Fig 4. GRA55 is a novel GRA which is not important for in vitro growth or short-term virulence.
(A) Complementation strategy of GRA55 into the UPRT locus. The complemented gene contains a C-terminal 3xHA epitope tag and is driven by the native promoter. (B) IFA of GRA55-HA, Δgra55, and GRA55c parasites. Rabbit anti-HA shows strong staining in the vacuole of GRA55-HA and GRA55c parasites, colocalizing with GRA14. HA signal is absent in Δgra55 parasites. Scale bar: 5 μm. (C) Western blot demonstrating GRA55-HA migrating at the predicted size of 35.2 kDa (the size of GRA55-HA without the predicted signal peptide) and showing similar expression levels to the endogenously tagged strain. As expected, there is no HA signal in the Δgra55 strain. RON5C is used as a loading control [50]. (D) Quantitation of plaques formed by GRA55-HA, Δgra55, and GRA55c parasites. Results are shown as box-whisker plot with the middle line representing the median, the bottom and top of box representing the 25th and 75th percentile, and whiskers corresponding to smallest and largest plaques. (E, F, G) C57BL/6 female mice were infected with indicated doses of GRA55HA, Δgra55, and GRA55c parasites and Kaplan-Meier survival curves were generated. There was no statistical difference between the virulence of Δgra55 (F) vs. GRA55-HA (E) or GRA55c (G) parasites. (H) CBA/J mice were infected with 250 parasites each of the indicated strain. Mice were euthanized and mouse brains were examined by IFA after 30 days infection to enumerate T. gondii cyst burden. Δgra55 caused 10-fold lower burden of cysts when compared to GRA55-HA infection and GRA55c infection (p < 0.05 by two-tailed T-test). (I) Numerical counts of cysts per mouse brain (* = mouse dead prior to end of experiment).