Biology subjects

Koshy, A. A.

Publications and source records attributed to Koshy, A. A..

5 recordsLinked to original sources

bioRxiv2022

ROP16-mediated activation of STAT6 facilitates encystment of type III Toxoplasma gondii in neurons

Toxoplasma gondii establishes a long-lived latent infection in the central nervous system (CNS) of its hosts. Reactivation in immunocompromised individuals can lead to life threatening disease. Latent infection is driven by the ability of the parasite to convert from the acute-stage tachyzoite to the latent-stage bradyzoite which resides in long-lived intracellular cysts. While much work has focused on the parasitic factors that drive cyst development, the host factors that influence encystment are not well defined. Here we show that a polymorphic secreted parasite kinase (ROP16), that phosphorylates host cell proteins, mediates efficient encystment of T. gondii in stress-induced models of encystment and primary neuronal cell cultures (PNCs) in a strain-specific manner. Using short-hairpin RNA (shRNA) knockdowns in human foreskin fibroblasts (HFFs) and PNCs from transgenic mice, we determined that ROP16s cyst enhancing abilities are mediated by phosphorylation of the host cell transcription factor STAT6. To test the role of STAT6 in vivo, we infected STAT6KO mice, finding that, compared to infected wild-type mice, infected STAT6KO mice have a decrease in cyst burden, but not overall parasite burden or dissemination to the CNS. Finally, we found a similar ROP16-dependent encystment defect in human pluripotent stem cell-derived neurons. Together, these findings identify a host cell factor (STAT6) that T. gondii manipulates in a strain-specific manner to generate a favorable encystment environment.

microbiology↗

bioRxiv2022

Impact of secondary TCR engagement on the heterogeneity of pathogen-specific CD8+ T cell response during acute and chronic toxoplasmosis

Initial TCR engagement of CD8+ T cells results in T cell expansion, and these early events influence the generation of diverse effector and memory populations. During infection, some activated T cells re-encounter cognate antigen, but how these events influence local effector responses or formation of memory populations is unclear. To address this issue, OT-I T cells which express the Nur77-GFP reporter of TCR activation were paired with T. gondii that express OVA to assess the impact of TCR activation on CD8+ T cell responses. During acute infection, TCR stimulation in affected tissues correlated with parasite burden and was associated with markers of effector cells while Nur77-GFP- OT-I showed signs of effector memory potential. However, adoptive transfer of Nur77-GFP negative or positive OT-I from infected mice into naive recipients resulted in formation of similar memory populations. During the chronic stage of infection in the CNS, TCR activation was associated with large scale transcriptional changes and the acquisition of an effector T cell phenotype as well as the generation of a population of CD103+ CD69+ Trm like cells. However, while inhibition of parasite replication resulted in reduced effector responses it did not alter the Trm population. These data sets highlight the contribution of recent TCR activation on the phenotypic heterogeneity of the CD8+ T cell response but suggest that this process has a limited impact on memory populations at acute and chronic stages of infection. Author SummaryCD8+ T cells are important to control many acute and chronic infections, however the role that recent T cell receptor stimulation plays in the formation of ongoing T cell responses is unclear. Here, we utilize a genetic reporter of TCR stimulation and high parameter flow cytometry to characterize TCR-driven phenotypes of pathogen specific T cell responses to the parasite Toxoplasma gondii during acute and chronic infection in the periphery and central nervous system. This work demonstrates the importance of recent TCR stimulation in driving local effector CD8+ T cell responses in peripheral tissues during infection, as well as the plasticity of the formation of memory T cells. Additionally, we utilize static and live imaging to investigate how Toxoplasma gondii life cycle impacts the ability to present antigen to CD8+ T cells. These studies aid in our understanding of how effector and memory CD8+ T cell responses are generated and maintained during an infection.

immunology↗

bioRxiv2021

IFN-γ stimulated murine and human neurons mount anti-parasitic defenses against the intracellular parasite Toxoplasma gondii

Dogma holds that Toxoplasma gondii persists in neurons because neurons cannot clear intracellular parasites, even with IFN-{gamma} stimulation. As several recent studies questioned this idea, we used primary murine neuronal cultures from wild-type and transgenic mice in combination with IFN-{gamma} stimulation and parental and transgenic parasites to reassess IFN-{gamma} dependent neuronal clearance of intracellular parasites. We found that neurons respond to IFN-{gamma} and that a subset of neurons clear intracellular parasites via immunity regulated GTPases. Whole neuron reconstructions from mice infected with parasites that trigger neuron GFP expression only after full invasion revealed that [~]40% of these T. gondii-invaded neurons no longer harbor parasites. Finally, IFN-{gamma} stimulated human stem cell derived neurons showed a [~] 50% decrease in parasite infection rate when compared to unstimulated cultures. This work highlights the capability of human and murine neurons to mount cytokine-dependent anti-T. gondii defense mechanisms in vitro and in vivo.

neuroscience↗

bioRxiv2021

Toxoplasma gondii injected neurons localize to the cortex and striatum and have altered firing

Toxoplasma gondii is an intracellular parasite that causes a long-term latent infection of neurons. Using a custom MATLAB-based mapping program in combination with a mouse model that allows us to permanently mark neurons injected with parasite proteins, we found that Toxoplasma-injected neurons (TINs) are heterogeneously distributed in the brain, primarily localizing to the cortex followed by the striatum. Using immunofluorescence co-localization assays, we determined that cortical TINs are commonly (>50%) excitatory neurons (FoxP2+) and that striatal TINs are often (>65%) medium spiny neurons (MSNs) (FoxP2+). As MSNs have highly characterized electrophysiology, we used ex vivo slices from infected mice to perform single neuron patch-clamping on striatal TINs and neighboring uninfected MSNs (bystander MSNs). These studies demonstrated that TINs have highly abnormal electrophysiology, while the electrophysiology of bystander MSNs was akin to that of MSNs from uninfected mice. Collectively, these data offer new neuroanatomic and electrophysiologic insights into CNS toxoplasmosis.

microbiology↗

bioRxiv2020

Transcriptional profiling reveals T cells cluster around neurons injected with Toxoplasma gondii proteins

Toxoplasma gondiis tropism for and persistence in the CNS underlies the symptomatic disease Toxoplasma causes in humans. Our recent work has shown that neurons are the primary CNS cell with which Toxoplasma interacts and infects in vivo. This predilection for neurons suggests that Toxoplasmas persistence in the CNS depends specifically upon parasite manipulation of the host neurons. Yet, most work on Toxoplasma-host cell interactions has been done in vitro and in non-neuronal cells. We address this gap by utilizing our Toxoplasma-Cre system that allows permanent marking and tracking of neurons injected with parasite effector proteins in vivo. Using laser capture microdissection (LCM) and RNA-seq, we isolated and transcriptionally profiled Toxoplasma-injected neurons (TINs), Bystander neurons (nearby non-Toxoplasma injected neurons), and neurons from uninfected mice (controls). These profiles show that TINs 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 Toxoplasma transcripts were primarily found in the TINs 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.

bioinformatics↗