Forward Genetics Screens Using Macrophages to Identify <em>Toxoplasma gondii </em>Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

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.

Macrophages facilitate the excystation and differentiation of Toxoplasma gondii sporozoites into tachyzoites following oocyst internalisation

“…The capacity of naïve macrophages to ingest large particles (10–20 μm in diameter) 23 besides their inability to eliminate the sporozoite and tachyzoite forms of T. gondii 11 13 14 24 25 prompted us to investigate whether the macrophages were able to internalise T. gondii oocysts, open their wall, hence allowing the sporozoites to excyst prior to their differentiation into tachyzoites. To address this question, we investigated the direct interactions between T. gondii oocysts and RAW murine macrophages at different time points by using single cell and parasite micropipette micromanipulations and a combination of real-time and time-point imaging techniques.…”

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in <em>Toxoplasma gondii</em>

“…To find the causal gene, mutants can either be complemented with cosmid libraries 44 or genome resequencing methods can be used to find the causal mutation 45 . For more on this, see two JoVE articles by Coleman et al and Walwyn et al that outline these approaches 46 47 .…”

A Comparison of Transcriptional Diversity of Swine Macrophages Infected With TgHB1 Strain of Toxoplasma gondii Isolated in China

“…Toxoplasma gondii replicating in immune cells, such as chicken macrophages and dendritic cells, can serve as a potential source of tachyzoites (Malkwitz et al, 2013 ; Quéré et al, 2013 ). Macrophages can ingest the parasite but, in some circumstances, fail to eliminate the sporozoites and tachyzoites (Dubey et al, 1997 , 1998 ; Speer and Dubey, 1998 ; Zhao et al, 2014 ; Walwyn et al, 2015 ), but mouse RAW macrophages are able to facilitate the excystation and differentiation of sporozoites into tachyzoites following oocyst internalization (Freppel et al, 2016 ). On the other hand, macrophages can also help fight T. gondii infection through a purinergic signaling pathway (Petit-Jentreau et al, 2017 ).…”