Regulation of the Complement System by Toxoplasma gondii Surface Coat Proteins SRS25, SRS29C, and SRS57

Abstract

Very little is known about how the protozoan parasite Toxoplasma gondii overcomes the complement system to establish infection. In this dissertation, I found significant differences in C3b deposition between T. gondii Type I and Type II strains. Taking both forward and reverse genetic approaches, I identified three Toxoplasma surface proteins that regulate complement deposition: SRS29C and SRS57 were identified as C3b acceptors, whereas SRS25 limited the level of C3b on the parasite surface. Serum resistance was achieved by the recruitment of host regulator proteins C4b-binding protein (C4BP) and Factor H (FH), facilitated cooperatively by SRS29C and SRS25. SRS57 was also shown to bind FH. Rapid death of Δsrs29CΔsrs25 DKO parasites upon exposure to human serum established that both FH and C4BP are essential for serum resistance. FH conferred a majority of parasite protection by suppressing the alternative pathway amplification loop to abrogate C5b-9 formation and susceptibility to non-immune human serum (NHS).

Host sulfated proteoglycans (SPGs) are “self” factors that stabilize FH on host cells to confer protection against complement attack. We hypothesized that an SRS lectin-specific activity for SPGs catalyzes FH recruitment to the parasite surface. The three-dimensional structure of SRS29C alongside biochemical analyses established that SRS29C binds SPGs whereas SRS57 does not, indicating that T. gondii employs both SPG-dependent and -independent FH recruitment mechanisms. We engineered a K16/17S mutation in the basic groove of the SRS29C homodimer that facilitates SPG binding and showed that the mutant failed to bind either SPGs or FH. Transgenic overexpression of SRS29C in Type I strains enhanced FH recruitment and protected mice from lethal infection by altering inflammatory cytokine production, protecting mice from the cytokine storm induced by Type I strains. Infection with the K16/17S mutant restored virulence, indicating that SPG-dependent FH recruitment appeared necessary to alter induction of the cytokine storm. Type II srs29C parasites were more virulent and surviving mice exhibited greater inflammatory infiltrate in the brain. To establish whether C3 was responsible for these phenotypes, studies using C3-/- mice showed that SRS29C and SRS57 controlled T. gondii virulence in a C3-dependent manner. This work has provided significant mechanistic insight into complement regulation by T. gondii and established the first biological role for an SRS protein expressed on the surface of Toxoplasma.