Deficiency of a Niemann-Pick, Type C1-related Protein in Toxoplasma Is Associated with Multiple Lipidoses and Increased Pathogenicity

Several proteins that play key roles in cholesterol synthesis, regulation, trafficking and signaling are united by sharing the phylogenetically conserved ‘sterol-sensing domain’ (SSD). The intracellular parasite Toxoplasma possesses at least one gene coding for a protein containing the canonical SSD. We investigated the role of this protein to provide information on lipid regulatory mechanisms in the parasite. The protein sequence predicts an uncharacterized Niemann-Pick, type C1-related protein (NPC1) with significant identity to human NPC1, and it contains many residues implicated in human NPC disease. We named this NPC1-related protein, TgNCR1. Mammalian NPC1 localizes to endo-lysosomes and promotes the movement of sterols and sphingolipids across the membranes of these organelles. Miscoding patient mutations in NPC1 cause overloading of these lipids in endo-lysosomes. TgNCR1, however, lacks endosomal targeting signals, and localizes to flattened vesicles beneath the plasma membrane of Toxoplasma. When expressed in mammalian NPC1 mutant cells and properly addressed to endo-lysosomes, TgNCR1 restores cholesterol and GM1 clearance from these organelles. To clarify the role of TgNCR1 in the parasite, we genetically disrupted NCR1; mutant parasites were viable. Quantitative lipidomic analyses on the ΔNCR1 strain reveal normal cholesterol levels but an overaccumulation of several species of cholesteryl esters, sphingomyelins and ceramides. ΔNCR1 parasites are also characterized by abundant storage lipid bodies and long membranous tubules derived from their parasitophorous vacuoles. Interestingly, these mutants can generate multiple daughters per single mother cell at high frequencies, allowing fast replication in vitro, and they are slightly more virulent in mice than the parental strain. These data suggest that the ΔNCR1 strain has lost the ability to control the intracellular levels of several lipids, which subsequently results in the stimulation of lipid storage, membrane biosynthesis and parasite division. Based on these observations, we ascribe a role for TgNCR1 in lipid homeostasis in Toxoplasma

Insects in the feed of rainbow trout, Oncorhynchus mykiss (Actinopterygii, Salmonidae): Effect on growth, fatty acid composition, and sensory attributes.

Background. An ongoing quest for alternative feed sources in global aquaculture includes insect breeding for feed and food production. The aim of this study was to evaluate the effect of partial to full replacement of commercial diets with live insects on growth and health parameters of rainbow trout, as well as on sensory and texture attributes and fatty acid composition of fish muscle. Materials and methods. Five isocaloric diets containing commercial pellets and live insects were evaluated in rainbow trout, Oncorhynchus mykiss (Walbaum, 1792), in a 60-day feeding trial. Control Group (K) was fed commercial pellets only. In other groups, 25% gross energy of pellets was replaced by live adult house cricket, Acheta domestica (Group C), live superworm, Zophobas morio larvae (Group L), or a combination of 12.5% crude energy of each (group LC). The insect-only group (I) was fed live cricket and superworm only (50% by 50% crude energy). Results. No significant differences were found in growth, survival, feed conversion ratio (dry basis), or energy utilization between groups. The protein efficiency ratio was highest in Group K and decreased with increasing cricket proportion. Insect inclusion was associated with lower content of nutritionally valuable n-3 fatty acid in fish muscle. Subjective sensory evaluation of cooked fillets revealed significantly less acceptable taste, aroma, and aftertaste in Group I than for Groups K, L, and LC. Some differences were found in the whiteness and redness of fillets between groups. The control group had significantly lower hardness compared to those receiving insect diets. No gross morphological or histopathological anomalies of viscera in any group and no significant differences in 7-ethoxyresorufin-O-deethylase activity were observed. Conclusion. Live insects replaced the commercial diet of the equivalent caloric level without negative effects on the growth or health of rainbow trout. The lower content of n-3 fatty acids and differences in color and texture of fillets from fish fed insects may influence acceptability to consumers. The high cost of insects compared to commercial feed currently limits their widespread use in trout production

CRISPR/cas9-mediated generation of tetracycline repressor-based inducible knockdown in toxoplasma gondii

The phylum Apicomplexa groups numerous pathogenic protozoan parasites including Plasmodium, the causative agent of malaria, Cryptosporidium which can cause severe gastrointestinal infections, as well as Babesia, Eimeria, and Theileria that account for considerable economic burdens to poultry and cattle industry. Toxoplasma gondii is the most ubiquitous and opportunistic member of this phylum able to infect all warm-blooded animals and responsible for severe disease in immunocompromised individuals and unborn fetuses.Due to its ease of cultivation and genetic tractability T. gondii has served as recipient for the transfer and adaptation of multiple genetic tools developed to control gene expression. In these parasites, a collection of tight conditional systems exists to control gene expression at the levels of transcription, RNA degradation or protein stability. The recent implementation of the CRISPR/Cas9 technology considerably reduces time and effort to generate transgenic parasites and at the same time increases to an ultimate level of precision the editing of the parasite genome. Here, we provide a step-by-step protocol for CRISPR/Cas9-mediated generation of tetracycline repressor-based inducible knockdown in T. gondii