Dou, Z. — Page 2 | bioRxiv Science

bioRxiv2019

Erosion of the Epigenetic Landscape and Loss of Cellular Identity as a Cause of Aging in Mammals

All living things experience entropy, manifested as a loss of inherited genetic and epigenetic information over time. As budding yeast cells age, epigenetic changes result in a loss of cell identity and sterility, both hallmarks of yeast aging. In mammals, epigenetic information is also lost over time, but what causes it to be lost and whether it is a cause or a consequence of aging is not known. Here we show that the transient induction of genomic instability, in the form of a low number of non-mutagenic DNA breaks, accelerates many of the chromatin and tissue changes seen during aging, including the erosion of the epigenetic landscape, a loss of cellular identity, advancement of the DNA methylation clock and cellular senescence. These data support a model in which a loss of epigenetic information is a cause of aging in mammals.\n\nOne Sentence SummaryThe act of repairing DNA breaks induces chromatin reorganization and a loss of cell identity that may contribute to mammalian aging

molecular biology↗

bioRxiv2019

Modulation of de novo heme production in Toxoplasma essentially controls its infection.

Heme, an iron-enclosed organic ring, is essential for virtually all living organisms by serving as a prosthetic group in proteins that function in diverse cellular activities ranging from diatomic gas transport and detection to mitochondrial respiration to detoxification. Cellular heme levels in microbial pathogens can be a composite of endogenous de novo synthesis or exogenous uptake of heme or heme synthesis intermediates1,2. Intracellular pathogenic microbes switch routes for heme supply when heme availability in their replicative environment fluctuates through infections2. Here, we show that the Toxoplasma gondii, an obligate intracellular human pathogen, encodes a functional heme biosynthesis pathway. A chloroplast-derived organelle, termed apicoplast, is involved in the heme production. Genetic and chemical manipulation revealed that de novo heme production is essential for T. gondii intracellular growth and pathogenesis. Surprisingly, the herbicide oxadiazon significantly impaired Toxoplasma growth, consistent with phylogenetic analyses that show T. gondii protoporphyrinogen oxidase is more closely related to plants than mammals. We further improve upon this inhibition by 15-to 25-fold with two oxadiazon derivatives, providing therapeutic proof that Toxoplasma heme biosynthesis is a druggable target. As T. gondii has been used to model other apicomplexan parasites3, our study underscores the utility of targeting heme biosynthesis in other pathogenic apicomplexans.

microbiology↗

bioRxiv2018

The ortholog of chloroquine resistance transporter (TgCRT) plays a key role in maintaining the integrity of the endolysosomal system in Toxoplasma gondii to facilitate host invasion

Toxoplasma gondii is an apicomplexan parasite with the ability to use foodborne, zoonotic, and congenital routes of transmission that causes severe disease in immunocompromised patients. The parasites harbor a lysosome-like digestive vacuole, termed the \"Vacuolar Compartment/Plant-Like Vacuole\" (VAC/PLV), which plays an important role in maintaining the lytic cycle and virulence of T. gondii. The VAC supplies proteolytic enzymes that are required to mature the parasites invasion effectors and that digest autophagosomes and endocytosed host proteins. Previous work identified a T. gondii ortholog of the Plasmodium falciparum chloroquine resistance transporter (PfCRT) that localized to the VAC. Here, we show that TgCRT is a membrane transporter that is functionally similar to PfCRT. We also genetically ablate TgCRT and reveal that TgCRT protein plays a key role in maintaining the integrity of the parasites endolysosomal system by controlling morphology of the VAC. When TgCRT is absent, the VAC dramatically increases in size by ~15-fold and co-localizes with its adjacent endosome-like compartment. Presumably to reduce aberrant swelling, transcription and translation of endolysosomal proteases are decreased in {Delta}TgCRT parasites. Expression of one endolysosomal subtilisin protease is quite significantly reduced, which impedes trimming of micronemal proteins, and significantly decreases parasite invasion. Chemical and genetic inhibition of proteolysis within the VAC reverses these effects, reducing VAC size and partially restoring the endolysosomal system, micronemal protein trimming, and invasion. Taken together, these findings reveal for the first time a physiological role of TgCRT in controlling VAC volume and the integrity of the endolysosomal system in T. gondii.\n\nAuthor SummaryToxoplasma gondii is an obligate intracellular protozoan parasite that belongs to the phylum Apicomplexa and that infects virtually all warm-blooded organisms. Approximately one-third of the human population is infected with Toxoplasma. The parasites invade host cells via processed invasion effectors in order to disseminate infection. A lysosome-like digestive vacuole (VAC) is involved in refining these invasion effectors to reach their final forms. A T. gondii ortholog of the malarial chloroquine resistance transporter protein (TgCRT) was found to be localized to the VAC membrane. Although the mutated version of the malarial chloroquine resistance transporter (PfCRT) has been shown to confer resistance to chloroquine treatment, its physiologic function remains poorly understood. Comparison between the related PfCRT and TgCRT proteins facilitates definition of the physiologic role of CRT proteins. In this study, we report that TgCRT plays a key role in regulating the integrity and proteolytic activity of the VAC and adjacent organelles, the secretion of invasion effectors, and parasite invasion and virulence. To relieve osmotic stress caused by VAC swelling when TgCRT is deleted, parasites repress proteolytic activities within this organelle to decrease solute accumulation, which then has secondary effects on parasite invasion. Our findings highlight a common function for PfCRT and TgCRT proteins in regulating apicomplexan parasite vacuolar size and function.

microbiology↗