Identification and characterization of T helper cell epitopes of the major outer membrane protein of Chlamydia trachomatis.

Chlamydia trachomatis serovars A, B, and C are the causative agents of trachoma, the world's leading cause of preventable blindness. Immunoprophylaxis is a possible approach to control trachoma. The chlamydial major outer membrane protein (MOMP) is thought to play an important role in the development of protective immunity against chlamydial infection, and is therefore considered to be a promising candidate antigen in the development of a trachoma vaccine. Much effort has been focused on the molecular characterization of B cell sites of the MOMP that elicit neutralizing antibodies. Neutralizing sites have been identified as linear epitopes that reside within variable domains (VDs) of the protein whose primary sequences vary among different serovars. No information exists on MOMP T helper (Th) cell antigenic determinants, which are likely critical components for the development of a successful chlamydial vaccine. We used overlapping synthetic peptides (25 mers) representing the entire primary sequence of serovar A MOMP in T cell proliferation assays to identify T cell antigenic determinants of this molecule. Eight synthetic peptides (A-2, A-3, A-7, A-8, A-11, A-22, A-23, and A-24) stimulated proliferative responses of splenic T cells isolated from MOMP-immunized A/J mice. To ascertain if these peptides functioned as Th cell antigens, we determined their ability to prime A/J mice in vivo to produce an anamnestic IgG response specific to the MOMP. Mice primed with synthetic peptides A-8 (106-130) or A-23 (331-355) produced IgG antibodies reactive with the native MOMP and with the synthetic peptides corresponding to surface-accessible serovar-specific epitopes located in VD I and serogroup-specific epitopes located in VD IV of the protein. We synthesized the A-8 and A-23 peptides with the VD I sequence as colinear chimeric peptides. Immunization of mice with the T/B cell peptides produced high titered antibodies against the VD I sequence, and these antibodies reacted with the native MOMP and intact chlamydiae. The MOMP sequences containing these Th cell epitopes are conserved among the MOMP genes of different C. trachomatis serovars, indicating that they are common Th cell antigenic sites. Thus, the Th cell epitopes contained within these peptides, in combination with different trachoma serovar-specific B cell neutralizing determinants, may be useful in the development of a synthetic or recombinant trivalent trachoma vaccine.

Chlamydia trachomatis-host cell interactions: role of the chlamydial major outer membrane protein as an adhesin.

The major outer membrane protein (MOMP) of Chlamydia trachomatis is characterized by four symmetrically spaced variable domains (VDs I to IV) whose sequences vary among serotypes. The surface-exposed portions of these VDs contain contiguous sequences that are both serotyping determinants and in vivo target sites for neutralizing antibodies. Previous studies using surface proteolysis of C. trachomatis B implicated VDs II and IV of the MOMP of this serotype in the attachment of chlamydiae to host cells. In this study, we used monoclonal antibodies (MAbs) specific to antigenic determinants located in VDs II and IV of the MOMP of serotype B to further investigate the role of the MOMP in the attachment of chlamydiae to host cells. MABs specific to serotype- and subspecies-specific epitopes located in exposed VDs II and IV, respectively, neutralized chlamydial infectivity for hamster kidney cells by blocking chlamydial attachment. We radioiodinated these MAbs and used them to determine the number and topology of the surface-exposed VDs II and IV epitopes on chlamydial elementary bodies. VDs II and IV each comprised approximately 2.86 x 10(4) negatively charged sites and were in proximity on the chlamydial cell surface. These studies suggest that the MAbs blocked chlamydial attachment by inhibiting electrostatic interactions with host cells. We examined the effects of thermal inactivation on both chlamydial attachment and conformation of the MOMP. Heat-inactivated chlamydiae failed to attach to host cells and exhibited a conformational change in an inaccessible invariant hydrophobic nonapeptide sequence located within VD IV of the MOMPs of C. trachomatis serotypes. These findings suggest that in addition to electrostatic interactions, a common hydrophobic component of the MOMP also contributes to the binding of chlamydiae to host cells. Thus, we propose that the MOMP functions as a chlamydial adhesin by promoting nonspecific (electrostatic and hydrophobic) interactions with host cells. Surface-accessible negatively charged VDs appear to be important in electrostatic binding, while the invariant region of VD IV may provide a subsurface hydrophobic depression which further promotes binding of chlamydiae to host cells through hydrophobic interactions.

A recombinant Rickettsia conorii vaccine protects guinea pigs from experimental boutonneuse fever and Rocky Mountain spotted fever.

There are no vaccines against boutonneuse fever and Rocky Mountain spotted fever. Previous studies have identified a Rickettsia rickettsii surface protein as a vaccine candidate and shown that an antigenically related protein is present in R. conorii, which causes boutonneuse fever. The gene encoding the R. rickettsii protein has been cloned and expressed in Escherichia coli. We confirmed by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis of rickettsial lysates followed by immunoblotting with a monoclonal antibody raised against the R. rickettsii protein that an analogous protein exists in R. conorii. Although these proteins were previously called 155-kilodalton (kDa) proteins, we found that their apparent molecular masses were 198 kDa for R. conorii Kenya tick typhus and 190 kDa for R. rickettsii R. Using the R. rickettsii gene probe, we cloned and expressed a 5.5-kilobase HindIII fragment from R. conorii Kenya tick typhus genomic DNA in E. coli JM107. The expressed recombinant product was recognized by a monospecific polyclonal rabbit antiserum prepared against the 198-kDa protein. Guinea pigs immunized with sonic lysates of the E. coli strain expressing the recombinant gene product developed antibodies recognizing R. conorii when tested by a microimmunofluorescence antibody assay. Upon immunoblotting of rickettsial lysates, those antisera specifically recognized the 198-kDa R. conorii protein and its 190-kDa analog in R. rickettsii. Guinea pigs immunized with sonic lysates of the recombinant E. coli expressing the 198-kDa protein were protected from experimental infections with the homologous R. conorii strain and partially protected from experimental infections with a strain of the heterologous species R. rickettsii. These findings show that the 198-kDa R. conorii protein is a candidate for a vaccine against boutonneuse fever.

Nucleotide and deduced amino acid sequences for the four variable domains of the major outer membrane proteins of the 15 Chlamydia trachomatis serovars.

The amino acid sequences of major outer membrane proteins (MOMPs) from Chlamydia trachomatis serovars A, B, C, L1, and L2 are predominantly conserved but have four variable domains (VDs) in which major neutralizing and serotyping antigenic determinants are located. Because these MOMP VDs are primarily responsible for antigenic differences between serovars and are associated with important immunological and biological properties, we undertook studies focused on defining these sequences within the MOMPs of all 15 C. trachomatis serovars. We used oligonucleotide primer extension sequencing of MOMP mRNA to determine the nucleotide and deduced amino acid sequences of the four MOMP VDs of the 15 C. trachomatis serovars. Comparative amino acid sequence homologies of all four domains separated the serovars into three groups: group 1, serovars B, Ba, D, E, L1, and L2; group 2, serovars G and F; and group 3, serovars A, C, H, I, J, K, and L3. Hydrophilicity and charge values for each domain were determined. The MOMP VDs of given serovars with the greatest total hydrophilicity and charge values were found to be the location of antigenic determinants recognized by MOMP-specific monoclonal antibodies. These findings should be useful for predicting MOMP antigenic determinants and testing the antigenic properties of these VDs by using synthetic peptides corresponding to each MOMP VD. The potential usefulness of the VD sequence information is discussed in relation to the development of defined synthetic peptides and oligonucleotides that may be used to develop new serological and diagnostic assays for C. trachomatis infections.

The Amadori product on protein: structure and reactions.

The Amadori Rearrangement Product is the first stable adduct formed during glycation (nonenzymatic glycosylation) of protein. This review deals with the structure of the Amadori adduct on protein, factors affecting the kinetics and specificity of glycation of protein, measurements of the extent of glycation of proteins in vivo, and the possible significance of glycation itself, versus post-glycation reactions, in the development of pathophysiology in diabetes.

Differential effect of trypsin on infectivity of Chlamydia trachomatis: loss of infectivity requires cleavage of major outer membrane protein variable domains II and IV.

The initial interaction of chlamydiae with host cells is not well understood. Chlamydial cell surface components that function in attachment are key virulence factors, and their identification is critical for understanding the pathogenic strategies of this very successful parasite. We used trypsin proteolysis of chlamydiae to define surface components that function in chlamydia-host cell interactions. We found that trypsin had a differential effect on the infectivity of Chlamydia trachomatis serovars B and L2 for HeLa 229 cells. Trypsin treatment resulted in a significant loss of attachment and infectivity of serovar B but had no effect on the infectivity of serovar L2. Fluorograms of chlamydiae metabolically labeled with 14C-amino acids and treated with trypsin showed that the major outer membrane protein (MOMP) of both serovars was cleaved. Evidence for two trypsin cleavage sites was found for the serovar B MOMP. One cleavage site was located between lysine 145 and valine 146 in variable domain (VD) II of the protein. The second site was located between lysine 309 and threonine 310 in VD IV. In contrast, the serovar L2 MOMP was cleaved only at lysine 309 in VD IV. These results suggest a functional role for MOMP in chlamydial attachment and implicate VDs II and IV of MOMP in this interaction.

The low-molecular-mass, cysteine-rich outer membrane protein of Chlamydia trachomatis possesses both biovar- and species-specific epitopes.

We isolated, by hydroxylapatite high-performance liquid chromatography, 14- and 15-kilodalton (kDa), cysteine-rich outer membrane proteins from Chlamydia trachomatis TW-5/OT (serovar B) and LGV-434 (serovar L2), respectively. Monoclonal antibodies (MAbs) were generated against the purified proteins, and their specificities were determined by immunoblotting. MAb B-14k recognized an epitope located on the 14-kDa cysteine-rich protein of the TW-5/OT strain and was immunoreactive with a comigrating 14-kDa protein that was common to all trachoma biovar strains, but it did not react with the 15-kDa, cysteine-rich protein of LGV biovar strains. In contrast, MAb L2-15k, which recognized an epitope located on the 15-kDa protein of the LGV-434 strain, reacted with the 15- and 14-kDa, cysteine-rich proteins of both LGV and trachoma biovar strains, but did not react with related proteins of two Chlamydia psittaci strains. Thus, the low-molecular-mass, cysteine-rich outer membrane proteins of C. trachomatis possess antigenic determinants that are both biovar and species specific. Neither MAbB-14k nor MAb L2-15k was reactive by dot-blot assay when viable chlamydiae were used as test antigens, indicating that the cysteine-rich proteins are not accessible to antibody on the native chlamydial cell surface.

Chlamydial hemagglutinin identified as lipopolysaccharide.

Chlamydial lipopolysaccharide (LPS) agglutinated mouse and rabbit erythrocytes but not human, guinea pig, or pronghorn antelope erythrocytes. Hemagglutination was not specific for Chlamydia spp., as rough LPSs from Coxiella burnetii and Escherichia coli also agglutinated erythrocytes from the same animal species. Nonagglutinated and agglutinated erythrocytes bound equivalent amounts of LPS, indicating that hemagglutination was not due to a specific interaction of chlamydial LPS with erythrocytes. Thus, hemagglutination by chlamydial LPS is not mediated by specific receptor-ligand interactions but is a property of the altered surface of the LPS-coated erythrocytes.

Effect of phosphate on the kinetics and specificity of glycation of protein.

The glycation (nonenzymatic glycosylation) of several proteins was studied in various buffers in order to assess the effects of buffering ions on the kinetics and specificity of glycation of protein. Incubation of RNase with glucose in phosphate buffer resulted in inactivation of the enzyme because of preferential modification of lysine residues in or near the active site. In contrast, in the cationic buffers, 3-(N-morpholino)propane-sulfonic acid and 3-(N-tris(hydroxymethyl)methyl-amino)-2-hydroxypropanesulfonic acid, the kinetics of glycation of RNase were decreased 2- to 3-fold, there was a decrease in glycation of active site versus peripheral lysines, and the enzyme was resistant to inactivation by glucose. The extent of Schiff base formation on RNAse was comparable in the three buffers, suggesting that phosphate, bound in the active site of RNase, catalyzed the Amadori rearrangement at active site lysines, leading to the enhanced rate of inactivation of the enzyme. Phosphate catalysis of glycation was concentration-dependent and could be mimicked by arsenate. Phosphate also stimulated the rate of glycation of other proteins, such as lysozyme, cytochrome c, albumin, and hemoglobin. As with RNase, phosphate affected the specificity of glycation of hemoglobin, resulting in increased glycation of amino-terminal valine versus intrachain lysine residues. 2,3-Diphosphoglycerate exerted similar effects on the glycation of hemoglobin, suggesting that inorganic and organic phosphates may play an important role in determining the kinetics and specificity of glycation of hemoglobin in the red cell. Overall, these studies establish that buffering ions or ligands can exert significant effects on the kinetics and specificity of glycation of proteins.

Ocular delayed hypersensitivity: a pathogenetic mechanism of chlamydial-conjunctivitis in guinea pigs.

We used a naturally occurring, Chlamydia psittaci-caused eye disease in guinea pigs, guinea pig inclusion conjunctivitis, as an animal model to understand both the immune response and the pathogenesis of chlamydial eye infections. When instilled into the conjunctival sac of guinea pigs that had been previously infected and were immune, viable chlamydiae or a Triton X-100-soluble extract of them produced a short-lived (12-48 hr) eye disease indistinguishable clinically and histologically from that observed during primary chlamydial eye infection. The clinical and histologic findings were consistent with those of ocular delayed hypersensitivity. Ocular delayed hypersensitivity was induced by primary chlamydial infection at mucosal sites other than conjunctival, such as vaginal and intestinal. Preliminary characterization of the hypersensitivity allergen shows that it is heat sensitive and common to the genus Chlamydia. The allergen is apparently not surface-exposed on chlamydiae and requires viable but not replicating organisms for activity. Our observation should be useful in understanding pathogenetic mechanisms of Chlamydia trachomatis-caused infections in humans, in particular those that produce chronic inflammatory diseases, such as blinding trachoma and urogenital diseases.

Glycation of amino groups in protein. Studies on the specificity of modification of RNase by glucose.

Ribonuclease A has been used as a model protein for studying the specificity of glycation of amino groups in protein under physiological conditions (phosphate buffer, pH 7.4, 37 degrees C). Incubation of RNase with glucose led to an enhanced rate of inactivation of the enzyme relative to the rate of modification of lysine residues, suggesting preferential modification of active site lysine residues. Sites of glycation of RNase were identified by amino acid analysis of tryptic peptides isolated by reverse-phase high pressure liquid chromatography and phenylboronate affinity chromatography. Schiff base adducts were trapped with Na-BH3CN and the alpha-amino group of Lys-1 was identified as the primary site (80-90%) of initial Schiff base formation on RNase. In contrast, Lys-41 and Lys-7 in the active site accounted for about 38 and 29%, respectively, of ketoamine adducts formed via the Amadori rearrangement. Other sites reactive in ketoamine formation included N alpha-Lys-1 (15%), N epsilon-Lys-1 (9%), and Lys-37 (9%) which are adjacent to acidic amino acids. The remaining six lysine residues in RNase, which are located on the surface of the protein, were relatively inactive in forming either the Schiff base or Amadori adduct. Both the equilibrium Schiff base concentration and the rate of the Amadori rearrangement at each site were found to be important in determining the specificity of glycation of RNase.