title: Metabolic footprinting of extracellular metabolites of brain endothelium infected with Neospora caninum in vitro.
abstract: BACKGROUND
  The 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.
  
  
  METHODS
  HBMECs 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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  The 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.
authors: School of Veterinary Medicine and Science, Faculty of Medicine and Health Sciences, University of Nottingham, Sutton Bonington Campus, Leicestershire LE12 5RD, UK. hany.elsheikha@nottingham.ac.uk.
authors: Hany M
authors: HM
authors: Elsheikha
authors: Mamdowh
authors: M
authors: Alkurashi
authors: Kenny
authors: K
authors: Kong
authors: Xing-Quan
authors: XQ
authors: Zhu
mesh_terms: D020155:3-Hydroxybutyric Acid / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001921:Brain / Q000098:blood supply / Q000166:cytology; D002470:Cell Survival; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism* / Q000469:parasitology / Q000473:pathology; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000469:parasitology / Q000473:pathology; D005230:Fatty Acids, Nonesterified / Q000378:metabolism; D006790:Host-Parasite Interactions; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D055442:Metabolome; D018688:Neospora / Q000254:growth & development / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism; D013059:Spectrum Analysis, Raman; D014280:Triglycerides / Q000378:metabolism
chemical_list: D005230:Fatty Acids, Nonesterified; D014280:Triglycerides; D019289:Pyruvic Acid; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D020155:3-Hydroxybutyric Acid