Besnoitia besnoiti infection alters both endogenous cholesterol de novo synthesis and exogenous LDL uptake in host endothelial cells

Besnoitia besnoiti, an apicomplexan parasite of cattle being considered as emergent in Europe, replicates fast in host endothelial cells during acute infection and is in considerable need for energy, lipids and other building blocks for offspring formation. Apicomplexa are generally considered as defective in cholesterol synthesis and have to scavenge cholesterol from their host cells for successful replication. Therefore, we here analysed the influence of B. besnoiti on host cellular endogenous cholesterol synthesis and on sterol uptake from exogenous sources. GC-MS-based profiling of cholesterol-related sterols revealed enhanced cholesterol synthesis rates in B. besnoiti-infected cells. Accordingly, lovastatin and zaragozic acid treatments diminished tachyzoite production. Moreover, increased lipid droplet contents and enhanced cholesterol esterification was detected and inhibition of the latter significantly blocked parasite proliferation. Furthermore, artificial increase of host cellular lipid droplet disposability boosted parasite proliferation. Interestingly, lectin-like oxidized low density lipoprotein receptor 1 expression was upregulated in infected endothelial hostcells, whilst low density lipoproteins (LDL) receptor was not affected by parasite infection. However, exogenous supplementations with non-modified and acetylated LDL both boosted B. besnoiti proliferation. Overall, current data show that B. besnoiti simultaneously exploits both, endogenous cholesterol biosynthesis and cholesterol uptake from exogenous sources, during asexual replication

3D holotomographic monitoring of Ca++ dynamics during ionophore-induced Neospora caninum tachyzoite egress from primary bovine host endothelial cells

Neospora caninum represents an obligate intracellular parasite that belongs to the phylum Apicomplexa and is a major abortive agent in bovines. During merogony, N. caninum tachyzoites invade and proliferate in host cells in vivo, including endothelial cells of lymphatic and blood vessels. The egress at the end of the lytic cycle is tightly regulated in apicomplexans. Evidence in Toxoplasma gondii shows that Ca⁺⁺ signalling governs tachyzoite egress. Much less is known on egress mechanisms of N. caninum. Here, we show, using 3D live cell holotomographic microscopy in fluo-4 AM-loaded N. caninum-infected BUVEC, that treatments with the calcium ionophore A23187 at 24- and 42-h post-infection (h p. i.) induced a fast and sustained increase in Ca⁺⁺ signals in parallel to tachyzoite egress. A23187 treatments exclusively triggered tachyzoite release at 42-h p. i. but failed to do so at 24-h p. i. indicating a role for meront maturation in calcium-induced tachyzoite egress. Overall, we show that live cell 3D holotomographic analysis in combination with epifluorescence is a suitable tool to study calcium dynamics related to coccidian egress or other important cell functions

Thiosemicarbazone Copper Chelator BLT-1 Blocks Apicomplexan Parasite Replication by Selective Inhibition of Scavenger Receptor B Type 1 (SR-BI)

Coccidian parasites are obligate intracellular pathogens that affect humans and animals. Apicomplexans are defective in de novo synthesis of cholesterol, which is required for membrane biosynthesis and offspring formation. In consequence, cholesterol has to be scavenged from host cells. It is mainly taken up from extracellular sources via LDL particles; however, little is known on the role of HDL and its receptor SR-BI in this process. Here, we studied effects of the SR-BI-specific blocker BLT-1 on the development of different fast (Toxoplasma gondii, Neospora caninum, Besnoitia besnoiti) and slow (Eimeria bovis and Eimeria arloingi) replicating coccidian species. Overall, development of all these parasites was significantly inhibited by BLT-1 treatment indicating a common SR-BI-related key mechanism in the replication process. However, SR-BI gene transcription was not affected by T. gondii, N. caninum and B. besnoiti infections. Interestingly, BLT-1 treatment of infective stages reduced invasive capacities of all fast replicating parasites paralleled by a sustained increase in cytoplasmic Ca++ levels. Moreover, BLT1-mediated blockage of SR-BI led to enhanced host cell lipid droplet abundance and neutral lipid content, thereby confirming the importance of this receptor in general lipid metabolism. Finally, the current data suggest a conserved role of SR-BI for successful coccidian infections

Thiosemicarbazone Copper Chelator BLT-1 Blocks Apicomplexan Parasite Replication by Selective Inhibition of Scavenger Receptor B Type 1 (SR-BI)

Coccidian parasites are obligate intracellular pathogens that affect humans and animals. Apicomplexans are defective in de novo synthesis of cholesterol, which is required for membrane biosynthesis and offspring formation. In consequence, cholesterol has to be scavenged from host cells. It is mainly taken up from extracellular sources via LDL particles; however, little is known on the role of HDL and its receptor SR-BI in this process. Here, we studied effects of the SR-BI-specific blocker BLT-1 on the development of different fast (Toxoplasma gondii, Neospora caninum, Besnoitia besnoiti) and slow (Eimeria bovis and Eimeria arloingi) replicating coccidian species. Overall, development of all these parasites was significantly inhibited by BLT-1 treatment indicating a common SR-BI-related key mechanism in the replication process. However, SR-BI gene transcription was not affected by T. gondii, N. caninum and B. besnoiti infections. Interestingly, BLT-1 treatment of infective stages reduced invasive capacities of all fast replicating parasites paralleled by a sustained increase in cytoplasmic Ca++ levels. Moreover, BLT1-mediated blockage of SR-BI led to enhanced host cell lipid droplet abundance and neutral lipid content, thereby confirming the importance of this receptor in general lipid metabolism. Finally, the current data suggest a conserved role of SR-BI for successful coccidian infections

video2_Neospora caninum Infection Triggers S-phase Arrest and Alters Nuclear Characteristics in Primary Bovine Endothelial Host Cells.mp4

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video3_Neospora caninum Infection Triggers S-phase Arrest and Alters Nuclear Characteristics in Primary Bovine Endothelial Host Cells.mp4

Graphical Abstract</p

Image1_Neospora caninum Infection Triggers S-phase Arrest and Alters Nuclear Characteristics in Primary Bovine Endothelial Host Cells.tiff

Graphical Abstract</p

video1_Neospora caninum Infection Triggers S-phase Arrest and Alters Nuclear Characteristics in Primary Bovine Endothelial Host Cells.mp4

Graphical Abstract</p