Secretion of Rhoptry and Dense Granule Effector Proteins by Nonreplicating Toxoplasma gondii Uracil Auxotrophs Controls the Development of Antitumor Immunity

Nonreplicating type I uracil auxotrophic mutants of Toxoplasma gondii possess a potent ability to activate therapeutic immunity to established solid tumors by reversing immune suppression in the tumor microenvironment. Here we engineered targeted deletions of parasite secreted effector proteins using a genetically tractable Δku80 vaccine strain to show that the secretion of specific rhoptry (ROP) and dense granule (GRA) proteins by uracil auxotrophic mutants of T. gondii in conjunction with host cell invasion activates antitumor immunity through host responses involving CD8α+ dendritic cells, the IL-12/interferon-gamma (IFN-γ) TH1 axis, as well as CD4+ and CD8+ T cells. Deletion of parasitophorous vacuole membrane (PVM) associated proteins ROP5, ROP17, ROP18, ROP35 or ROP38, intravacuolar network associated dense granule proteins GRA2 or GRA12, and GRA24 which traffics past the PVM to the host cell nucleus severely abrogated the antitumor response. In contrast, deletion of other secreted effector molecules such as GRA15, GRA16, or ROP16 that manipulate host cell signaling and transcriptional pathways, or deletion of PVM associated ROP21 or GRA3 molecules did not affect the antitumor activity. Association of ROP18 with the PVM was found to be essential for the development of the antitumor responses. Surprisingly, the ROP18 kinase activity required for resistance to IFN-γ activated host innate immunity related GTPases and virulence was not essential for the antitumor response. These data show that PVM functions of parasite secreted effector molecules, including ROP18, manipulate host cell responses through ROP18 kinase virulence independent mechanisms to activate potent antitumor responses. Our results demonstrate that PVM associated rhoptry effector proteins secreted prior to host cell invasion and dense granule effector proteins localized to the intravacuolar network and host nucleus that are secreted after host cell invasion coordinately control the development of host immune responses that provide effective antitumor immunity against established ovarian cancer.
Author Summary Toxoplasma gondii extensively manipulates cellular signaling pathways and host immune responses through secreted effector proteins, yet the host rapidly establishes T cell immunity to control acute infection thereby permitting survival of the host as well as survival of the parasite in latent infection. Recently, vaccination of mice bearing highly aggressive ovarian cancer with a safe nonreplicating, noncyst forming, vaccine strain of T. gondii was shown to effectively reverse tumor associated immune suppression and activate potent antitumor immunity. Using a new genetically tractable Δku80 vaccine strain of T. gondii we deleted multiple parasite secreted effector molecules to explore parasite specific mechanisms associated with the development of potent antitumor immunity. Our results demonstrate that specialized effector proteins secreted by T. gondii both before and after host cell invasion trigger and coordinately control the development of a potent antitumor response. Consequently, tracking and understanding the host cell pathways manipulated by these T. gondii secreted effector proteins can reveal fundamental mechanisms controlling immunity to infection and can also identify relevant mammalian cell mechanisms as new targets for devising more effective therapies against highly aggressive solid tumors.