Phospholipid translocation and miltefosine potency require both L. donovani miltefosine transporter and the new protein LdRos3 in Leishmania parasites

The antitumor drug miltefosine has been recently approved as the first oral drug active against visceral leishmaniasis. We have previously identified the L. donovani miltefosine transporter (LdMT) as a P-type ATPase involved in phospholipid translocation at the plasma membrane of Leishmania parasites. Here we show that this protein is essential but not sufficient for the phospholipid translocation activity and, thus, for the potency of the drug. Based on recent findings in yeast, we have identified the putative beta subunit of LdMT, named LdRos3, as another protein factor required for the translocation activity. LdRos3 belongs to the CDC50/Lem3 family, proposed as likely beta subunits for P4-ATPases. The phenotype of LdRos3-defective parasites was identical to that of the LdMT-/-, including a defect in the uptake of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)-phosphatidylserine, generally considered as not affected in Lem3p-deficient yeast. Both LdMT and LdRos3 normally localized to the plasma membrane but were retained inside the endoplasmic reticulum in the absence of the other protein or when inactivating point mutations were introduced in LdMT. Modulating the expression levels of either protein independently, we show that any one of them could behave as the protein limiting the level of flippase activity. Thus, LdMT and LdRos3 seem to form part of the same translocation machinery that determines flippase activity and miltefosine sensitivity in Leishmania, further supporting the consideration of CDC50/Lem3 proteins as beta subunits required for the normal functioning of P4-ATPases.     

Breakdown in membrane asymmetry regulation leads to monocyte recognition of P. falciparum-infected red blood cells

The human malaria parasite Plasmodium falciparum relies on lipids to survive; this makes its lipid metabolism an attractive drug target. The lipid phosphatidylserine (PS) is usually confined to the inner leaflet of the red blood cell membrane (RBC) bilayer; however, some studies suggest that infection with the intracellular parasite results in the presence of this lipid in the RBC membrane outer leaflet, where it could act as a recognition signal to phagocytes. Here, we used fluorescent lipid analogues and probes to investigate the enzymatic reactions responsible for maintaining asymmetry between membrane leaflets, and found that in parasitised RBCs the maintenance of membrane asymmetry was partly disrupted, and PS was increased in the outer leaflet. We examined the underlying causes for the differences between uninfected and infected RBCs using fluorescent dyes and probes, and found that calcium levels increased in the infected RBC cytoplasm, whereas membrane cholesterol was depleted from the erythrocyte plasma membrane. We explored the resulting effect of PS exposure on enhanced phagocytosis by monocytes, and show that infected RBCs must expend energy to limit phagocyte recognition, and provide experimental evidence that PS exposure contributes to phagocytic recognition of P. falciparum-infected RBCs. Together, these findings underscore the pivotal role for PS exposure on the surface of Plasmodium falciparum-infected erythrocytes for in vivo interactions with the host immune system, and provide a rationale for targeted antimalarial drug design.     

Loss of P4 ATPases Drs2p and Dnf3p disrupts aminophospholipid transport and asymmetry in yeast post-Golgi secretory vesicles

Eukaryotic plasma membranes generally display asymmetric lipid distributions with the aminophospholipids concentrated in the cytosolic leaflet. This arrangement is maintained by aminophospholipid translocases (APLTs) that use ATP hydrolysis to flip phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the external to the cytosolic leaflet. The identity of APLTs has not been established, but prime candidates are members of the P4 subfamily of P-type ATPases. Removal of P4 ATPases Dnf1p and Dnf2p from budding yeast abolishes inward translocation of 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] (NBD)-labeled PS, PE, and phosphatidylcholine (PC) across the plasma membrane and causes cell surface exposure of endogenous PE. Here, we show that yeast post-Golgi secretory vesicles (SVs) contain a translocase activity that flips NBD-PS, NBD-PE, and NBD-PC to the cytosolic leaflet. This activity is independent of Dnf1p and Dnf2p but requires two other P4 ATPases, Drs2p and Dnf3p, that reside primarily in the trans-Golgi network. Moreover, SVs have an asymmetric PE arrangement that is lost upon removal of Drs2p and Dnf3p. Our results indicate that aminophospholipid asymmetry is created when membrane flows through the Golgi and that P4-ATPases are essential for this process.     

Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity,a process that is counteracted by sandfly saliva

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host''s immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.     

LABCG2, a New ABC Transporter Implicated in Phosphatidylserine Exposure,Is Involved in the Infectivity and Pathogenicity of Leishmania

Leishmaniasis is a neglected disease produced by the intracellular protozoan parasite Leishmania. In the present study, we show that LABCG2, a new ATP-binding cassette half-transporter (ABCG subfamily) from Leishmania, is involved in parasite virulence. Down-regulation of LABCG2 function upon expression of an inactive mutant version of this half-transporter (LABCG2^K/M) is shown to reduce the translocation of short-chain analogues of phosphatidylserine (PS). This dominant-negative phenotype is specific for the headgroup of the phospholipid, as the movement of phospholipid analogues of phosphatidylcholine, phosphatidylethanolamine or sphingomyelin is not affected. In addition, promastigotes expressing LABCG2^K/M expose less endogenous PS in the stationary phase than control parasites. Transient exposure of PS at the outer leaflet of the plasma membrane is known to be one of the mechanisms used by Leishmania to infect macrophages and to silence their immune response. Stationary phase/metacyclic promastigotes expressing LABCG2^K/M are less infective for macrophages and show decreased pathogenesis in a mouse model of cutaneous leishmaniasis. Thus, mice infected with parasites expressing LABCG2^K/M did not develop any lesion and showed significantly lower inflammation and parasite burden than mice infected with control parasites. Our results indicate that LABCG2 function is required for the externalization of PS in Leishmania promastigotes, a process that is involved in the virulence of the parasite.     

Topological distribution of aminophospholipid fatty acids in trout intestinal brush-border membrane

The transbilayer distribution of aminophospholipids in trout intestinal brush-border membrane has been investigated using trinitrobenzene sulfonic acid (TNBS). In the middle intestine, phosphatidylethanolamine (PE) is symmetrically distributed between the two leaflets while 68% of the phosphatidylserine (PS) are located in the inner membrane leaflet. In the posterior intestine, 64% of the PE and 69% of the PS are located in the inner membrane leaflet. When asymmetrically distributed, the inner species of PE and PS have a higher content of 22:6(n − 3) than the outer ones. This asymmetric distribution of docosahexaenoic acid in trout intestinal brush-border membrane might be related to the rod-like shape of the microvillus membrane and to its metabolism to hydroxylated derivatives.     

Mechanisms of apoptotic phosphatidylserine exposure

It has been a long-standing enigma which scramblase causes phosphatidylserine residues to be exposed on the surface of apoptotic cells, thereby facilitating the phagocytic recognition, engulfment and destruction of apoptotic corpses. In a recent paper in Science, Nagata and coworkers reveal that the scramblases Xkr8 and its C. elegans ortholog, CED-8, are activated by caspase cleavage in apoptotic cells.All cells are separated from the extracellular environment by the plasma membrane, a phospholipid bilayer that prevents diffusion of proteins, ions and other essential molecules into the extracellular space and constitutes the structure in which membrane proteins are embedded. In animal cells, the lipid composition of the outer and inner leaflets of the plasma membrane is not symmetrical. Phosphatidylcholine (PC) and sphingomyelin (SM) are mainly present in the outer leaflet of the plasma membrane, whereas phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylethanolamine (PE) are restricted to the inner leaflet. This lipid asymmetry is maintained by the combined action of ATP-dependent enzymes called flippases and floppases, which specifically translocate phospholipids and other molecules from the outer to the inner membrane leaflet and from the inner to the outer membrane leaflet, respectively¹. Lipid composition asymmetry not only defines the curvature and electrochemical properties of the plasma membrane, but is also essential for the correct function of determined lipids, as for instance, PI, which only functions as a second messenger if present in the inner leaflet². Nonetheless, several physiologically relevant processes as diverse as platelet activation, neurotransmitter release, sperm capacitation or apoptosis, require dissipation of plasma membrane lipid asymmetry, a process known as scrambling. The enzymes responsible for this activity are called scramblases, and function to randomize the distribution of phospholipids between both membrane leaflets in an ATP-independent manner^2,3,4.Although plasma membrane asymmetry and the existence of flippases, floppases and scramblases have been known for decades, the identity of the specific enzymes involved in these activities has only begun to be revealed during the last few years. Very recently, the group of Shigekazu Nagata identified TMEM16F as the long sought-after calcium-dependent phospholipid scramblase³. However, to date, the identity of the scramblase(s) involved in apoptosis-related (and calcium-independent) PS exposure had remained elusive. Cell surface PS exposure is a classic feature of apoptotic cells and acts as an “eat me” signal allowing phagocytosis of post-apoptotic bodies. In a recent paper in Science, Nagata''s group identified Xk-Related Protein 8 (Xkr8) as the enzyme responsible for this activity and demonstrated an evolutionarily conserved role of this protein in apoptosis-induced lipid scrambling⁵.To identify enzymes involved in membrane lipid scrambling, Nagata''s group took advantage of their previously generated mouse Ba/F3 pro-B cell line³, which presented a high basal level of PS exposure. They then generated a cDNA library from Ba/F3 cells and overexpressed it in the parental cell line. Through sequential enrichment of cells with increased PS exposure, they were able to isolate a cDNA encoding the Xkr8 protein, which enhanced PS scrambling when overexpressed. Xkr8 overexpression (but not that of TMEM16F) was able to increase apoptosis-associated PS exposure. The authors then noticed that both impaired apoptosis-induced PS exposure and deficient post-apoptotic body clearance were correlated with low Xkr8 expression in leukemia and lymphoma cell lines, which was linked to hypermethylation of its promoter. Interestingly, these two alterations were reverted either by overexpressing Xkr8 or by restitution of endogenous Xkr8 expression after treatment with the demethylating agent 5-aza-2′-deoxycytidine (DAC), suggesting that methylation of the Xrk8 promoter may be a mechanism by which tumor cells evade their phagocytosis after apoptotic death, which may result in increased local inflammation, thus favoring tumor progression. Far from being restricted only to PS exposure, Xrk8 overexpression was able to promote scrambling of multiple lipid species during apoptosis, which was demonstrated by incorporation of fluorescent PC and SM analogues. This scrambling activity was restricted to apoptotic events, as Xkr8 overexpression had no effect on Ca²⁺-induced PS exposure. This specificity may be explained by the presence of an evolutionarily conserved caspase recognition site near Xkr8 C-terminal region, whose mutation prevented both Xkr8 cleavage by caspase-3 or -7 and PS exposure during the course of apoptosis (Figure 1). These results from human cell lines were confirmed in Xkr8^−/− mouse embryonic fibroblasts and fetal thymocytes, which were unable to expose PS upon induction of apoptosis, underscoring the broad physiological relevance of Xkr8 in the apoptotic process. Finally, the authors moved to the nematode Caenorhabditis elegans to analyze whether the role of Xpr8 as lipid scramblase is evolutionarily conserved. C. elegans harbors only one ortholog of Xk proteins, CED-8, known to participate in the phagocytic removal of apoptotic corpses⁶. To determine the role of CED-8 in PS exposure, the authors took advantage of the “floater” assay, which is based on the appearance of floating cells (“floaters”) that have detached from developing C. elegans embryos defective for apoptotic cell phagocytosis⁷. Nagata''s group discovered that ced-8 deficiency leads to the accumulation of floaters. Moreover, ced-8 deficiency synergistically enhanced the number of floaters found in other engulfment mutants, which suggests that CED-8 function is not redundant to that developed by previously known engulfment mutants. This enhancing effect of ced-8 deletion was dependent on CED-3, the C. elegans ortholog of caspase-3, confirming the aforementioned results in mammalian cells. The authors then characterized that floaters resulting from ced-8 deletion show a largely deficient PS exposure after developmental apoptosis, confirming the evolutionarily conserved role of Xk-related proteins in apoptosis-induced lipid scrambling. However, they observed that ced-8 deletion does not lead to a total impairment in apoptotic PS presentation, suggesting that additional proteins must be involved in this process. Indeed, apoptosis-inducing factor can induce PS exposure in mammalian cells in a caspase-independent fashion⁸, and the C. elegans AIF ortholog, WAF-1, physically interacts with and activates another scramblase, SCRM-1⁴.Open in a separate windowFigure 1Xrp8 acts as apoptosis-induced lipid scramblase. Under normal conditions, the combined action of multiple mechanisms, including the activity of flippases and floppases, maintains lipid asymmetry between the outer and inner leaflets of the plasma membrane. Once apoptotic program is activated, caspases-3 and -7 are able to cleave and activate Xrp8 protein, which acts as a lipid scramblase and leads to the loss of lipid asymmetry, resulting in PS exposure to the extracellular space. This acts as the “eat-me” signal that will allow phagocytosis of post-apoptotic cell corpses. PC, phosphatidylcholine; SM, sphingomyelin; PE, phosphatidylethanolamine; PS, phosphatidylserine.In summary, through a series of elegant manipulations, Nagata''s group has found the long-sought caspase-activated lipid scramblase that mediates the exposure of “eat-me” signals in post-apoptotic cell corpses. Further studies involving Xkr8 protein, including the mechanisms participating in its epigenetic repression may open new roads for the study of autoimmune diseases, such as lupus erythematosus, which is associated with failure in the post-apoptotic corpse clearance system.     

In vitro effects of triterpenic acids from olive leaf extracts on the mitochondrial membrane potential of promastigote stage of Leishmania spp

Protozoan diseases, such as leishmaniasis, are a cause of considerable morbidity throughout the world, affecting millions every year. In this study, two triterpenic acids (maslinic and oleanolic acids) were isolated from Tunisian olive leaf extracts and their in vitro activity against the promastigotes stage of Leishmania (L.) infantum and Leishmania (L.) amazonensis was investigated. Maslinic acid showed the highest activity with an IC₅₀ of 9.32 ± 1.654 and 12.460 ± 1.25 μg/ml against L. infantum and L. amazonensis, respectively. The mechanism of action of these drugs was investigated by detecting changes in the phosphatidylserine (PS) exposure, the plasma membrane permeability, the mitochondrial membrane potential and the ATP level production in the treated parasites. By using the fluorescent probe SYTOX® Green, both triterpenic acids showed that they produce a time-dependent plasma membrane permeabilization in the treated Leishmania species. In addition, spectrofluorimeteric data revealed the surface exposure of PS in promastigotes. Both molecules reduced the mitochondrial membrane potential and decreased the ATP levels to 15% in parasites treated with IC₉₀ for 24 h. We conclude that the triterpenic acids tested in this study, show potential as future therapeutic alternative against leishmaniasis. Further studies are needed to confirm this.     

An in vitro study of apoptotic like death in Leishmania donovani promastigotes by withanolides

The aim of this study was to isolate and evaluate the withanolides in inducing apoptotic like death in Leishmania donovani in vitro. Withanolides were fractionated and isolated from the leaves of Withania somnifera and LC-MS/MS analysis of two fractions namely, F5 and F6 of ethanolic extracts, obtained through column chromatography with silica gel, was performed. The antileishmanial effect of withanolides on L. donovani promastigotes was assessed in vitro using PI dye exclusion test. The effect of withanolides on promastigote morphology was determined by scanning electron microscopy. To understand their mode of action against L. donovani, DNA fragmentation, quantification of parasites at sub G₀/G₁ phase, determination of phosphatidylserine externalization, measurement of reactive oxygen species (ROS) and mitochondrial membrane potential (Ψ_m) were done. Results showed that LC–MS/MS analysis confirmed the presence of withanolides in isolated fractions. Treatment with withanolides resulted in morphological alterations from spindle to round shape and loss of flagella/cell integrity in promastigotes. Moreover, it induced DNA nicks, cell cycle arrest at sub G₀/G₁ phase and externalization of phosphatidylserine in dose and time dependent manner via increase in ROS and decrease in Ψ_m. Results of this study indicate that withanolides induce apoptotic like death through the production of ROS from mitochondria and disruption of Ψ_m in promastigotes of L donovani.     

Phosphatidylserine exposure in living cells

Abstract

P4-ATPases, a subfamily of P-type ATPases, translocate cell membrane phospholipids from the exoplasmic/luminal leaflet to the cytoplasmic leaflet to generate and maintain membrane lipid asymmetry. Exposure of phosphatidylserine (PS) in the exoplasmic leaflet is well known to transduce critical signals for apoptotic cell clearance and platelet coagulation. PS exposure is also involved in many other biological processes, including myoblast and osteoclast fusion, and the immune response. Moreover, mounting evidence suggest that PS exposure is critical for neuronal regeneration and degeneration. In apoptotic cells, PS exposure is induced by irreversible activation of scramblases and inactivation of P4-ATPases. However, how PS is reversibly exposed and restored in viable cells during other biological processes remains poorly understood. In the present review, we discuss the physiological significance of reversible PS exposure in living cells, and the putative roles of flippases, floppases, and scramblases.     

Genomic and Molecular Characterization of Miltefosine Resistance in Leishmania infantum Strains with Either Natural or Acquired Resistance through Experimental Selection of Intracellular Amastigotes

During the last decade miltefosine (MIL) has been used as first-line treatment for visceral leishmaniasis in endemic areas with antimonial resistance, but a decline in clinical effectiveness is now being reported. While only two MIL-resistant Leishmania infantum strains from HIV co-infected patients have been documented, phenotypic MIL-resistance for L. donovani has not yet been identified in the laboratory. Hence, a better understanding of the factors contributing to increased MIL-treatment failure is necessary. Given the paucity of defined MIL-resistant L. donovani clinical isolates, this study used an experimental amastigote-selected MIL-resistant L. infantum isolate (LEM3323). In-depth exploration of the MIL-resistant phenotype was performed by coupling genomic with phenotypic data to gain insight into gene function and the mutant phenotype. A naturally MIL-resistant L. infantum clinical isolate (LEM5159) was included to compare both datasets. Phenotypically, resistance was evaluated by determining intracellular amastigote susceptibility in vitro and actual MIL-uptake. Genomic analysis provided supportive evidence that the resistance selection model on intracellular amastigotes can be a good proxy for the in vivo field situation since both resistant strains showed mutations in the same inward transporter system responsible for the acquired MIL-resistant phenotype. In line with previous literature findings in promastigotes, our data confirm a defective import machinery through inactivation of the LiMT/LiRos3 protein complex as the main mechanism for MIL-resistance also in intracellular amastigotes. Whole genome sequencing analysis of LEM3323 revealed a 2 base pair deletion in the LiMT gene that led to the formation an early stop codon and a truncation of the LiMT protein. Interestingly, LEM5159 revealed mutations in both the LiMT and LiRos3 genes, resulting in an aberrant expression of the LiMT protein. To verify that these mutations were indeed accountable for the acquired resistance, transfection experiments were performed to re-establish MIL-susceptibility. In LEM3323, susceptibility was restored upon expression of a LiMT wild-type gene, whereas the MIL-susceptibility of LEM5159 could be reversed after expression of the LiRos3 wild-type gene. The aberrant expression profile of the LiMT protein could be restored upon rescue of the LiRos3 gene both in the LEM5159 clinical isolate and a ΔLiRos3 strain, showing that expression of LdMT is dependent on LdRos3 expression. The present findings clearly corroborate the pivotal role of the LiMT/LiRos3 complex in resistance towards MIL.     

Integrity of the Actin Cytoskeleton of Host Macrophages is Essential for Leishmania donovani Infection

Visceral leishmaniasis is a vector-borne disease caused by an obligate intracellular protozoan parasite Leishmania donovani. The molecular mechanism involved in internalization of Leishmania is poorly understood. The entry of Leishmania involves interaction with the plasma membrane of host cells. We have previously demonstrated the requirement of host membrane cholesterol in the binding and internalization of L. donovani into macrophages. In the present work, we explored the role of the host actin cytoskeleton in leishmanial infection. We observed a dose-dependent reduction in the attachment of Leishmania promastigotes to host macrophages upon destabilization of the actin cytoskeleton by cytochalasin D. This is accompanied by a concomitant reduction in the intracellular amastigote load. We utilized a recently developed high resolution microscopy-based method to quantitate cellular F-actin content upon treatment with cytochalasin D. A striking feature of our results is that binding of Leishmania promastigotes and intracellular amastigote load show close correlation with cellular F-actin level. Importantly, the binding of Escherichia coli remained invariant upon actin destabilization of host cells, thereby implying specific involvement of the actin cytoskeleton in Leishmania infection. To the best of our knowledge, these novel results constitute the first comprehensive demonstration on the specific role of the host actin cytoskeleton in Leishmania infection. Our results could be significant in developing future therapeutic strategies to tackle leishmaniasis.     

Insulin effect on the phospholipid organization and some enzyme activities of rat liver membrane fractions

1. The influence of insulin on rat liver membrane lipid composition, fluidity, some enzyme activities and asymmetry of microsomal phospholipids were investigated. 2. The total phospholipids and cholesterol were increased in microsomes and reduced in plasma membranes from insulin-treated rats. 3. Of all the investigated enzymes participating in the lipid metabolism, only the neutral sphingomyelinase activity was observed to be enhanced, whereas the ceramide-phosphatidylethanolamine (PE) synthetase and phospholipase A2 activities remained unchanged. 4. Insulin administration caused translocation of phosphatidylserine (PS) and PE to the outer leaflet and of phosphatidylinositol (PI) to the inner leaflet of microsomal membranes.     

Fertilization Induces a Transient Exposure of Phosphatidylserine in Mouse Eggs

Phosphatidylserine (PS) is normally localized to the inner leaflet of the plasma membrane and the requirement of PS translocation to the outer leaflet in cellular processes other than apoptosis has been demonstrated recently. In this work we investigated the occurrence of PS mobilization in mouse eggs, which express flippase Atp8a1 and scramblases Plscr1 and 3, as determined by RT-PCR; these enzyme are responsible for PS distribution in cell membranes. We find a dramatic increase in binding of flouresceinated-Annexin-V, which specifically binds to PS, following fertilization or parthenogenetic activation induced by SrCl₂ treatment. This increase was not observed when eggs were first treated with BAPTA-AM, indicating that an increase in intracellular Ca²⁺ concentration was required for PS exposure. Fluorescence was observed over the entire egg surface with the exception of the regions overlying the meiotic spindle and sperm entry site. PS exposure was also observed in activated eggs obtained from CaMKIIγ null females, which are unable to exit metaphase II arrest despite displaying Ca²⁺ spikes. In contrast, PS exposure was not observed in TPEN-activated eggs, which exit metaphase II arrest in the absence of Ca²⁺ release. PS exposure was also observed when eggs were activated with ethanol but not with a Ca²⁺ ionophore, suggesting that the Ca²⁺ source and concentration are relevant for PS exposure. Last, treatment with cytochalasin D, which disrupts microfilaments, or jasplakinolide, which stabilizes microfilaments, prior to egg activation showed that PS externalization is an actin-dependent process. Thus, the Ca²⁺ rise during egg activation results in a transient exposure of PS in fertilized eggs that is not associated with apoptosis.     

Phosphatidylethanolamine synthesized by four different pathways is supplied to the plasma membrane of the yeast Saccharomyces cerevisiae

In this study, we examined the contribution of the four different pathways of phosphatidylethanolamine (PE) synthesis in the yeast Saccharomyces cerevisiae to the supply of this phospholipid to the plasma membrane. These pathways of PE formation are decarboxylation of phosphatidylserine (PS) by (i) phosphatidylserine decarboxylase 1 (Psd1p) in mitochondria and (ii) phosphatidylserine decarboxylase 2 (Psd2p) in a Golgi/vacuolar compartment, (iii) incorporation of exogenous ethanolamine and ethanolamine phosphate derived from sphingolipid catabolism via the CDP-ethanolamine pathway in the endoplasmic reticulum (ER), and (iv) synthesis of PE through acylation of lyso-PE catalyzed by the acyl-CoA-dependent acyltransferase Ale1p in the mitochondria associated endoplasmic reticulum membrane (MAM). Deletion of PSD1 and/or PSD2 led to depletion of total cellular and plasma membrane PE level, whereas mutation in the other pathways had practically no effect. Analysis of wild type and mutants, however, revealed that all four routes of PE synthesis contributed not only to PE formation but also to the supply of PE to the plasma membrane. Pulse-chase labeling experiments with L[³H(G)]serine and [¹⁴C]ethanolamine confirmed the latter finding. Fatty acid profiling demonstrated a rather balanced incorporation of PE species into the plasma membrane irrespective of mutations suggesting that all four pathways of PE synthesis provide at least a basic portion of “correct” PE species required for plasma membrane biogenesis. In summary, the PE level in the plasma membrane is strongly influenced by total cellular PE synthesis, but fine tuned by selective assembly mechanisms.     

Drug susceptibility in Leishmania isolates following miltefosine treatment in cases of visceral leishmaniasis and post kala-azar dermal leishmaniasis

Background

With widespread resistance to antimonials in Visceral Leishmaniasis (VL) in the Indian subcontinent, Miltefosine (MIL) has been introduced as the first line therapy. Surveillance of MIL susceptibility in natural populations of Leishmania donovani is vital to preserve it and support the VL elimination program.

Methodology and Principal Findings

We measured in vitro susceptibility towards MIL and paromomycin (PMM) in L. donovani isolated from VL and PKDL, pre- and post-treatment cases, using an amastigote-macrophage model. MIL susceptibility of post-treatment isolates from cured VL cases (n = 13, mean IC₅₀±SD = 2.43±1.44 µM), was comparable (p>0.05) whereas that from relapses (n = 3, mean IC₅₀ = 4.72±1.99 µM) was significantly higher (p = 0.04) to that of the pre-treatment group (n = 6, mean IC₅₀ = 1.86±0.75 µM). In PKDL, post-treatment isolates (n = 3, mean IC₅₀ = 16.13±2.64 µM) exhibited significantly lower susceptibility (p = 0.03) than pre-treatment isolates (n = 5, mean IC₅₀ = 8.63±0.94 µM). Overall, PKDL isolates (n = 8, mean IC₅₀ = 11.45±4.19 µM) exhibited significantly higher tolerance (p<0.0001) to MIL than VL isolates (n = 22, mean IC₅₀ = 2.58±1.58 µM). Point mutations in the miltefosine transporter (LdMT) and its beta subunit (LdRos3) genes previously reported in parasites with experimentally induced MIL resistance were not present in the clinical isolates. Further, the mRNA expression profile of these genes was comparable in the pre- and post-treatment isolates. Parasite isolates from VL and PKDL cases were uniformly susceptible to PMM with respective mean IC₅₀ = 7.05±2.24 µM and 6.18±1.51 µM.

Conclusion

The in vitro susceptibility of VL isolates remained unchanged at the end of MIL treatment; however, isolates from relapsed VL and PKDL cases had lower susceptibility than the pre-treatment isolates. PKDL isolates were more tolerant towards MIL in comparison with VL isolates. All parasite isolates were uniformly susceptible to PMM. Mutations in the LdMT and LdRos3 genes as well as changes in the expression of these genes previously correlated with experimental resistance to MIL could not be verified for the field isolates.     

Coincident exposure of phosphatidylethanolamine and anionic phospholipids on the surface of irradiated cells

The major anionic phospholipid, phosphatidylserine (PS), and the neutral phospholipid, phosphatidylethanolamine (PE), are largely confined to the inner leaflet of the plasma membrane bilayer in mammalian cells under normal conditions. This asymmetry is lost when cells undergo apoptosis, become activated, or are exposed to irradiation, reactive oxygen species or certain drugs. It is not known whether exposure of anionic phospholipids (APLs) and PE occurs simultaneously or in the same region of the plasma membrane. Here we examined the coincidence of exposure of APLs and PE on the surface of bovine aortic endothelial cells and NS0 myeloma cells after irradiation. The cells were irradiated (5 Gy) and stained for APLs and PE using liposomes coated with either an Fab′ fragment of a PS-binding antibody (bavituximab) or a PE-binding peptide (duramycin). Using live cell imaging and flow cytometry, we showed that irradiation leads to synchronous externalization of APLs and PE. The time course of appearance of APLs and PE on the cell surface was the same and the two phospholipid types remained colocalized over time. Distinct patches double positive for APLs and PE were visible. Larger areas of APLs and PE appeared to have detached from the cytoskeleton to form membrane blebs which protruded and drifted on the cell surface. We conclude that APLs and PE coincidently appear on the external leaflet of the plasma membrane of cells after irradiation. Probably, this is because PE and the major APL, PS, share common regulatory mechanisms of translocation.     

The dependence of lipid asymmetry upon polar headgroup structure

The effect of lipid headgroup structure upon the stability of lipid asymmetry was investigated. Using methyl-β-cyclodextrin -induced lipid exchange, sphingomyelin (SM) was introduced into the outer leaflets of lipid vesicles composed of phosphatidylglycerol, phosphatidylserine (PS), phosphatidylinositol, or cardiolipin, in mixtures of all of these lipids with phosphatidylethanolamine (PE), and in a phosphatidylcholine/phosphatidic acid mixture. Efficient SM exchange (>85% of that expected for complete replacement of the outer leaflet) was obtained for every lipid composition studied. Vesicles containing PE mixed with anionic lipids showed nearly complete asymmetry which did not decay after 1 day of incubation. However, vesicles containing anionic lipids without PE generally only exhibited partial asymmetry, which further decayed after 1 day of incubation. Vesicles containing the anionic lipid PS were an exception, showing nearly complete and stable asymmetry. It is likely that the combination of multiple charged groups on PE and PS inhibit transverse diffusion of these lipids across membranes relative to those lipids that only have one anionic group. Possible explanations of this behavior are discussed. The asymmetry properties of PE and PS may explain some of their functions in plasma membranes.     

Bidirectional transbilayer movement of phospholipid analogs in human red blood cells. Evidence for an ATP-dependent and protein-mediated process.

The transbilayer movement of fluorescent and isotopically labeled analogs of phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) from the outer to the inner leaflet (flip) and from the inner to the outer leaflet (flop) of human red blood cells (RBC) was examined. The inward movement of 1-oleoyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole-aminocaproyl)- (C6-NBD-), 1-oleoyl-2-(N-(3-(3-[125I]iodo-4-hydroxyphenyl)propionyl)aminocaproyl)- (C6-125I-), or 1-oleoyl-2-(N-(3-3-[125I]iodo-4-azido-phenyl)propionyl)aminocaproyl- (C6-125I-N3-) analogs of PC and PE were relatively slow. In contrast, all analogs of PS and PE analogs containing aminododecanoic acid (C12 lipids) were rapidly transported to the cell's inner leaflet. Analysis of 125I-N3 lipids cross-linked to membrane proteins revealed labeling of 32-kDa Rh polypeptides that was dependent on the lipid's capacity to be transported to the inner leaflet but was independent of lipid species. To investigate whether lipids could also be transported from the inner to the outer leaflet, lipid probes residing exclusively in the inner leaflet were monitored for their appearance in the outer leaflet. Lipid movement could not be detected at 0 degrees C. At 37 degrees C, however, approximately 70% of the PC, 40% of the PE, and 15% of the PS redistributed to the cells outer leaflet, thereby attaining their normal asymmetric distribution. Continuous incubation in the presence of bovine serum albumin depleted the cells of the analogs (t1/2 approximately 1.5 h) in a manner that was independent of lipid species. Similar to the inward movement of aminophospholipids, the outward movement of PC, PE, and PS was ATP-dependent and could be blocked by oxidation of membrane sulfhydryls and by the histidine reagent bromophenacyl bromide. Evidence is presented which suggests that the outward movement of lipids is an intrinsic property of the cells unrelated to compensatory mechanisms due to an imbalance in lipid distribution.