Neospora caninum hijacks host PFKFB3-driven glycolysis to facilitate intracellular propagation of parasites.

Infection with Neospora caninum leads to reproductive failure in ruminants, such as cattle and goats; however, no effective vaccines or treatments are currently available to control this infection. Carefully regulating the glycolysis of host cells is essential for the intracellular survival of pathogens. Nonetheless, the impact of N. caninum infection on host cell glycolysis and the effects and mechanisms of host cell glycolysis on the intracellular survival of this parasite remains unclear. In this study, the analysis of metabolomics and transcriptomics revealed that N. caninum infection increases the expression of glycolysis-related enzymes and lactate production in caprine endometrial epithelial cells (EECs). The study's findings demonstrate that the inhibition of host cell glycolysis using 2-DG or sodium oxamate (an LDH-A inhibitor) inhibits host cell glycolysis and the intracellular propagation of N. caninum tachyzoites. Moreover, the addition of lactate further promotes the replication of N. caninum tachyzoites both in vivo and in vitro. Further investigation found that N. caninum infection induces host cell glycolysis via up-regulating 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) expression, while knockdown of PFKFB3 with small-interfering RNA or 3-PO significantly inhibits host cell glycolysis and the propagation of N. caninum tachyzoites both in vivo and in vitro. Additionally, a mechanistic study showed that N. caninum infection activates the JNK signalling pathway and inhibits the ubiquitination degradation of HIF-1α. Chromatin immunoprecipitation and dual-luciferase reporter assays revealed that N. caninum infection induces the expression of HIF-1α, which binds to the promoter region of pfkfb3. Our findings indicate that cellular glycolysis may serve as a potential therapeutic target for neosporosis, offering a novel insight for further investigating the intracellular survival mechanisms of N. caninum.