Intersection of phosphate transport, oxidative stress and TOR signalling in Candida albicans virulence

Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells’ oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 did not ameliorate these cells’ hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/- null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing C. albicans to oxidative stress

Effects of in vitro adult platelet transfusions on neonatal hemostasis

BACKGROUND: Thrombocytopenia is frequent among neonates, and 20-25% of affected infants are treated with platelet transfusions. These are frequently given for mild thrombocytopenia (platelets: 50-100 × 10(9) L(-1)), largely because of the known hyporeactivity of neonatal platelets. In tests of primary hemostasis, however, neonates have shorter bleeding and closure times (CTs) than adults. This has been attributed to their higher hematocrits, higher von Willebrand factor (VWF) concentrations, and predominance of longer VWF polymers. OBJECTIVE: To determine whether the 'transfusion' of adult (relatively hyperreactive) platelets into neonatal blood results in a hypercoagulable profile. METHODS: Cord blood (CB) and adult peripheral blood (PB) were separated (with a modified buffy coat method) to generate miniaturized platelet concentrates (PCs) and thrombocytopenic blood. PB-derived and CB-derived PCs (n = 7 per group) were then 'transfused'in vitro into thrombocytopenic CB and PB. The effects of autologous vs. allogeneic (developmentally mismatched) 'transfusions' were evaluated with whole blood aggregometry, a platelet function analyzer (PFA-100), and thromboelastography (TEG). RESULTS: Adult platelets aggregated significantly better than neonatal platelets in response to thrombin receptor-activating peptide, ADP, and collagen, regardless of the blood into which they were transfused. The 'transfusion' of adult platelets into thrombocytopenic CB resulted in shorter CTs-EPI (PFA-100) and higher clot strength and firmness (TEG) than 'transfusion' of neonatal autologous platelets. CONCLUSIONS: In vitro'transfusion' of adult platelets into neonatal blood results in shorter CTs than 'transfusion' with neonatal platelets. Our findings should raise awareness of the differences between the neonatal and adult hemostatic system and the potential 'developmental mismatch' associated with platelet transfusions for neonatal hemostasis

Eltrombopag: a powerful chelator of cellular or extracellular iron(III) alone or combined with a second chelator.

Eltrombopag (ELT) is a thrombopoietin receptor agonist reported to decrease labile iron in leukemia cells. Here we examine the previously undescribed iron(III)-coordinating and cellular iron-mobilizing properties of ELT. We find a high binding constant for iron(III) (log β2=35). Clinically achievable concentrations (1 µM) progressively mobilized cellular iron from hepatocyte, cardiomyocyte, and pancreatic cell lines, rapidly decreasing intracellular reactive oxygen species (ROS) and also restoring insulin secretion in pancreatic cells. Decrements in cellular ferritin paralleled total cellular iron removal, particularly in hepatocytes. Iron mobilization from cardiomyocytes exceeded that obtained with deferiprone, desferrioxamine, or deferasirox at similar iron-binding equivalents. When combined with these chelators, ELT enhanced cellular iron mobilization more than additive (synergistic) with deferasirox. Iron-binding speciation plots are consistent with ELT donating iron to deferasirox at clinically relevant concentrations. ELT scavenges iron citrate species faster than deferasirox, but rapidly donates the chelated iron to deferasirox, consistent with a shuttling mechanism. Shuttling is also suggested by enhanced cellular iron mobilization by ELT when combined with the otherwise ineffective extracellular hydroxypyridinone chelator, CP40. We conclude that ELT is a powerful iron chelator that decreases cellular iron and further enhances iron mobilization when combined with clinically available chelators

Eltrombopag: a powerful chelator of cellular or extracellular iron(III) alone or combined with a second chelator.

Eltrombopag (ELT) is a thrombopoietin receptor agonist reported to decrease labile iron in leukemia cells. Here we examine the previously undescribed iron(III)-coordinating and cellular iron-mobilizing properties of ELT. We find a high binding constant for iron(III) (log β2=35). Clinically achievable concentrations (1 µM) progressively mobilized cellular iron from hepatocyte, cardiomyocyte, and pancreatic cell lines, rapidly decreasing intracellular reactive oxygen species (ROS) and also restoring insulin secretion in pancreatic cells. Decrements in cellular ferritin paralleled total cellular iron removal, particularly in hepatocytes. Iron mobilization from cardiomyocytes exceeded that obtained with deferiprone, desferrioxamine, or deferasirox at similar iron-binding equivalents. When combined with these chelators, ELT enhanced cellular iron mobilization more than additive (synergistic) with deferasirox. Iron-binding speciation plots are consistent with ELT donating iron to deferasirox at clinically relevant concentrations. ELT scavenges iron citrate species faster than deferasirox, but rapidly donates the chelated iron to deferasirox, consistent with a shuttling mechanism. Shuttling is also suggested by enhanced cellular iron mobilization by ELT when combined with the otherwise ineffective extracellular hydroxypyridinone chelator, CP40. We conclude that ELT is a powerful iron chelator that decreases cellular iron and further enhances iron mobilization when combined with clinically available chelators