Transcriptional Analysis of Murine Macrophages Infected with Different Toxoplasma Strains Identifies Novel Regulation of Host Signaling Pathways

Abstract

Most isolates of Toxoplasma from Europe and North America fall into one of three genetically distinct clonal lineages, the type I, II and III lineages. However, in South America these strains are rarely isolated and instead a great variety of other strains are found. T. gondii strains differ widely in a number of phenotypes in mice, such as virulence, persistence, oral infectivity, migratory capacity, induction of cytokine expression and modulation of host gene expression. The outcome of toxoplasmosis in patients is also variable and we hypothesize that, besides host and environmental factors, the genotype of the parasite strain plays a major role. The molecular basis for these differences in pathogenesis, especially in strains other than the clonal lineages, remains largely unexplored. Macrophages play an essential role in the early immune response against T. gondii and are also the cell type preferentially infected in vivo. To determine if non-canonical Toxoplasma strains have unique interactions with the host cell, we infected murine macrophages with 29 different Toxoplasma strains, representing global diversity, and used RNA-sequencing to determine host and parasite transcriptomes. We identified large differences between strains in the expression level of known parasite effectors and large chromosomal structural variation in some strains. We also identified novel strain-specifically regulated host pathways, including the regulation of the type I interferon response by some atypical strains. IFNβ production by infected cells was associated with parasite killing, independent of interferon gamma activation, and dependent on endosomal Toll-like receptors in macrophages and the cytoplasmic receptor retinoic acid-inducible gene 1 (RIG-I) in fibroblasts.

STAT2 Is a Pervasive Cytokine Regulator due to Its Inhibition of STAT1 in Multiple Signaling Pathways

“…T . gondii infection, moreover, does not elicit type 1 IFNs and their potential disease-exacerbating comorbidities [33]; a phenomenon observed in the course of many infections and an important consideration, particularly in the context of STAT2 deficiency and hence disrupted type 1 IFN signaling [34]. At first, WT- and STAT2-deficient mice were injected with IFN-γ or PBS (controls), before peritoneal macrophages were collected for ex vivo infection experiments with T .…”

Unveiling the pathogen behind the vacuole

“…In support of this hypothesis, it has been shown that T. gondii infection of fibroblasts pre-stimulated with IFNγ triggers a stronger type I IFN response than infection of untreated fibroblasts 35 . ISG-encoded GTPases are essential for controlling the infection both ex vivo 42,44 and in vivo, as mice lacking genes of the IRG family or genes encoding several GBPs are more susceptible to T. gondii infection than their wild-type counterparts 42,[45][46][47] .…”

“…1c) in macrophages infected with less virulent T. gondii type II strains but not in macrophages infected with virulent type I strains 60 . This protein is highly polymorphic, and differences in its amino acid sequence and its expression levels seem to explain the variations observed between distinct T. gondii strains concerning the activation of pro-inflammatory host genes 35 . GRA15 is secreted from dense granules and traffics to the host cytosol during the invasion and intracellular growth of PV-enclosed T. gondii parasites.…”

“…This pathogen not only triggers the NAIP-NLRC4 pathway via the T3SS 6,7 , but also activates the NOD2 pathway 22 , a GBPdependent signalling pathway (resulting in caspase 11 activation) 24 and GAL8-induced autophagy 68 . Similarly, not only is T. gondii recognized through the reduction in IRGMs on the PV and the detection of effectors in the cytosol, but this pathogen also triggers inflammasome activation, and some non-canonical T. gondii strains induce a type I IFN response by exposing dsRNA to RIG-I or endosomal TLRs 26,35,61,[64][65][66] . This emerging appreciation for the complexity of mechanisms involved in the detection of a given pathogen will probably also expand the complexity of the analyses required for their study, as it will be crucial to carefully discriminate between the effects of the different modes of innate immune activation.…”

“…Their detection could be mediated by the cell in two ways: first, by inducing a loss of vacuolar-membrane integrity and subsequent leakage of pathogen ligands into the cytosol, where they activate cytosolic receptors; or second, by regaining control over the PV, prompting either vacuole acidification and endolyso somal fusion or a cellular autophagic response against the pathogen, which in both cases leads to pathogen ligands being accessible to endosomal TLRs 34 . These recognition mechanisms might occur for two closely related apicomplexan parasites: T. gondii and N. caninum 35,36 .…”