Extracellular vesicles from diverse fungal pathogens induce species-specific and endocytosis-dependent immunomodulation.

Microbial pathogens generate extracellular vesicles (EVs) for intercellular communication and quorum sensing. Microbial EVs also induce inflammatory pathways within host innate immune cells. We previously demonstrated that EVs secreted by trigger type I interferon signaling in host cells specifically via the cGAS-STING innate immune signaling pathway.

Protocol for iterative enrichment of integrated sgRNAs via derivative CRISPR-Cas9 libraries from genomic DNA of sorted fixed cells.

Here, we present a protocol for iterative enrichment of integrated single guide RNA (sgRNA) via derivative CRISPR-Cas9 from genomic DNA (gDNA) of phenotypically sorted fixed cells. We describe steps for high-scale lentiviral production, genome-wide screening, extracting gDNA from fixed cells, cloning of integrated sgRNAs, and high-scale transformation. This protocol introduces three key advantages: (1) applicability to fixed cells, (2) bypassing epigenetic drift, and (3) pause points lowering the contamination risk.

A genome-wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6α.

Activating transcription factor 6 (ATF6) is one of three endoplasmic reticulum (ER) transmembrane stress sensors that mediate the unfolded protein response (UPR). Despite its crucial role in long-term ER stress adaptation, regulation of ATF6 alpha (α) signalling remains poorly understood, possibly because its activation involves ER-to-Golgi and nuclear trafficking. Here, we generated an ATF6α/Inositol-requiring kinase 1 (IRE1) dual UPR reporter CHO-K1 cell line and performed an unbiased genome-wide CRISPR/Cas9 mutagenesis screen to systematically profile genetic factors that specifically contribute to ATF6α signalling in the presence and absence of ER stress.

Malassezia responds to environmental pH signals through the conserved Rim/Pal pathway.

During mammalian colonization and infection, microorganisms must be able to rapidly sense and adapt to changing environmental conditions including alterations in extracellular pH. The fungus-specific Rim/Pal signaling pathway is one process that supports microbial adaptation to alkaline pH. This cascading series of interacting proteins terminates in the proteolytic activation of the highly conserved Rim101/PacC protein, a transcription factor that mediates microbial responses that favor survival in neutral/alkaline pH growth conditions, including many mammalian tissues.