Time-resolved proximity biotinylation implicates a porin protein in export of transmembrane malaria parasite effectors.

The malaria-causing parasite, Plasmodium falciparum completely remodels its host red blood cell (RBC) through the export of several hundred parasite proteins, including transmembrane proteins, across multiple membranes to the RBC. However, the process by which these exported membrane proteins are extracted from the parasite plasma membrane for export remains unknown. To address this question, we fused the exported membrane protein, skeleton binding protein 1 (SBP1), with TurboID, a rapid, efficient and promiscuous biotin ligase (SBP1TbID).

Plastid biogenesis in malaria parasites requires the interactions and catalytic activity of the Clp proteolytic system.

The human malaria parasite, , contains an essential plastid called the apicoplast. Most apicoplast proteins are encoded by the nuclear genome and it is unclear how the plastid proteome is regulated. Here, we study an apicoplast-localized caseinolytic-protease (Clp) system and how it regulates organelle proteostasis.

Genetic Manipulation of Cryptosporidium parvum with CRISPR/Cas9.

Cryptosporidium parvum can be reliably genetically manipulated using CRISPR/Cas9-driven homologous repair coupled to in vivo propagation within immunodeficient mice. Recent modifications have simplified the initial protocol significantly. This chapter will guide through procedures for excystation, transfection, infection, collection, and purification of transgenic Cryptosporidium parvum.

A Genetically Tractable, Natural Mouse Model of Cryptosporidiosis Offers Insights into Host Protective Immunity.

Cryptosporidium is a leading cause of diarrheal disease and an important contributor to early childhood mortality, malnutrition, and growth faltering. Older children in high endemicity regions appear resistant to infection, while previously unexposed adults remain susceptible. Experimental studies in humans and animals support the development of disease resistance, but we do not understand the mechanisms that underlie protective immunity to Cryptosporidium.

Generating and Maintaining Transgenic Cryptosporidium parvum Parasites.

The apicomplexan parasite Cryptosporidium is a leading cause of diarrheal disease and an important contributor to overall global child mortality. We currently lack effective treatment and immune prophylaxis. Recent advances now permit genetic modification of this important pathogen.

A Cryptosporidium PI(4)K inhibitor is a drug candidate for cryptosporidiosis.

Diarrhoeal disease is responsible for 8.6% of global child mortality. Recent epidemiological studies found the protozoan parasite Cryptosporidium to be a leading cause of paediatric diarrhoea, with particularly grave impact on infants and immunocompromised individuals.

Bumped-Kinase Inhibitors for Cryptosporidiosis Therapy.

Bumped kinase inhibitors (BKIs) of Cryptosporidium parvum calcium-dependent protein kinase 1 (CpCDPK1) are leading candidates for treatment of cryptosporidiosis-associated diarrhea. Potential cardiotoxicity related to anti-human ether-à-go-go potassium channel (hERG) activity of the first-generation anti-Cryptosporidium BKIs triggered further testing for efficacy. A luminescence assay adapted for high-throughput screening was used to measure inhibitory activities of BKIs against C.

Toxoplasma gondii Toc75 Functions in Import of Stromal but not Peripheral Apicoplast Proteins.

Apicomplexa are unicellular parasites causing important human and animal diseases, including malaria and toxoplasmosis. Most of these pathogens possess a relict but essential plastid, the apicoplast. The apicoplast was acquired by secondary endosymbiosis between a red alga and a flagellated eukaryotic protist.

Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum.

Recent studies into the global causes of severe diarrhoea in young children have identified the protozoan parasite Cryptosporidium as the second most important diarrhoeal pathogen after rotavirus. Diarrhoeal disease is estimated to be responsible for 10.5% of overall child mortality.

Genetic manipulation of the Toxoplasma gondii genome by fosmid recombineering.

Unlabelled: Apicomplexa are obligate intracellular parasites that cause important diseases in humans and animals. Manipulating the pathogen genome is the most direct way to understand the functions of specific genes in parasite development and pathogenesis. In Toxoplasma gondii, nonhomologous recombination is typically highly favored over homologous recombination, a process required for precise gene targeting.

Antiapicoplast and gametocytocidal screening to identify the mechanisms of action of compounds within the malaria box.

Malaria remains a significant infectious disease that causes millions of clinical cases and >800,000 deaths per year. The Malaria Box is a collection of 400 commercially available chemical entities that have antimalarial activity. The collection contains 200 drug-like compounds, based on their oral absorption and the presence of known toxicophores, and 200 probe-like compounds, which are intended to represent a broad structural diversity.

An apicoplast localized ubiquitylation system is required for the import of nuclear-encoded plastid proteins.

Apicomplexan parasites are responsible for numerous important human diseases including toxoplasmosis, cryptosporidiosis, and most importantly malaria. There is a constant need for new antimalarials, and one of most keenly pursued drug targets is an ancient algal endosymbiont, the apicoplast. The apicoplast is essential for parasite survival, and several aspects of its metabolism and maintenance have been validated as targets of anti-parasitic drug treatment.

Tic22 is an essential chaperone required for protein import into the apicoplast.

Most plastids proteins are post-translationally imported into organelles through multisubunit translocons. The TIC and TOC complexes perform this role in the two membranes of the plant chloroplast and in the inner two membranes of the apicoplasts of the apicomplexan parasites, Toxoplasma gondii and Plasmodium falciparum. Tic22 is a ubiquitous intermembrane translocon component that interacts with translocating proteins.

Apicoplast and endoplasmic reticulum cooperate in fatty acid biosynthesis in apicomplexan parasite Toxoplasma gondii.

Apicomplexan parasites are responsible for high impact human diseases such as malaria, toxoplasmosis, and cryptosporidiosis. These obligate intracellular pathogens are dependent on both de novo lipid biosynthesis as well as the uptake of host lipids for biogenesis of parasite membranes. Genome annotations and biochemical studies indicate that apicomplexan parasites can synthesize fatty acids via a number of different biosynthetic pathways that are differentially compartmentalized.

Autophagy protein Atg3 is essential for maintaining mitochondrial integrity and for normal intracellular development of Toxoplasma gondii tachyzoites.

Autophagy is a cellular process that is highly conserved among eukaryotes and permits the degradation of cellular material. Autophagy is involved in multiple survival-promoting processes. It not only facilitates the maintenance of cell homeostasis by degrading long-lived proteins and damaged organelles, but it also plays a role in cell differentiation and cell development.

Apicoplast isoprenoid precursor synthesis and the molecular basis of fosmidomycin resistance in Toxoplasma gondii.

Apicomplexa are important pathogens that include the causative agents of malaria, toxoplasmosis, and cryptosporidiosis. Apicomplexan parasites contain a relict chloroplast, the apicoplast. The apicoplast is indispensable and an attractive drug target.

Toxoplasma gondii sequesters centromeres to a specific nuclear region throughout the cell cycle.

Members of the eukaryotic phylum Apicomplexa are the cause of important human diseases including malaria, toxoplasmosis, and cryptosporidiosis. These obligate intracellular parasites produce new invasive stages through a complex budding process. The budding cycle is remarkably flexible and can produce varied numbers of progeny to adapt to different host-cell niches.

A Toxoplasma MORN1 null mutant undergoes repeated divisions but is defective in basal assembly, apicoplast division and cytokinesis.

The membrane occupation and recognition nexus protein 1 (MORN1) is highly conserved among apicomplexan parasites and is associated with several structures that have a role in cell division. Here we dissected the role of MORN1 using the relatively simple budding process of Toxoplasma gondii as a model. Ablation of MORN1 in a conditional null mutant resulted in pronounced defects suggesting a central role for MORN1 in apicoplast segregation and in daughter cell budding.

The toxoplasma apicoplast phosphate translocator links cytosolic and apicoplast metabolism and is essential for parasite survival.

Apicomplexa are unicellular eukaryotic pathogens that carry a vestigial algal endosymbiont, the apicoplast. The physiological function of the apicoplast and its integration into parasite metabolism remain poorly understood and at times controversial. We establish that the Toxoplasma apicoplast membrane-localized phosphate translocator (TgAPT) is an essential metabolic link between the endosymbiont and the parasite cytoplasm.

Forward genetic analysis of the apicomplexan cell division cycle in Toxoplasma gondii.

Apicomplexa are obligate intracellular pathogens that have fine-tuned their proliferative strategies to match a large variety of host cells. A critical aspect of this adaptation is a flexible cell cycle that remains poorly understood at the mechanistic level. Here we describe a forward genetic dissection of the apicomplexan cell cycle using the Toxoplasma model.