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ABSTRACT Toxoplasma gondii’s tropism for and persistence in the central nervous system (CNS) underlies the symptomatic disease that T. gondii causes in humans. Our recent work has shown that neurons are the primary CNS cell with which Toxoplasma interacts and which it infects in vivo. This predilection for neurons suggests that T. gondii’s persistence in the CNS depends specifically upon parasite manipulation of the host neurons. Yet, most work on T. gondii-host cell interactions has been done in vitro and in nonneuronal cells. We address this gap by utilizing our T. gondii-Cre system that allows permanent marking and tracking of neurons injected with parasite effector proteins in vivo. Using laser capture microdissection (LCM) and RNA sequencing using RNA-seq, we isolated and transcriptionally profiled T. gondii-injected neurons (TINs), Bystander neurons (nearby non-T. gondii-injected neurons), and neurons from uninfected mice (controls). These profiles show that TIN transcriptomes significantly differ from the transcriptomes of Bystander and control neurons and that much of this difference is driven by increased levels of transcripts from immune cells, especially CD8+ T cells and monocytes. These data suggest that when we used LCM to isolate neurons from infected mice, we also picked up fragments of CD8+ T cells and monocytes clustering in extreme proximity around TINs and, to a lesser extent, Bystander neurons. In addition, we found that T. gondii transcripts were primarily found in the TIN transcriptome, not in the Bystander transcriptome. Collectively, these data suggest that, contrary to common perception, neurons that directly interact with or harbor parasites can be recognized by CD8+ T cells. IMPORTANCE Like other persistent intracellular pathogens, Toxoplasma gondii, a protozoan parasite, has evolved to evade the immune system and establish a chronic infection in specific cells and organs, including neurons in the CNS. Understanding T. gondii’s persistence in neurons holds the potential to identify novel, curative drug targets. The work presented here offers new insights into the neuron-T. gondii interaction in vivo. By transcriptionally profiling neurons manipulated by T. gondii, we unexpectedly revealed that immune cells, and specifically CD8+ T cells, appear to cluster around these neurons, suggesting that CD8+ T cells specifically recognize parasite-manipulated neurons. Such a possibility supports evidence from other labs that questions the long-standing dogma that neurons are often persistently infected because they are not directly recognized by immune cells such as CD8+ T cells. Collectively, these data suggest we reconsider the broader role of neurons in the context of infection and neuroinflammation.

Francisella tularensis is the causative agent of tularemia. We have previously shown that infection with F. tularensis Live Vaccine Strain (LVS) induces macrophages to synthesize prostaglandin E2 (PGE2). Synthesis of PGE2 by F. tularensis infected macrophages results in decreased T cell proliferation in vitro and increased bacterial survival in vivo. Although we understand some of the biological consequences of F. tularensis induced PGE2 synthesis by macrophages, we do not understand the cellular pathways (neither host nor bacterial) that result in up-regulation of the PGE2 biosynthetic pathway in F. tularensis infected macrophages. We took a genetic approach to begin to understand the molecular mechanisms of bacterial induction of PGE2 synthesis from infected macrophages. To identify F. tularensis genes necessary for the induction of PGE2 in primary macrophages, we infected cells with individual mutants from the closely related strain Francisella tularensis subspecies novicida U112 (U112) two allele mutant library. Twenty genes were identified that when disrupted resulted in U112 mutant strains unable to induce the synthesis of PGE2 by infected macrophages. Fourteen of the genes identified are located within the Francisella pathogenicity island (FPI). Genes in the FPI are required for F. tularensis to escape from the phagosome and replicate in the cytosol, which might account for the failure of U112 with transposon insertions within the FPI to induce PGE2. This implies that U112 mutant strains that do not grow intracellularly would also not induce PGE2. We found that U112 clpB::Tn grows within macrophages yet fails to induce PGE2, while U112 pdpA::Tn does not grow yet does induce PGE2. We also found that U112 iglC::Tn neither grows nor induces PGE2. These findings indicate that there is dissociation between intracellular growth and the ability of F. tularensis to induce PGE2 synthesis. These mutants provide a critical entrée into the pathways used in the