Forward genetics screens using macrophages to identify toxoplasma gondii genes important for resistance to IFN-γ-dependent cell autonomous immunity

Odaelys Walwyn, Sini Skariah, Brian Lynch, Nathaniel Kim, Yukari Ueda, Neal Vohora, Josh Choe, Dana G. Mordue

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependentinnate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in naïve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

Original languageEnglish
Article numbere52556
JournalJournal of Visualized Experiments
Volume2015
Issue number97
DOIs
Publication statusPublished - 12 Mar 2015
Externally publishedYes

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Keywords

  • Forward genetics
  • Immune evasion
  • Immunology
  • Infectious disease
  • Innate immunity
  • Intracellular pathogen
  • Issue 97
  • Macrophages
  • Parasite
  • Toxoplasma

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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