs with monoclonal antibodies, the present study assessed whether the same or different surface-exposed regions of the protein express these different epitopes. Results with both chymotrypsin and the staphylococcal V8 protease for MOMP of serovar F indicated that many of the shared sur face epitopes are clustered in one region whereas the serovar-reactive epitope is located in a separate region. Analysis of MOMPs from all the serovars with a species-specific monoclonal antibody indicated that each MOMP possesses a structurally analogous region that expresses this epitope. Such a region might play a role in pathogenic mechanisms that are shared by all serovars. Chlamydia trachomatis is a human pathogen that is responsible for a significant proportion of ocular and sexually transmitted diseases throughout the world. Although available antibiotics appear to be effective in eradicating disease, questions remain whether such treatment truly eliminates infection and whether Chlamydia can establish latent infections. Present data indicate that vaccination or infection may confer partial immunity to reinfection. However, vaccination has been shown to lead to a severe delayed hypersensitivity response with some vaccines. Thus, development of a useful vaccine will require a better understanding of the pathogenic mechanisms employed by Chlamydia, a more complete description of the host immune response to chlamydial infection, and the identification and isolation of the chlamydial antigens that can stimulate a protective response. One of the chlamydial antigens currently being considered for use in a vaccine is the major outermembrane protein (MOMP). This protein comprises about 60% of the outer-membrane protein mass the infectious form of the organism the elementary body [1]. Functionally, MOMP serves as a porin, allowing the passage of small molecules through the outer membrane [2]. MOMP also serves an important structural role that is mediated by disulfide cross-linking to other MOMP molecules as well as Please address requests for reprints to Dr. Wilbert J. Newhall V, Department of Medicine, Indiana University School of Medicine, 545 Barnhill Drive EM 435, Indianapolis, Indiana to other outer-membrane proteins [3, 4]. There is currently no direct evidence implicating MOMP in either the attachment or uptake stages of chlamydial pathogenesis; however, antibodies against MOMP are capable of neutralizing the infection cycle in vitro [5-7]. Antigenic analyses have shown MOMP to be a fairly complex surface antigen. On the basis of studies using polyclonal MOMP-specific antisera [8] and monoclonal antibodies [9-11], MOMP has been found to express a range of epitopes from serovarspecific to species-specific. Since the serovar-specific epitopes of C. trachomatis are thought to be involved in the generation of protective immune responses, one approach to the development of a potential vaccine is to isolate those epitopes from the others and to test their ability to stimulate protection against reinfection. As a step toward isolating the serovar-specific epitopes, the present study was performed to determine if the different surface-exposed epitopes of MOMP are clustered in a specific region of MOMP or are dispersed along the length of the molecule. The approach was to generate a number of peptide fragments from MOMP and to determine if the various MOMP epitopes are located on the same or different fragments. Materials and Methods All chlamydial strains were grown in HeLa 229 cell monolayers. Elementary bodies (EBs) were harvested S386

2 Surface Epitopes of Chlamydial MOMP S387 at the end of the growth cycle and purified on Percoll density gradients. Outer-membrane preparations were derived by extraction of EBs with sodium N- lauroylsarcosine (Sarkosyl). Outer-membrane proteins were solubilized with SDS and dithiothreitol and resolved by high-performance liquid chromatography (HPLC) [12]. Fractions containing purified MOMP were identified by SDS-PAGE, pooled, and stored at - 70 C before use. Monoclonal antibodies specific for MOMP have been characterized previously by their cross-reactivities among the different serovars by radioimmunoassay and immunoblotting [9, 13]. All antibodies were titered by immunoblotting and were used in excess to provide maximum sensitivity. Peptide mapping of MOMP was performed by SDS-PAGE analysis of limited proteolytic digests with use of the method of Cleveland et al. [14]. After digestion of purified MOMPs with either staphylococcal V8 protease or chymotrypsin, the resulting polypeptide fragments were resolved on a 16 07o polyacrylamide gel and visualized by silver staining. To probe the resolved polypeptides by immunoblotting, the fragments on nonstained polyacrylamide gels were electrophoretically transferred to a nitrocellulose membrane [9]. The adsorbed fragments were probed with a monoclonal antibody to MOMP having serovar, subspecies, species, or genus specificity. Bound antibody was detected by autoradiography after incubation with either 125 I-labeled protein A or 125I-labeled goat anti-mouse IgG or IgM. Results and Discussion Preliminary experiments were performed to assess the effects of time, temperature, and enzyme concentration on the generation of MOMP fragments. A maximum number of resolvable fragments, as determined by silver staining, was obtained with 80 gg of V8 protease/ml and 80 gg of chymotrypsin/ml using 5 gg of purified MOMP in a volume of 50 p,l and with incubation for 30 minutes at 37 C. These conditions were then used to evaluate the MOMPs from each of the 15 C. trachomatis serovars, the mouse pneumonitis strain of C. trachomatis, and the MN/Cal-10 strain of Chlamydia psittaci. The fragment patterns derived with the use of chymotrypsin are shown in figure 1. At this enzyme concentration (80 gg/ml), digestion was incomplete, as indicated by the presence of undigested MOMP in each sample. Below MOMP in the gel are a number of peptide fragments that were generated; these fragments are clearly distinguishable from the bands in the control (which contained enzyme without MOMP). All of the MOMPs appeared to yield some fragments of similar sizes; each also gave fragments that were often unique or that were shared by only one or a few of the other strains. From these patterns and a Figure 1. Silver-stained polyacrylamide gel of peptide fragments generated by chymotrypsin of the major outermembiane protein (MOMP) of Chlamydia trachomatis. The position of MOMP and molecular-weight markers are indicated on the left. The ENZ lane is chymotrypsin alone. MN represents the CAL-10 strain of Chlamydia psittaci.

3 S388 Newhall similar set of observations in experiments with the V8 protease, it appears that MOMPs are structurally variable but have some degree of structural homology even between species. In a similar study of five C. trachomatis serovars by Caldwell and Judd [15], the structural variation of surface-exposed regions of MOMP appeared to correlate with the classic serologic classifications that have been defined by microimmunofluorescence, including serovar, certain subspecies, and species. This suggested that the epitopes responsible for these conserved specificities are located in structurally conserved regions of the molecule while nonconserved epitopes are in other regions. The present study was designed to test this hypothesis by identifying the fragments generated by limited proteolytic digestion that react with a panel of monoclonal antibodies to MOMP that recognize different surface-exposed epitopes and comparing those fragments for different strains that have the same epitope. Using the MOMP from serovar F (F/MOMP), we compared the fragments generated with the V8 protease and chymotrypsin by assessing their reactivities with seven different mbnoclonal antibodies. The reaction patterns for these antibodies are shown in figure 2. With the V8 protease, three general patterns were seen. The species-specific and two subspeciesspecific antibodies generally reacted with the same fragments. The genus-specific antibody gave a similar pattern but lacked reactions at 10,000 molecular weight (10K) and just below the 17K fragment, and had two unique reactions: one at 12K and another with a low-molecular-weight fragment that was out of the range of the molecular-weight markers. The patterns of the three serovar-specific antibodies were indistinguishable, a finding suggesting that they may each recognize the same epitope. However, the patterns were clearly unique relative to the species and subspecies patterns, with the absence of reactions at 17K, 21K, and 24.5K and the presence of a unique reaction at 12K. A similar set of observations was obtained with chymotrypsin. It is interesting that all of the antibodies reacted with the 24K, 27K, and 29K fragments. However, while the species and subspecies epitopes all appeared to be present on the same 15K fragment, no fragments smaller than 24K reacted with the serovar-specific antibodies. These data suggest that the three species and subspecies epitopes are clustered together in a region of MOMP that is distinct from those containing the serovar or genus epitopes. The finding of clustering of some broadly crossreactive epitopes suggested that certain regions of MOMP may be structurally conserved among the Figure 2. Epitope mapping of the major outer-membrane protein of serovar F (F/MOMP) of Chlamydia trachomatis digested with V8 protease and chymotrypsin with seven monoclonal antibodies: lane 1, genus (not surface-exposed); lane 2, species; lane 3, species (except B and Ba); lane 4, B, Ba, D, F, 1_,1; lanes 5-7, F serovar. The position of undigested MOMP is indicated at 42.5K. The estimated masses of various fragments are indicated on the left side of each panel.

4 Surface Epitopes of Chlamydial MOMP S389 Figure 3. Epitope mapping of the major outer-membrane protein (MOMP) of Chlamydia trachomatis from different serovars with a species-specific monoclonal antibody. The positions of undigested MOMP and various fragments are indicated by arrows. different serovars. This possibility was tested by an epitope mapping analysis of the MOMPs from all the serovars with the species-specific monoclonal antibody. The reactivity patterns for each serovar were very similar (figure 3). For the V8 protease each MOMP gave a pattern of four different reactive fragments of nearly the same size in each serovar. Analysis of the fragments generated by chymotrypsin also indicated the conservation of certain fragments (28K and 16K). In addition, a 21K and a 10K fragment also appeared to be conserved, although the reactions with these fragments were very faint for some of the serovars seen in figure 3. When the MOMP digests of the B-complex serovars were probed with the B-complex-specific antibody, the patterns obtained were nearly identical as compared with those obtained with the species-specific antibody (data not shown). Thus, these data not only indicate structural conservation at the species and subspecies levels that correlates with established antigenic relationships, but also confirm the epitope clustering that was seen with the F/MOMP. On the basis of this information and studies with other serovar-specific antibodies, there appear to be at least two surface-exposed antigenic domains within all C. trachomatis MOMPs. One of these domains contains the species, B and C complex, and other subspecies epitopes. Another domain, which is less well characterized, appears to possess the serovar epitopes. Further studies are needed to determine if either of these or some other surface-exposed MOMP domain is involved in chlamydial pathogenesis, if any of these or other epitopes are capable of eliciting protective immunity or are involved in the generation of a delayed hypersensitivity response, and whether the whole molecule or one of these isolated domains should be considered for use as a potential vaccine. References 1. Caldwell HD, Kromhout J, Schachter J. Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis. Infect Immun 1981;3: Bavoil P, Ohlin A, Schachter J. Role of disulfide bonding in outer membrane structure and permeability in Chlamydia trachomatis. Infect Immun 1984;44: Hatch TP, Miceli M, Sublett JE. Synthesis of disulfide-bonded outer membrane proteins during the developmental cycle of Chlamydia psittaci and Chlamydia trachomatis. J Bacteriol 1986;165: Newhall WJ V, Jones RB. Disulfide-linked oligomers of the

5 S390 Newhall major outer membrane protein of chlamydiae. J Bacteriol 1983;154: Lucero ME, Kuo C-C. Neutralization of Chlamydia trachomatis cell culture infection by serovar-specific monoclonal antibodies. Infect Immun 1985;50: Caldwell HD, Perry LJ. Neutralization of Chlamydia trachomatis infectivity with antibodies to the major outer membrane protein. Infect Immun 1982;38: Peeling R, MacLean IW, Brunham RC. In vitro neutralization of Chlamydia trachomatis with monoclonal antibody to an epitope on the major outer membrane protein. Infect Immun 1984;46:: Caldwell HD, Schachter J. Antigenic analysis of the major outer membrane protein of Chlamydia spp. Infect Immun 1982;35: Newhall WJ V, Terho P, Wilde CE III, Batteiger BE, Jones RB. Serovar determination of Chlamydia trachomatis isolates by using type-specific monoclonal antibodies. J Clin Microbiol 1986;23: Stephens RS, Tam MR, Kuo C-C, Nowinski RC. Monoclonal antibodies to Chlamydia trachomatis: antibody specificities and antigen characterization. J Immunol 1982;128: Zhang Y-X, Stewart S, Joseph T, Taylor HR, Caldwell HD. Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of Chlamydia trachomatis. J Immunol 1987;138: Newhall WJ, Basinski MB. Purification and structural characterization of chlamydial outer membrane proteins. In: Oriel D, Ridgway G, Schachter J, Taylor-Robinson D, Ward M, eds. Chlamydial infections. Cambridge: Cambridge University Press, 1986: Batteiger BE, Newhall WJ, Terho P, Wilde CE III, Jones RB. Antigenic analysis of the major outer membrane protein of Chlamydia trachomatis with murine monoclonal antibodies. Infect Immun 1986;53: Cleveland DW, Fischer SG, Kirschner MW, Laemmli UK. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem 1977; 252: Caldwell HD, Judd RC. Structural analysis of chlamydial major outer membrane proteins. Infect Immun 1982;38:960-8