Biology subjects

Key, M.

Publications and source records attributed to Key, M..

3 recordsLinked to original sources

bioRxiv2023

Toxoplasma gondii harbors a hypoxia-responsive coproporphyrinogen dehydrogenase-like protein.

Toxoplasma gondii is an apicomplexan parasite that is the cause of toxoplasmosis, a potentially lethal disease for immunocompromised individuals. During in vivo infection, the parasites encounter various growth environments, such as hypoxia. Therefore, the metabolic enzymes in the parasites must adapt to such changes to fulfill their nutritional requirements. Toxoplasma can de novo biosynthesize some nutrients, such as heme. The parasites heavily rely on their own heme production for intracellular survival. Notably, the antepenultimate step within this pathway is facilitated by coproporphyrinogen III oxidase (CPOX), which employs oxygen to convert coproporphyrinogen III to protoporphyrinogen IX through oxidative decarboxylation. Conversely, some bacteria can accomplish this conversion independently of oxygen through coproporphyrinogen dehydrogenase (CPDH). Genome analysis found a CPDH ortholog in Toxoplasma. The mutant Toxoplasma lacking CPOX displays significantly reduced growth, implying that TgCPDH potentially functions as an alternative enzyme to perform the same reaction as CPOX under low oxygen conditions. In this study, we demonstrated that TgCPDH exhibits coproporphyrinogen dehydrogenase activity by complementing it in a heme synthesis-deficient Salmonella mutant. Additionally, we observed an increase in TgCPDH expression in Toxoplasma when it grew under hypoxic conditions. However, deleting TgCPDH in both wildtype and heme-deficient parasites did not alter their intracellular growth under both ambient and low oxygen conditions. This research marks the first report of a coproporphyrinogen dehydrogenase-like protein in eukaryotic cells. Although TgCPDH responds to hypoxic conditions and possesses enzymatic activity, our findings suggest that it does not directly affect intracellular infection or the pathogenesis of Toxoplasma parasites. IMPORTANCEToxoplasma gondii is a ubiquitous parasite capable of infecting a wide range of warm-blooded hosts, including humans. During its lifecycle, these parasites must adapt to varying environmental conditions, including situations with low oxygen levels. Our research, in conjunction with studies conducted by other laboratories, has revealed that Toxoplasma primarily relies on its own heme production during acute infections. Intriguingly, in addition to this classical heme biosynthetic pathway, the parasites encode a putative oxygen-independent coproporphyrinogen dehydrogenase, suggesting its potential contribution to heme production under varying oxygen conditions, a feature typically observed in simpler organisms like bacteria. Notably, so far, coproporphyrinogen dehydrogenase has only been identified in some bacteria for heme biosynthesis. Our study discovered that Toxoplasma harbors a functional enzyme displaying coproporphyrinogen dehydrogenase activity, which alters its expression in the parasites when they face fluctuating oxygen levels in their surroundings.

microbiology↗

bioRxiv2023

A cathepsin C-like protease post-translationally modifies Toxoplasma gondii secretory proteins for optimal invasion and egress.

Microbial pathogens use proteases for their infections, such as digestion of proteins for nutrients and activation of their virulence factors. As an obligate intracellular parasite, Toxoplasma gondii must invade host cells to establish its intracellular propagation. To facilitate invasion, the parasites secrete invasion effectors from microneme and rhoptry, two unique organelles in apicomplexans. Previous work has shown that some micronemal invasion effectors experience a series of proteolytic cleavages within the parasites secretion pathway for maturation, such as the aspartyl protease (TgASP3) and the cathepsin L-like protease (TgCPL), localized within the post-Golgi compartment (1) and the endolysosomal system (2), respectively. Furthermore, it has been shown that the precise maturation of micronemal effectors is critical for Toxoplasma invasion and egress (1). Here, we show that an endosome-like compartment (ELC)-residing cathepsin C-like protease (TgCPC1) mediates the final trimming of some micronemal effectors, and its loss further results in defects in the steps of invasion, egress, and migration throughout the parasites lytic cycle. Notably, the deletion of TgCPC1 completely blocks the activation of subtilisin-like protease 1 (TgSUB1) in the parasites, which globally impairs the surface-trimming of many key micronemal invasion and egress effectors. Additionally, we found that TgCPC1 was not efficiently inhibited by the chemical inhibitor targeting its malarial ortholog, suggesting that these cathepsin C-like orthologs are structurally different within the apicomplexan phylum. Taken together, our findings identify a novel function of TgCPC1 in the processing of micronemal proteins within the secretory pathway of Toxoplasma parasites and expand the understanding of the roles of cathepsin C protease. IMPORTANCEToxoplasma gondii is a microbial pathogen that is well adapted for disseminating infections. It can infect virtually all warm-blooded animals. Approximately one-third of the human population carries toxoplasmosis. During infection, the parasites sequentially secrete protein effectors from the microneme, rhoptry, and dense granule, three organelles exclusively found in apicomplexan parasites, to help establish their lytic cycle. Proteolytic cleavage of these secretory proteins is required for the parasites optimal function. Previous work has revealed that two proteases residing within the parasites secretory pathway cleave micronemal and rhoptry proteins, which mediate parasite invasion and egress. Here, we demonstrate that a cathepsin C-like protease (TgCPC1) is involved in processing several invasion and egress effectors. The genetic deletion of TgCPC1 prevented the complete maturation of some effectors in the parasites. Strikingly, the deletion led to a full inactivation of one surface-anchored protease, which globally impaired the trimming of some key micronemal proteins before secretion. Therefore, this finding represents a novel post-translational mechanism for the processing of virulence factors within microbial pathogens.

microbiology↗

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↗