FIGURE 4.

Mutations within the ROP18 basic pocket reveal biochemical functions of ROP18 in addition to its kinase activity that are required for virulence. A, representative blot showing relative levels of HA-tagged ROP18 constructs in parasite lysates as determined by probing with a rat anti-HA antibody. Toxoplasma Sag1 was used as a loading control. B, quantitation of the Western blot data averaged from two biological replicates, with HA-tagged ROP18 levels normalized to the corresponding Sag1 levels. The B5.2 clone was not used in our analyses due to its exceptionally high HA-tagged ROP18 expression level relative to the other WT-expressing clones. C, survival of C57BL/6 mice infected intraperitoneal with type III parasites, type III parasites complemented with wild-type ROP18, or type III parasites complemented with the indicated mutant ROP18 through day 30 after injection. D, mouse survival analysis as determined in C showing no significant differences in the outcome after including the B5.2 high level WT ROP18 expressing clone. E and F, in vivo growth of type III parasites complemented with wild-type ROP18 or with the indicated ROP18 mutants, 5 days post-injection, as measured by luciferase imaging. E, one representative mouse infected with each parasite strain. F, average total flux of all infected mice for each parasite strain. Error bars show S.E.