Mapping host-microbe transcriptional interactions by dual perturb-seq

Abstract

Intracellular pathogens and other endosymbionts reprogram host cell transcription to suppress immune responses and recalibrate biosynthetic pathways. This reprogramming is critical in determining the outcome of infection or colonisation. Here, we combine pooled CRISPR knockout screening with dual host–microbe single–cell RNA–sequencing to identify the molecular mediators of these transcriptional interactions, a method we term dual perturb–seq. Applying dual perturb–seq to the intracellular pathogenToxoplasma gondii, we are able to identify previously uncharacterised effector proteins and directly infer their function from the transcriptomic data. We show thatTgGRA59 contributes to the export of other effector proteins from the parasite into the host cell and identify a novel effector,TgSOS1, that is necessary for sustained host STAT6 signalling and thereby contributes to parasite immune evasion and persistence. Together, this work demonstrates a novel tool that can be broadly adapted to interrogate host–microbe transcriptional interactions and reveal mechanisms of infection and immune evasion.

mSphere of Influence: Systemic decoding gene function in Toxoplasma gondii pathogenesis—CRISPR screens and beyond

“…However, considering that a large portion of predicted proteins lacks a defined role in the biology of Toxoplasma , the need for a systemic analysis of all parasite protein-coding genes has emerged as an immediate need in the field. You can therefore imagine my excitement on reading the papers by Sidik et al ( 3 ), which shows the power of CRISPR/Cas9-mediated screens in unraveling the complex biology of Toxoplasma , and by Butterworth et al ( 4 ), which shows that combining CRISPR screens with single-cell RNA sequencing (RNA-seq) provides invaluable insights into Toxoplasma -induced host transcriptional modification.…”

“…In the paper by Butterworth et al ( 4 ), the Treeck lab applied a targeted CRISPR screen coupled with single-cell RNA-seq in parasite-infected cells to understand Toxoplasma -secreted effector-mediated host reprogramming at a systemic level. To successfully establish an infection, Toxoplasma utilizes its protein secreted from two unique organelles, known as rhoptry and dense granule, to alter the host gene expression, modulate the immune response, and mediate pathogenesis ( 7 ).…”