Defunctionalizing Intracellular Organelles with Genetically-Encoded Molecular Tools Based on Engineered Phospholipase A/Acyltransferases (PLAATs)

Organelles vitally achieve multifaceted functions to maintain cellular homeostasis. Genetic and pharmacological approaches to manipulate individual organelles are powerful in probing their physiological roles. However, many of them are either slow in action, limited to certain organelles, or rely on toxic agents. Here, we designed a generalizable molecular tool utilizing phospholipase A/acyltransferases (PLAATs) for rapid induction of organelle defunctionalization via remodeling of the membrane phospholipid composition. In particular, we identified a minimal, fully catalytic PLAAT with no unfavorable side effects. Chemically-induced translocation of the engineered PLAAT to the mitochondria surface resulted in their rapid deformation in a phospholipase activity dependent manner, followed by loss of luminal proteins as well as dissipated membrane potential, thus invalidating the functionality. To demonstrate wide applicability, we then adapted the molecular tool in peroxisomes, and observed leakage of matrix-resident functional proteins. The technique was compatible with optogenetic control, viral delivery and operation in primary neuronal cultures. Due to such versatility, the PLAAT strategy should present a novel utility in organelle biology of diverse contexts.

Inhibition of phospholipase A2, platelet aggregation and egg albumin induced rat paw oedema as anti-inflammatory effect of Peltophorun pterocarpus stem-bark

Background Most medicinal plants presently employed in traditional medicine are used without scientific evidence, thereby suggesting a need to explore efficient and reliable investigations of their potential. We, therefore, conducted the present study to ascertain the efficacy of flavonoid-rich extract of Peltophorum pterocarpum sterm-bark in the treatment and management of inflammatory disorders as employed in folk medicine. Materials and methods Flavonoid-rich extract of Peltophorum pterocarpum sterm-bark and a total of fifty-five (55) Wistar rats were used for this study. Eighteen (18) mice were used for toxicity testing, and the phytochemical analysis was done using the Trease and Evans method, while the acute toxicity was done using Lorke’s method. In vivo anti-inflammatory study was done using the egg albumin-induced paw oedema method, while the in vitro anti-inflammatory studies were performed for the extract using phospholipase A2 inhibition and calcium chloride-induced platelet aggregation assays. Results The phytochemical analysis revealed that the extract of Peltophorum pterocarpum sterm-bark contains tannins, terpenoids, steroids, phenols, alkaloids, flavonoids, glycosides, and saponins ranging from 0.307 ± 0.02 to 1279.567 ± 149.868. The acute toxicity test of the extract showed no toxicity up to 5000 mg/kg body weight. In the systemic oedema of the rat paw, scalar doses of the extract significantly (p < 0.05) suppressed the development of paw oedema induced by egg albumin, particularly with the Indomethacin (1.77 ± 0.41) when compared with the control (5.50 ± 0.26). However, varying doses of the extract significantly (p < 0.05) inhibited phospholipase A2 activity and CaCl2-Induced platelet aggregation in a concentration, dose, and time-dependent manner, in comparison to prednisolone. Conclusion These results indicate that the extract exhibited anti-inflammatory potential, and the mechanism of this activity has a promising ability to inhibit phospholipase A2 activity and platelet aggregation in rats inflicted with paw oedema.

Deletion of Yersinia pestis ail causes temperature sensitive pleiotropic effects including cell lysis that are suppressed by carbon source, cations, or loss of phospholipase A activity

Maintenance of phospholipid (PL) and lipopoly- or lipooligo-saccharide (LPS or LOS) asymmetry in the outer membrane (OM) of Gram-negative bacteria is essential but poorly understood. The Yersinia pestis OM Ail protein was required to maintain lipid homeostasis and cell integrity at elevated temperature (37° C). Loss of this protein had pleiotropic effects. A Y. pestis Δail mutant and KIM6 + wild- type were systematically compared for (i) growth requirements at 37° C, (ii) cell structure, (iii) antibiotic and detergent sensitivity, (iv) proteins released into supernates, (v) induction of the heat shock response, and (vi) physiological and genetic suppressors that restored the wild- type phenotype. The Δail mutant grew normally at 28° C but lysed at 37° C when it entered stationary phase as shown by cell count, SDS-PAGE of cell supernatants, and electron microscopy. Immuno-fluorescent microscopy showed that the Δail mutant did not assemble Caf1 capsule. Expression of heat shock promoters rpoE or rpoH fused to a lux operon reporter were not induced when the Δail mutant was shifted from the 28° C to 37° C (p<0.001 and p<0.01 respectively). Mutant lysis was suppressed by addition of 11 mM glucose, 22 or 44 mM glycerol, 2.5 mM Ca 2+ , or 2.5 mM Mg 2+ to the growth medium, or by a mutation in the phospholipase A gene ( pldA ::miniTn 5 , ΔpldA, or PldA S164A ). A model, accounting for the temperature-sensitive lysis of the Δail mutant and the Ail-dependent stabilization of the OM tetraacylated LOS at 37°C is presented. IMPORTANCE The Gram-negative pathogen, Yersinia pestis , transitions between a flea vector (ambient temperature) and a mammalian host (37° C). In response to 37° C, Y. pestis modifies its outer membrane (OM) by reducing the fatty acid content in lipid A, changing the outer leaflet from being predominantly hexaacylated to being predominantly tetraacylated. It also increases the Ail concentration, so it becomes the most prominent OM protein. Both measures are needed for Y. pestis to evade the host innate immune response. Deletion of ail destabilizes the OM at 37° C causing the cells to lyse. These results show that a protein is essential for maintaining lipid asymmetry and lipid homeostasis in the bacterial OM.