Abstract
Toxoplasma gondii (T. gondii) has evolved into a highly successful intracellular parasite capable of infecting nearly all warm-blooded animals. Its ability to replicate within host cells depends on the formation of a specialized compartment known as the parasitophorous vacuole (PV) and a diverse array of secreted effectors that protect the parasite from, and even modulate, host immune defenses. On the host side, interferon-gamma (IFNγ)-induced immunity restricts T. gondii replication and eventually clears parasites through the coordinated activity of interferon-inducible GTPases (IIGs) and additional antimicrobial pathways. The primary countermeasure of T. gondii against this response is the secretion of polymorphic effector proteins that vary across T. gondii strains. Rhoptry protein 5 (ROP5) expression is a key determinant of virulence between strains, depending on the allelic type expressed. In hypervirulent strains, ROP5 functions in coordination with additional rhoptry proteins (ROP17, -18) to strip IIGs off the PV. In contrast, the mechanism by which the hypo-virulent ROP5 allelic type contributes to parasite survival is less well understood.
In this work, I investigated how reactive nitrogen species (RNS) generated by inducible nitric oxide synthase (iNOS) contribute to host control of T. gondii. In early data, we demonstrated that iNOS is enriched around IIG-targeted PVs, that effective parasite restriction requires both RNS and IIGs, that IIG-mediated PV collapse depends on RNS, and that RNS-dependent damage occurs heterogeneously across PVs within the same host cell. Next, using biochemical enrichment and liquid chromatography mass spectrometry, I demonstrate that ROP5 undergoes S-nitrosylation during infection. Using both in vivo and in vitro models, we further demonstrate that ROP5 confers parasite survival in an iNOS-dependent manner, supporting a role for host-derived RNS in disabling parasite virulence during infection. Differential centrifugation and image-based analysis further show RNS lead to the loss of ROP5 association with the PV membrane. These findings support a model in which host-derived RNS nitrosylate parasite effector proteins and disable ROP5-mediated immune evasion mechanisms. Future studies will identify if nitrosylation of a specific cysteine on ROP5 leads to these identified phenotypes. Additionally, the effects of RNS-induced cytosolic ROP5 on host cell function should be explored and may provide insights into the unknown, IIG-independent ROP5 effector functions in hypo-virulent ROP5 allelic strains.
In conclusion, this thesis presents new insight into how iNOS contributes to the control of T. gondii. Importantly, it identifies a previously unexplored relationship between ROP5 expression and the susceptibility of T. gondii to iNOS.
