Enzymatic Characterization of Leishmania major Phosphatidylethanolamine Methyltransferases LmjPEM1 and LmjPEM2

Phosphatidylcholine (PC) is the most abundant phospholipid in the membranes of the human parasite Leishmania. It is synthesized via two metabolic routes, the de novo pathway that starts with the uptake of choline, and the threefold methylation of phosphatidylethanolamine. Choline was shown to be dispensable for Leishmania; thus, the methylation pathway likely represents the primary route for PC production. Here, we have identified and characterized two phosphatidylethanolamine methyltransferases, LmjPEM1and LmjPEM2. Both enzymes are expressed in promastigotes as well as in the vertebrate form amastigotes, suggesting that these methyltransferases are important for the development of the parasite throughout its life cycle. Heterologous expression in yeast has demonstrated that LmjPEM1 and LmjPEM2 complement the choline auxotrophy phenotype of a yeast double null mutant lacking phosphatidylethanolamine methyltransferase activity. LmjPEM1 catalyzes the first, and to a lesser extent, the second methylation reaction. In contrast, LmjPEM2 has the capacity to add the second and third methyl group onto phosphatidylethanolamine to yield (lyso)PC; it can also add the first methyl group, albeit with very low efficiency

The N-Terminal Domain and Glycosomal Localization of Leishmania Initial Acyltransferase LmDAT Are Important for Lipophosphoglycan Synthesis

Ether glycerolipids of Leishmania major are important membrane components as well as building blocks of various virulence factors. In L. major, the first enzyme of the ether glycerolipid biosynthetic pathway, LmDAT, is an unusual, glycosomal dihydroxyacetonephosphate acyltransferase important for parasite's growth and survival during the stationary phase, synthesis of ether lipids, and virulence. The present work extends our knowledge of this important biosynthetic enzyme in parasite biology. Site-directed mutagenesis of LmDAT demonstrated that an active enzyme was critical for normal growth and survival during the stationary phase. Deletion analyses showed that the large N-terminal extension of this initial acyltransferase may be important for its stability or activity. Further, abrogation of the C-terminal glycosomal targeting signal sequence of LmDAT led to extraglycosomal localization, did not impair its enzymatic activity but affected synthesis of the ether glycerolipid-based virulence factor lipophosphoglycan. In addition, expression of this recombinant form of LmDAT in a null mutant of LmDAT did not restore normal growth and survival during the stationary phase. These results emphasize the importance of this enzyme's compartmentalization in the glycosome for the generation of lipophosphoglycan and parasite's biology

Candida albicans Csy1p Is a Nutrient Sensor Important for Activation of Amino Acid Uptake and Hyphal Morphogenesis

Candida albicans is an important human pathogen that displays a remarkable ability to detect changes in its environment and to respond appropriately by changing its cell morphology and physiology. Serum- and amino acid-based media are known to induce filamentous growth in this organism. However, the mechanism by which amino acids induce filamentation is not yet known. Here, we describe the identification and characterization of the primary amino acid sensor of C. albicans, Csy1. We show that Csy1p plays an important role in amino acid sensing and filamentation. Loss of Csy1p results in a lack of amino acid-mediated activation of amino acid transport and a lack of induction of transcription of specific amino acid permease genes. Furthermore, a csy1Δ/csy1Δ strain, lacking Csy1p, is defective in filamentation and displays altered colony morphology in serum- and amino acid-based media. These data provide the first evidence that C. albicans utilizes the amino acid sensor Csy1p to probe its environment, coordinate its nutritional requirements, and determine its morphological state

Lipidomics and anti-trypanosomatid chemotherapy

Abstract Background Trypanosomatids such as Leishmania, Trypanosoma brucei and Trypanosoma cruzi belong to the order Kinetoplastida and are the source of many significant human and animal diseases. Current treatment is unsatisfactory and is compromised by the rising appearance of drug resistant parasites. Novel and more effective chemotherapeutics are urgently needed to treat and prevent these devastating diseases, which relies on the identification of essential, parasite specific targets that are absent in the host. Lipids constitute essential components of the cell and carry out multiple critical functions from building blocks of biological membranes to regulatory roles in signal transduction, organellar biogenesis, energy storage, and virulence. The recent technological advances of lipidomics has facilitated the broadening of our knowledge in the field of cellular lipid content, structure, functions, and metabolic pathways. Main body This review highlights the application of lipidomics (i) in the characterization of the lipidome of kinetoplastid parasites or of their subcellular structure(s), (ii) in the identification of unique lipid species or metabolic pathways that can be targeted for novel drug therapies, (iii) as an analytic tool to gain a deeper insight into the roles of specific enzymes in lipid metabolism using genetically modified microorganisms, and (iv) in deciphering the mechanism of action of anti-microbial drugs on lipid metabolism. Lastly, an outlook stating where the field is evolving is presented. Conclusion Lipidomics has contributed to the expanding knowledge related to lipid metabolism, mechanism of drug action and resistance, and pathogen–host interaction of trypanosomatids, which provides a solid basis for the development of better anti-parasitic pharmaceuticals

Unraveling the Mode of Action of the Antimalarial Choline Analog G25 in Plasmodium falciparum and Saccharomyces cerevisiae

Pharmacological studies have indicated that the choline analog G25 is a potent inhibitor of Plasmodium falciparum growth in vitro and in vivo. Although choline transport has been suggested to be the target of G25, the exact mode of action of this compound is not known. Here we show that, similar to its effects on P. falciparum, G25 prevents choline entry into Saccharomyces cerevisiae cells and inhibits S. cerevisiae growth. However, we show that the uptake of this compound is not mediated by the choline carrier Hnm1. An hnm1Δ yeast mutant, which lacks the only choline transporter gene HNM1, was not altered in the transport of a labeled analog of this compound. Eleven yeast mutants lacking genes involved in different steps of phospholipid biosynthesis were analyzed for their sensitivity to G25. Four mutants affected in the de novo cytidyldiphosphate-choline-dependent phosphatidylcholine biosynthetic pathway and, surprisingly, a mutant strain lacking the phosphatidylserine decarboxylase-encoding gene PSD1 (but not PSD2) were found to be highly resistant to this compound. Based on these data for S. cerevisiae, labeling studies in P. falciparum were performed to examine the effect of G25 on the biosynthetic pathways of the major phospholipids phosphatidylcholine and phosphatidylethanolamine. Labeling studies in P. falciparum and in vitro studies with recombinant P. falciparum phosphatidylserine decarboxylase further supported the inhibition of both the de novo phosphatidylcholine metabolic pathway and the synthesis of phosphatidylethanolamine from phosphatidylserine. Together, our data indicate that G25 specifically targets the pathways for synthesis of the two major phospholipids, phosphatidylcholine and phosphatidylethanolamine, to exert its antimalarial activity

The Trypanosoma brucei dihydroxyacetonephosphate acyltransferase TbDAT is dispensable for normal growth but important for synthesis of ether glycerophospholipids.

Glycerophospholipids are the most abundant constituents of biological membranes in Trypanosoma brucei, which causes sleeping sickness in humans and nagana in cattle. They are essential cellular components that fulfill various important functions beyond their structural role in biological membranes such as in signal transduction, regulation of membrane trafficking or control of cell cycle progression. Our previous studies have established that the glycerol-3-phosphate acyltransferase TbGAT is dispensable for growth, viability, and ester lipid biosynthesis suggesting the existence of another initial acyltransferase(s). This work presents the characterization of the alternative, dihydroxyacetonephosphate acyltransferase TbDAT, which acylates primarily dihydroxyacetonephosphate and prefers palmitoyl-CoA as an acyl-CoA donor. TbDAT restores the viability of a yeast double null mutant that lacks glycerol-3-phosphate and dihydroxyacetonephosphate acyltransferase activities. A conditional null mutant of TbDAT in T. brucei procyclic form was created and characterized. TbDAT was important for survival during stationary phase and synthesis of ether lipids. In contrast, TbDAT was dispensable for normal growth. Our results show that in T. brucei procyclic forms i) TbDAT but not TbGAT is the physiologically relevant initial acyltransferase and ii) ether lipid precursors are primarily made by TbDAT

HV-<i>Lm</i>DAT-ΔC₃ does not localize in the glycosomes.

<p>Wild type expressing recombinant HV-<i>Lm</i>DAT-ΔC₃ was analyzed by phase contrast (panel 1) or immunofluorescence microscopy using anti-V5 antibody (panel 2) or polyclonal antiserum specific to hypoxanthine guanine phosphoribosyltransferase (panel 3). Panel 4 shows the merge of panels 2 and 3.</p

Characterization of mutant forms of <i>Lm</i>DAT.

<p>(A) Schematic representation of human DHAPAT (<i>h</i>DHAPAT) and mutant forms of <i>Lm</i>DAT. The grey rectangle, the black rectangle and the hatched area depict the HV tag, the conserved domain, and the C-terminal glycosomal targeting tripeptide, respectively, and the asterisk depicts the point mutation. B) DHAPAT activity was quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027802#s2" target="_blank">Materials and Methods</a>. Equivalent of 0.5 mg protein extracts were applied for the assay. Null mutant alone or expressing HV-tagged wild-type and mutant forms of <i>Lm</i>DAT were used as a source of protein extracts. Activity is expressed as percentage of the positive control, the wild type (WT). The assay was performed twice in duplicate, and the graph depicts one representative experiment. Standard deviations are shown. (C) Western blot analyses in the presence of V5-specific (upper; V5) and hypoxanthine guanine phosphoribosyltransferase specific (lower; HGPRT; loading control) antibodies. Equivalent of 5×10⁷ cells were loaded in each lane. The apparent molecular weight is shown on the left. (D) Western blot analysis in the presence of WIC79.3 antibody to detect LPG. Equivalent of 10⁶ cells were loaded in each lane. (B, C, D): 1, <i>Δlmdat/Δlmdat</i>; 2, <i>Δlmdat/Δlmdat [HV-LmDAT NEO]</i>; 3, <i>Δlmdat/Δlmdat [HV-ΔN₅₄₆-LmDAT NEO]</i>; 4, <i>Δlmdat/Δlmdat [HV-ΔN₆₈₆-LmDAT NEO]</i>; 5, <i>Δlmdat/Δlmdat [HV-LmDAT-ΔC₇₃₃ NEO]</i>; 6, <i>Δlmdat/Δlmdat [HV-LmDAT-ΔC₃ NEO]</i>; 7, <i>Δlmdat/Δlmdat [HV-LmDAT^K852L NEO]</i>; WT, wild type.</p

Growth curves.

<p>Cells were inoculated at a cell density of 5×10⁵/ml and were enumerated with a hemacytometer as a function of time. The assay was performed twice and the graphs represent a typical experiment. Standard deviations are shown. (A) Black circles, wild type; grey circles, complemented line <i>Δlmdat</i>/<i>Δlmdat [HV-LmDAT NEO]</i>; white circles, <i>Δlmdat</i>/<i>Δlmdat</i>; white triangles, <i>Δlmdat/Δlmdat [HV-ΔN₅₄₆-LmDAT NEO]</i>; grey triangles, <i>Δlmdat/Δlmdat [HV-ΔN₆₈₆-LmDAT NEO]</i>; black triangles, <i>Δlmdat/Δlmdat [HV-LmDAT-ΔC₇₃₃ NEO]</i>. (B) Black circles, wild type; grey circles, complemented line <i>Δlmdat</i>/<i>Δlmdat [HV-LmDAT NEO]</i>; white circles, <i>Δlmdat</i>/<i>Δlmdat</i>; white triangles, <i>Δlmdat</i>/<i>Δlmdat [HV-LmDAT-ΔC₃ NEO]</i>; grey triangles, <i>Δlmdat</i>/<i>Δlmdat [HV-LmDAT^K852L NEO]</i>.</p