Phospholipase D (PLD) is Present in Leishmania donovani and its Activity Increases in Response to Acute Osmotic Stress

Abstract

We report here that the signaling molecule phospholipase D (PLD) is present in the parasitic protozoan Leishmania donovani. In vitro enzymatic activity is dependent on Ca2+ and Mg2+ ions, its basal activity is stimulated by phosphatidyl-inositol-4,5-bisphosphate (PIP2) and its pH optima are pH 8.0 and pH 6.0. PLD activity increases 3-fold about 5 min after an abrupt decrease in osmolality from 317 mOsm (isosmotic) to 155 mOsm and increases 1.5-fold in response to an abrupt increase in osmolality to 617 mOsM. Cells grown for> 24 h under the anisosmotic conditions showed only marginal changes in activity compared to the controls grown under isosmotic conditions, indicating an adaptation to long-term exposure to hypo- or hyper-osmolarity. Immunologically, two isoforms, PLD1 and PLD2, are present. An analysis of in vitro PLD activity in anti-PLD immunocomplexes revealed that either hypotonic (cell swelling) or hypertonic stress (cell shrinking) causes an increase in PLD1 activation but a reduction in PLD2 activity. The interplay between these two isoforms results in a predominance for PLD1 in the observed increase when measuring total PLD activity. Finally, the increase in enzymatic activity in acute hyposmotic shock is accompanied by tyrosyl phosphorylation of the PLD1 isoform, suggesting a role for protein tyrosine kinase in the control of PLD activity in response to osmotic stress.

Phospholipid and sphingolipid metabolism in Leishmania

“…The activity of phospholipase D has been detected in L. donovani and this activity increases in response to acute osmotic stress [130]. T. brucei contains a membrane bound glycosyl-phosphatidylinositol phospholipase C (GPI-PLC) which can cleave dimyristoylglycerol from the variant surface glycoprotein and protein-GPI precursors [131, 132].…”

The exquisite regulation of PLD2 by a wealth of interacting proteins: S6K, Grb2, Sos, WASp and Rac2 (And a surprise discovery: PLD2 is a GEF)

“…In addition to tyrosine kinases, PLD2 could be phosphorylated by the serine/threonine kinase AKT at residue T 175 that serves to upregulate de novo DNA synthesis [129]. Phosphorylation can be seen in response to osmotic stress [177] and in cell proliferation [178]. …”

Mechanisms Sensing and Modulating Signals Arising From Cell Swelling

“…Two isoforms, PLD1 and PLD2, are present and an analysis of their in vitro activities revealed that either hypotonicity or hypertonicity causes an increase in PLD1 activation but a reduction in PLD2 activity with predominance for PLD1. The enzymatic activity during acute hyposmotic shock is accompanied by tyrosyl phosphorylation of the PLD1 isoform, suggesting osmotic control by protein tyrosine kinase [21].…”

“…Two mammalian PLD enzymes (PLD1 and PLD2) have been cloned and their intracellular regulators identified as ARF and Rho proteins, PKCα as well as PI(4,5)P 2 [20]. Although in mammalian cells no reports on the influence of cell swelling on PLD activity are available yet, in the protozoon Leishmania donovani PLD activity is reported to increase upon both cell swelling and cell shrinkage [21]. Two isoforms, PLD1 and PLD2, are present and an analysis of their in vitro activities revealed that either hypotonicity or hypertonicity causes an increase in PLD1 activation but a reduction in PLD2 activity with predominance for PLD1.…”