Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/867705

A genome-wide loss-of-function screen identifies Toxoplasma gondii genes that determine fitness in interferon gamma-activated murine macrophages

Wang, Y.·Sangare, L. O.·Paredes-Santos, T. C.·Krishnamurthy, S.·Hassan, M. A.·Furuta, A. M.·Markus, B. M.·Lourido, S.·Saeij, J. P. J.
View original source ↗Export metadata ↓

Abstract

Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFN{gamma}) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identified [~]130 Toxoplasma genes that determine parasite fitness in naive macrophages and [~]466 genes that determine fitness in IFN{gamma}-stimulated murine macrophages, seven of which we investigated and confirmed. We show that one of these genes encodes dense granule protein GRA45, which contains a putative chaperone-like domain, and which we show is critical in preventing other GRA effectors from aggregating. Parasites lacking GRA45 mislocalize GRA effectors upon secretion, are more susceptible to IFN{gamma}-mediated growth inhibition, and have reduced virulence in mice. Our results provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFN{gamma}-stimulated macrophages. IMPORTANCEThe intracellular parasite Toxoplasma gondii can cause congenital infections and severe disease in immunocompromised patients. The cytokine IFN{gamma} can block parasite replication by upregulating a variety of toxoplasmacidal mechanisms in many cells, including macrophages. Toxoplasma preferentially infects macrophages. Therefore, the parasite has evolved mechanisms to survive in these cells in the presence of IFN{gamma}. Here, we generated pools of Toxoplasma mutants for every gene and determined which mutants were specifically depleted in IFN{gamma}-stimulated macrophages, thus identifying parasite genes determining fitness in these cells. We show that one of these genes encodes for a dense granule protein (GRA45) that plays an important role in preventing other GRA effectors from aggregating. Parasites without GRA45 mislocalize GRA effectors upon secretion, have enhanced susceptibility to IFN{gamma}-mediated growth inhibition, and are avirulent in mice. Thus, our screen provides a resource to the Toxoplasma community to determine the function of Toxoplasma genes that affect its fitness in IFN{gamma}-stimulated macrophages.

Explore related subjects

microbiologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Wang, Y., Sangare, L. O., Paredes-Santos, T. C., Krishnamurthy, S., Hassan, M. A., Furuta, A. M., Markus, B. M., Lourido, S., Saeij, J. P. J.. 2019-12-06. A genome-wide loss-of-function screen identifies Toxoplasma gondii genes that determine fitness in interferon gamma-activated murine macrophages. https://doi.org/10.1101/867705

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Microbially conjugated bile acids differentially affect the lifecycle and pathogenesis of Clostridioides difficile

Clostridioides difficile is a Gram-positive bacterium that causes C. difficile infection (CDI). In healthy adults, it has been proposed that the gut microbiota provides colonization resistance against C. difficile by competition through nutrient limitation and antagonism through secondary bile acid metabolism, the latter of which is mediated by the deconjugation of host-derived bile acids with bacterial bile salt hydrolases (BSH). Recently, BSHs have also shown an ability to reconjugate non-canonical (i.e., not glycine or taurine) amino acids to sterol cores, resulting in novel microbially conjugated bile acids (MCBAs). MCBAs have been associated with the resolution of recurrent CDI, though the mechanism underlying these effects are not well understood. To address this gap, we synthesized 36 MCBAs with nine amino acids (cysteine, isoleucine, leucine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine) conjugated to the primary and secondary bile acids found in the human gut (CA, DCA, CDCA, and LCA) and tested their impact on various stages of C. difficiles lifecycle including bacterial outgrowth, membrane integrity, sporulation, and toxin activity. We show that BSH-mediated amidation of bile acid sterol cores can either increase or decrease inhibitory effects on C. difficile in an amino acid-dependent manner. For example, serine MCBAs enhanced C. difficiles ability to grow in otherwise inhibitory bile acid concentrations, increased spore production, and elevated toxin activity. Tryptophan MCBAs increased inhibition. By demonstrating these variable effects, our work clarifies the molecular determinants of MCBA activity and highlights specific MCBAs as promising targets for the development of C. difficile microbial-based therapeutics.

microbiology↗
bioRxiv2026

Magnetic organelle gene clusters enable bacterial growth and ATP homeostasis under oxidizing conditions

Biological organisms can produce magnetic nanoparticles made of magnetite to detect the Earth's magnetic field for orientation. The evolutionary mechanisms that led to the acquisition of this capacity remain unknown. The most ancient organisms known to perform magnetic orientation are magnetotactic bacteria. Under tight genetic control, they produce chains of magnetite nanoparticles in dedicated organelles to allow efficient navigation in aquatic environments containing molecular oxygen. Yet, these bacteria have been proposed to emerge before molecular oxygen became abundant on Earth, at a time when this navigation system could not have served its current function. Here, we show that gene clusters responsible of magnetite formation and organization enable resistance against hydrogen peroxide, a toxic oxygen-derived compound that was produced during the early stages of Earth's oxygenation when iron oxidation consumed molecular oxygen. Characterization of the nanoparticle's oxidation state supported the involvement of magnetite in resistance to hydrogen peroxide-induced oxidative stress. These findings are compatible with an early emergence of magnetotactic bacteria, before natural waters became oxygenated. We propose a conceptual framework to explain the origin of magnetic orientation in bacteria, and offer tools to identify the initiation of Earth's oxygenation in a time-resolved manner.

microbiology↗
bioRxiv2026

Identification and characterization of new P. aeruginosa phages using a surface receptor platform

Bacteriophages are of increasing interest for their potential use as alternatives to antibiotics for treating drug-resistant infections. Phage steering, specifically receptor-based steering, involves use of phages that bias the development of resistance in bacteria towards phenotypes such as reduced fitness. Phage therapy typically involves administering cocktails of phages with different targets to reduce the likelihood of resistance emerging. To ensure effective steering and cocktail design, there is a need to identify the receptors used by phages of interest, and to isolate phages that use diverse receptors. Here we describe a SURFAce Receptor (SURFAR) platform consisting of a set of defined Pseudomonas aeruginosa strains and mutants for rapid characterization of phage receptors. This platform can also be used to enrich for and isolate diverse phages from environmental samples, yielding novel phage genomes from wastewater. Using the SURFAR platform, two new phages were isolated and characterized: Dalewood, which targets both flagella and the conserved LPS outer core, and Bowman, a biofilm targeting phage capable of supressing growth of a subset of carbapenem-resistant clinical isolates. This tool helps to standardize the laborious but important task of phage receptor characterization, with the goal of expanding the known repertoire of P. aeruginosa phage receptors and phages.

microbiology↗