| Summary: | Chiamydia trachomatis is an obligate intracellular human pathogen responsible for
important diseases, including trachoma, the leading cause of preventable blindness,
infecting more than 500 million people worldwide. It is also the etiologic agent of
a sexually transmitted disease causing serious sequelae such as salpingitis, leading to
ectopic pregnancy or infertility.
Chiamydial infectivity of tissue culture cells can be neutralized by anti-chiamydial
antibody, however, the presence of complement with antibody results in maximal
neutralization. This thesis investigated the interaction of complement and anti
chiamydial antibody, and sought to provide a mechanism for the enhancement effect
in complement-dependent neutralization of chiamydial infectivity. Three approaches
were used to study this problem: 1) flow cytometry was optimized for detection of
purified chiamydial elementary bodies, and in situ binding experiments were
performed; 2) outer membrane protein-complement C3b complexes were extracted
and immunoblotted in order to study in situ binding qualitatively; and 3) in vitro
neutralization assays were used to determine the step in the complement cascade at
which antibody interacts with complement in mediating neutralization of infectivity.
The flow cytometry experiments tested the hypothesis that antibody affects the
quantity of C3b bound to the surface of whole elementary bodies in situ. The results
indicated that antibody did not augment either the rate or magnitude of C3b binding, and that complement was fixed predominantly via the alternative pathway.
Immunoblotting of outer membrane protein-C3b complexes tested the hypothesis
that antibody was determining the specific C3 target proteins on the chiamydial cell
surface. However, the results showed that the major outer membrane protein was
the primary C3b target protein in either the presence or absence of antibody.
Immunoblotting experiments were repeated with outer membrane protein-C3b
complexes treated with hydroxylamine. C3b appeared to be bound by hydroxyl
esters, not amino esters, to the outer membrane proteins, and this was unchanged by
antibody.
The final set of experiments utilized in vitro neutralization assays. The results
demonstrated that antibody must be present before the formation of C5 convertase,
and that if antibody was added at later stages of activation of the complement
cascade, neutralization did not occur. The data also showed that neutralization
occurred via activation of the alternative complement pathway, and infectivity was
not neutralized when the classical pathway alone was isolated. Interestingly,
neutralization occurred, although to a lesser degree, when the terminal complement
components C7 and C8 were missing, suggesting that terminal components are not
essential for neutralization. Flow cytometry binding experiments were repeated to
measure the effect of anti-chlamydial antibody on in situ binding of terminal
components C9 and C5b-9 neoantigen. The results indicated that the presence of antibody significantly increased C9 and, to a lesser extent, C5b-9 neoantigen binding.
In conclusion, the experiments in this thesis demonstrated that antibody mediated
complement-dependent neutralization of C. trachomatis serovar L2 at the stage of
alternative pathway C5 convertase formation. Anti-chlamydial antibody probably
configures the C3b molecules in specific locations on the major outer membrane
protein; however, the binding experiments were unable to detect any augmentation
of C3b binding by antibody. Neutralization may occur as a result of the covalently
bound complement complex on the major outer membrane protein inhibiting
reorganization of the EB into an RB. The role of terminal complement components
in neutralization is unclear. These data showed an increase in terminal components
bound in the presence of antibody, yet neutralization occurred when C7 and C8 were
excluded. It is likely that there are several mechanisms of complement-dependent
neutralization, some requiring the terminal components, and others not.
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