Functional analysis of Ca ²⁺ signalling in Besnoitia besnoiti tachyzoites

Besnoitia besnoiti is an apicomplexan parasite, the causal agent of bovine besnoitiosis. This disease is characterised by cyst formation in the skin and mucosa. During early infection, fast proliferating tachyzoites invade and replicate within host endothelial cells. In non-excitable cells, extracellular signals activate the inositol-triphosphate/calcium (InsP3/Ca 2+ ) pathway, which depends on phospholipase C (PLC) activation, inducing an increase in IP 3 levels, followed by intracellular Ca 2+ release. Despite the understanding of Ca 2+ signalling, this process in B. besnoiti tachyzoites is unclear. This work aimed to study Ca 2+ dynamics during B. besnoiti infection in bovine umbilical vein endothelial cells (BUVEC) and the role of the InsP 3 /Ca 2+ pathway during B. besnoiti infection. Ca 2+ dynamics during tachyzoite replication were determined in B. besnoiti- infected BUVEC loaded with fluo-4-AM. The role of InsP 3 /Ca 2+ signalling for parasite invasion was evaluated by treatments with Ca 2+ chelators (BAPTA, EGTA) or PLC inhibitors (U73122, D609). PLC activation was studied in fluo-4-loaded free tachyzoites using the PLC activator m-3M3FBS, in the presence or absence of PLC inhibitors. Current data show an infection-driven increase in total Ca 2+ signals in B. besnoiti -infected BUVEC over time. BAPTA, but not EGTA, treatments of tachyzoites affected their invasion, reducing infection rates by 85.4 ± 9.3%, suggesting that intracellular Ca 2+ sources are necessary for B. besnoiti invasion. In line, treatments with U73122 and D609 reduced infection rates by 79.3 ± 9.4% and 49.7 ± 8.9%, respectively, demonstrating that PLC participation is required for host cell infection. Finally, m-3M3FBS treatments induced a PLC-independent Ca 2+ flux in B. besnoiti tachyzoites.