Eimeria bovis-infected host cell-bystander cell interactions are essential for effective macromeront formation

Eimeria bovis is an obligate intracellular apicomplexan parasite and the major agent of bovine coccidiosis. Intracellular E. bovis first merogony results in the formation of large macromeronts (≤ 400 µm) containing up to 140,000 merozoites I. This extensive replication imposes substantial metabolic stress on bovine endothelial host cells, leading to mitochondrial dysfunction and premature senescence. During macromeront development, non-infected bystander cells (BCs) accumulate around infected host cells and previous findings provided evidence for tunneling nanotube (TNT)-based interactions between infected cells and BCs. In the current study, we aimed to further analyze interactions between infected cells and BCs, potentially forming part of a multicellular support mechanism promoting macromeront development. To dissect BC-based reactions, we used a meront-transfer-system, which allowed for selective treatments of non-infected cells, serving as BCs in mixed cultures with E. bovis meront-carrying host cells (MCHCs). When addressing gap junction formation, protein expression profiles of E. bovis-infected cell layers revealed a significant upregulation of the connexins Cx37 and Cx40, but not of Cx43 during macromeront formation. In addition, E. bovis infection altered the subcellular distribution of Cx37, Cx40 and Cx43. The functional relevance of connexins was supported by blocking gap junction formation in BCs with carbenoxolone or Gap26 (inhibits Cx37, Cx40 and Cx43), both of which impaired parasite development. Besides connexins, we also studied TNT-based BC-MCHC-interactions in more detail. When quantitatively estimating BC-mediated donation of mitochondria to E. bovis-infected cells to improve MCHC bioenergetics, we showed that a high proportion of MCHCs (89%) obtained mitochondria from BCs. In line, the disruption of microtubule integrity by nocodazole treatments in BCs diminished both TNT formation and merozoite I production. Moreover, inhibitor-based impairment of BC metabolism demonstrated that intact glycolytic and glutaminolytic activities in addition to functional cholesterol esterification and trafficking are required in BCs to sustain effective merozoite I production in BC-MCHC-cultures. Collectively, the current findings support the hypothesis that effective intracellular E. bovis macromeront formation depends on an environmental BC-based support network involving BC metabolic integrity, short-range gap-junctional small molecule transfer, and long-range TNT-mediated mitochondria donation.