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BackgroundThe survival of the intracellular protozoan parasite Neospora caninum depends on its ability to adapt to changing metabolic conditions of the host cell. Thus, defining cellular and metabolic changes in affected target tissues may aid in delineating pathogenetic mechanism. We undertook this study to assess the metabolic response of human brain microvascular endothelial cells (HBMECs) to N. caninum infection in vitro.MethodsHBMECs were exposed to N. caninum infection and the cytotoxic effects of infection were analyzed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromidin (MTT) assay and lactate dehydrogenase (LDH) release assay. Metabolic footprinting of the extracellular metabolites of parasite-infected and non-infected culture supernatant was determined by using targeted (Randox RX Imola clinical chemistry analyser) and unbiased RS (Raman microspectroscopy) approaches.ResultsThe MTT assay did not reveal any cytotoxic effect of N. caninum challenge on host cell viability. Measurement of LDH activity showed that N. caninum significantly induced loss of cell membrane integrity in a time-dependent and dose-dependent manner compared to control cells. Targeted biochemical analysis revealed that beta hydroxybutyrate, pyruvate, ATP, total protein, non-esterified fatty acids, and triglycerides are significantly different in infected cells compared to controls. RS-based footprinting with principal component analysis (PCA) were able to correctly distinguish extracellular metabolites obtained from infected and control cultures, and revealed infection-related spectral signatures at 865 cm−1, 984 cm−1, 1046 cm−1, and 1420 cm−1, which are attributed to variations in the content of lipids and nucleic acids in infected cultures.ConclusionsThe changing pattern of extracellular metabolites suggests that HBMECs are target of metabolic alterations in N. caninum infection, which seem to reflect the changing metabolic state of infected cells and constitute a level of information exchange that host and parasite use to coordinate activities.

In this work, the effects of the protozoan Neospora caninum on the bioenergetics, chemical composition, and elemental content of human brain microvascular endothelial cells (hBMECs) were investigated. We showed that N. caninum can impair cell mitochondrial (Mt) function and causes an arrest in host cell cycling at S and G2 phases. These adverse effects were also associated with altered expression of genes involved in Mt energy metabolism, suggesting Mt dysfunction caused by N. caninum infection. Fourier Transform Infrared (FTIR) spectroscopy analysis of hBMECs revealed alterations in the FTIR bands as a function of infection, where infected cells showed alterations in the absorption bands of lipid (2924 cm−1), amide I protein (1649 cm−1), amide II protein (1537 cm−1), nucleic acids and carbohydrates (1092 cm−1, 1047 cm−1, and 939 cm−1). By using quantitative synchrotron radiation X-ray fluorescence (μSR-XRF) imaging and quantification of the trace elements Zn, Cu and Fe, we detected an increase in the levels of Zn and Cu from 3 to 24 h post infection (hpi) in infected cells compared to control cells, but there were no changes in the level of Fe. We also used Affymetrix array technology to investigate the global alteration in gene expression of hBMECs and rat brain microvascular endothelial cells (rBMVECs) in response to N. caninum infection at 24 hpi. The result of transcriptome profiling identified differentially expressed genes involved mainly in immune response, lipid metabolism and apoptosis. These data further our understanding of the molecular events that shape the interaction between N. caninum and blood-brain-barrier endothelial cells.