Universität zu Köln

Mehra, Chahat (2025). An organelle pathogen sensor reveals factors required for mediating trans-kingdom membrane contact sites. PhD thesis, Universität zu Köln.

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Abstract

Toxoplasma gondii (Toxoplasma) is a human parasite that establishes a life-long infection in one-third of the world’s population although prevalence can vary depending on the country. In Germany for example more than 50% of the population over age 50 are estimated to be seropositive for Toxoplasma. Thus, defining the mechanisms by which Toxoplasma engages with the host cells can lead to the development of better therapeutics. A common consequence of infection with Toxoplasma is the formation of trans-kingdom membrane contact sites (MCS) between the vacuole of Toxoplasma and host mitochondria and host endoplasmic reticulum (ER). Although the association of host ER with the vacuole was first described in the early 1970s neither of the molecular components that mediate this interaction have been identified thus far. Here, we investigated the molecular machinery that mediates the MCS between Toxoplasma and ER. To this end, we developed a split-GFP based sensor where GFP reconstitution indicates successful formation of MCS between the pathogen and host organelles. To validate our sensor for FACS based CRISPR-Cas9 screening, I first applied it to monitor the known mitochondria- Toxoplasma MCS. As expected, GFP reconstitution occurred at these contact sites but failed to do so in the absence of the Toxoplasma tether TgMAF1 or the host counterpart TOM70, confirming the sensors specificity. I then adapted our sensor to study host ER-Toxoplasma MCS and performed a Toxoplasma effector protein targeted loss-of-function CRISPR screen. I found that Toxoplasma rhoptry protein 1 (TgROP1) is required for mediating Toxoplasma-ER MCS. Interestingly, TgROP1 contains putative FFAT [(two phenylalanines (FF) in an acidic tract (AT)] motifs, that are regions known to interact with ER membrane proteins VAPA/VAPB (VAPs). Subsequent work identified VAPs as required to mediate MCS with the Toxoplasma vacuole and mutating the FFAT-binding domain of VAPs reduced this interaction. Our findings reveal that Toxoplasma exploits a mechanism like host proteins to establish MCS with host organelles. This work advances our current understanding of host pathogen MCS and lays the foundation for future studies investigating their functional consequences on infection outcomes.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Mehra, Chahat chahat95@gmail.com UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-792772
Date:	2025
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Außeruniversitäre Forschungseinrichtungen > MPI for Biology of Ageing
Subjects:	Natural sciences and mathematics
Uncontrolled Keywords:	Keywords Language Membrane contact sites English Host pathogen interactions English
Date of oral exam:	6 June 2025
Referee:	Name Academic Title Pernas, Lena Prof. Dr. Lemberg, Marius Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/79277