Regulated expression of the Leishmania major surface virulence factor lipophosphoglycan using conditionally destabilized fusion proteins.
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ABSTRACT:
SUBMITTER: Madeira da Silva L
PROVIDER: S-EPMC2678649 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Protozoan parasites of the genus Leishmania undergo a complex life cycle involving transmission by biting sand flies and replication within mammalian macrophage phagolysosomes. A major component of the Leishmania surface coat is the glycosylphosphatidylinositol (GPI)-anchored polysaccharide called lipophosphoglycan (LPG). LPG has been proposed to play many roles in the infectious cycle, including protection against complement and oxidants, serving as the major ligand for macrophage adhesion, and as a key factor mitigating host responses by deactivation of macrophage signaling pathways. However, all structural domains of LPG are shared by other major surface or secretory products, providing a biochemical redundancy that compromises the ability of in vitro tests to establish whether LPG itself is a virulence factor. To study truly lpg(-) parasites, we generated Leishmania major lacking the gene LPG1 [encoding a putative galactofuranosyl (Gal(f)) transferase] by targeted gene disruption. The lpg1(-) parasites lacked LPG but contained normal levels of related glycoconjugates and GPI-anchored proteins. Infections of susceptible mice and macrophages in vitro showed that these lpg(-) Leishmania were highly attenuated. Significantly and in contrast to previous LPG mutants, reintroduction of LPG1 into the lpg(-) parasites restored virulence. Thus, genetic approaches allow dissection of the roles of this complex family of interrelated parasite virulence factors, and definitively establish the role of LPG itself as a parasite virulence factor. Because the lpg1(-) mutant continue to synthesize bulk GPI-anchored Gal(f)-containing glycolipids other than LPG, a second pathway distinct from the Golgi-associated LPG synthetic compartment must exist.
| S-EPMC16855 | biostudies-literature
Project description:Immediately following their deposition into the mammalian host by an infected sand fly vector, Leishmania parasites encounter and are engulfed by a variety of cell types. From there, parasites may transit to other cell types, primarily macrophages or dendritic cells, where they replicate and induce pathology. During this time, Leishmania cells undergo a dramatic transformation from the motile non-replicating metacyclic stage to the non-motile replicative amastigote stage, a differentiative process that can be termed amastigogenesis. To follow this at the single cell level, we identified a suite of experimental 'landmarks' delineating different stages of amastigogenesis qualitatively or quantitatively, including new uses of amastigote-specific markers that showed interesting cellular localizations at the anterior or posterior ends. We compared amastigogenesis in synchronous infections of peritoneal and bone-marrow derived macrophages (PEM, BMM) or dendritic cells (BMDC). Overall, the marker suite expression showed an orderly transition post-infection with similar kinetics between host cell types, with the emergence of several amastigote traits within 12 hours, followed by parasite replication after 24 hours, with parasites in BMM or BMDC initiating DNA replication more slowly. Lipophosphoglycan (LPG) is a Leishmania virulence factor that facilitates metacyclic establishment in host cells but declines in amastigotes. Whereas LPG expression was lost by parasites within PEM by 48 hours, >40% of the parasites infecting BMM or BMDC retained metacyclic-level LPG expression at 72 hr. Thus L. major may prolong LPG expression in different intracellular environments, thereby extending its efficacy in promoting infectivity in situ and during cell-to-cell transfer of parasites expressing this key virulence factor.
| S-EPMC9642900 | biostudies-literature
Project description:Lipophosphoglycan (LPG), is the major cell surface molecule of promastigotes of all Leishmania species. It is comprised of three domains: a conserved glycosylphosphatidylinositol anchor linked to a repeating phosphorylated disaccharide (P2; PO4-6Gal beta 1-4Man alpha 1-) backbone and capped with a neutral oligosaccharide. In Leishmania major the backbone is substituted at the C(O)3 of the Galp residue with side chains containing Galp, Glcp and Arap residues whereas in Leishmania donovani the backbone is unsubstituted. We report the solubilization of a (beta 1-3) galactosyltransferase [(beta 1-3)GalT] from a L. major microsomal preparation using Triton X-100. Solubilization occurs with a 10-fold stimulation of enzyme activity. This (beta 1-3)GalT specifically transfers Gal residues from UDP-Gal to exogenously added L. donovani LPG acceptor. Depolymerization of the [14C]Gal-labelled LPG product with mild acid and analysis by high-performance anion-exchange chromatography detected only the phosphotrisaccharide. (P3; PO4-6([14C]Gal beta 1-3-4Man alpha 1-) found in L. major LPG. This contrasts with the activity of the membrane-bound enzyme which also synthesizes the larger phosphosaccharide units[Ng, Handman and Bacic (1994) Glycobiology 4, 845-853]. This suggests that more than one (beta 1-3)GalT is involved in the addition of these Gal units and that the solubilized activity is the (beta 1-3)GalT that adds the first beta Gal residue to the acceptor. The (beta 1-3)GalT was partially purified by lectin-affinity chromatography and used to establish the K(m) values for UDP-Gal (445 microM) and L. donovani acceptor (280 microM as P2 molar equivalent) in kinetic assays. Inhibition studies with various glycosides and mono- and di-saccharides established the P2 repeating unit as the minimum acceptor structure recognized by (beta 1-3)GalT. The detergent-solubilized (beta 1-3)GalT was reversibly inactivated by millimolar concentrations of univalent anionic salts. The (beta 1-3)GalT had an absolute requirement for Mn2+ and also required Mg2+ for optimum activity; Mg2+ cannot substitute for Mn2+, which is loosely bound to beta (1-3)GalT and is probably involved in the correct folding of the enzyme. The (beta 1-3)GalT was unaffected by Ca2+ ions, but were irreversibly inactivated by micromolar levels of transition metal ions (Cu2+ > Zn2+ > Ni2 > Co2+). The (beta 1-3)GalT activity was also inhibited by diethyl pyrocarbonate, but not by N-ethylmaleimide or iodoacetamide, suggesting that active-site histidine residues, rather than cysteine residue(s), are important for enzyme activity.
| S-EPMC1217470 | biostudies-literature
Project description:Parasitic protozoa, such as Leishmania species, are thought to express a number of surface and secreted nucleoside triphosphate diphosphohydrolases (NTPDases) which hydrolyze a broad range of nucleoside tri- and diphosphates. However, the functional significance of NTPDases in parasite virulence is poorly defined. The Leishmania major genome was found to contain two putative NTPDases, termed LmNTPDase1 and 2, with predicted NTPDase catalytic domains and either an N-terminal signal sequence and/or transmembrane domain, respectively. Expression of both proteins as C-terminal GFP fusion proteins revealed that LmNTPDase1 was exclusively targeted to the Golgi apparatus, while LmNTPDase2 was predominantly secreted. An L. major LmNTPDase1 null mutant displayed increased sensitivity to serum complement lysis and exhibited a lag in lesion development when infections in susceptible BALB/c mice were initiated with promastigotes, but not with the obligate intracellular amastigote stage. This phenotype is characteristic of L. major strains lacking lipophosphoglycan (LPG), the major surface glycoconjugate of promastigote stages. Biochemical studies showed that the L. major NTPDase1 null mutant synthesized normal levels of LPG that was structurally identical to wild type LPG, with the exception of having shorter phosphoglycan chains. These data suggest that the Golgi-localized NTPase1 is involved in regulating the normal sugar-nucleotide dependent elongation of LPG and assembly of protective surface glycocalyx. In contrast, deletion of the gene encoding LmNTPDase2 had no measurable impact on parasite virulence in BALB/c mice. These data suggest that the Leishmania major NTPDase enzymes have potentially important roles in the insect stage, but only play a transient or non-major role in pathogenesis in the mammalian host.
| S-EPMC4270689 | biostudies-other
Project description:BACKGROUND:The Leishmania spp. protozoa are introduced into humans through a sand fly blood meal, depositing the infectious metacyclic promastigote form of the parasite into human skin. Parasites enter a variety of host cells, although a majority are found in macrophages where they replicate intracellularly during chronic leishmaniasis. Symptomatic leishmaniasis causes considerable human morbidity in endemic regions. The Leishmania spp. evade host microbicidal mechanisms partially through virulence-associated proteins such as the major surface protease (MSP or GP63), to inactivate immune factors in the host environment. MSP is a metalloprotease encoded by a tandem array of genes belonging to three msp gene classes, whose mRNAs are differentially expressed in different life stages of the parasite. Like other cells, Leishmania spp. release small membrane-bound vesicles called exosomes into their environment. The purpose of this study was to detect MSP proteins in exosomal vesicles of Leishmania spp. protozoa. METHODS:Using mass spectrometry data we determined the profile of MSP class proteins released in L. infantum exosomes derived from promastigotes in their avirulent procyclic (logarithmic) stage and virulent stationary and metacyclic stages. MSP protein isoforms belonging to each of the three msp gene classes could be identified by unique peptides. RESULTS:Metacyclic promastigote exosomes contained the highest, and logarithmic exosomes had the lowest abundance of total MSP. Among the MSP classes, MSPC class had the greatest variety of isoforms, but was least abundant in all exosomes. Nonetheless, all MSP classes were present at higher levels in exosomes released from stationary or metacyclic promastigotes than logarithmic promastigotes. CONCLUSIONS:The data suggest the efficiency of exosome release may be more important than the identity of MSP isoform in determining the MSP content of Leishmania spp. exosomes.
| S-EPMC6006689 | biostudies-literature
Project description:BackgroundLeishmania spp. are digenetic parasites capable of infecting humans and causing a range of diseases collectively known as leishmaniasis. The main mechanisms involved in the development and permanence of this pathology are linked to evasion of the immune response. Crosstalk between the immune system and particularities of each pathogenic species is associated with diverse disease manifestations. Lipophosphoglycan (LPG), one of the most important molecules present on the surface of Leishmania parasites, is divided into four regions with high molecular variability. Although LPG plays an important role in host-pathogen and vector-parasite interactions, the distribution and phylogenetic relatedness of the genes responsible for its synthesis remain poorly explored. The recent availability of full genomes and transcriptomes of Leishmania parasites offers an opportunity to leverage insight on how LPG-related genes are distributed and expressed by these pathogens.ResultsUsing a phylogenomics-based framework, we identified a catalog of genes involved in LPG biosynthesis across 22 species of Leishmania from the subgenera Viannia and Leishmania, as well as 5 non-Leishmania trypanosomatids. The evolutionary relationships of these genes across species were also evaluated. Nine genes related to the production of the glycosylphosphatidylinositol (GPI)-anchor were highly conserved among compared species, whereas 22 genes related to the synthesis of the repeat unit presented variable conservation. Extensive gain/loss events were verified, particularly in genes SCG1-4 and SCA1-2. These genes act, respectively, on the synthesis of the side chain attached to phosphoglycans and in the transfer of arabinose residues. Phylogenetic analyses disclosed evolutionary patterns reflective of differences in host specialization, geographic origin and disease manifestation.ConclusionsThe multiple gene gain/loss events identified by genomic data mining help to explain some of the observed intra- and interspecies variation in LPG structure. Collectively, our results provide a comprehensive catalog that details how LPG-related genes evolved in the Leishmania parasite specialization process.
| S-EPMC6993435 | biostudies-literature
Project description:Leishmania parasites alternate between extracellular promastigotes in sandflies and intracellular amastigotes in mammals. These protozoans acquire sphingolipids (SLs) through de novo synthesis (to produce inositol phosphorylceramide) and salvage (to obtain sphingomyelin from the host). A single ISCL (Inositol phosphoSphingolipid phospholipase C-Like) enzyme is responsible for the degradation of both inositol phosphorylceramide (the IPC hydrolase or IPCase activity) and sphingomyelin (the SMase activity). Recent studies of a L. major ISCL-null mutant (iscl(-)) indicate that SL degradation is required for promastigote survival in stationary phase, especially under acidic pH. ISCL is also essential for L. major proliferation in mammals. To further understand the role of ISCL in Leishmania growth and virulence, we introduced a sole IPCase or a sole SMase into the iscl(-) mutant. Results showed that restoration of IPCase only complemented the acid resistance defect in iscl(-) promastigotes and improved their survival in macrophages, but failed to recover virulence in mice. In contrast, a sole SMase fully restored parasite infectivity in mice but was unable to reverse the promastigote defects in iscl(-). These findings suggest that SL degradation in Leishmania possesses separate roles in different stages: while the IPCase activity is important for promastigote survival and acid tolerance, the SMase activity is required for amastigote proliferation in mammals. Consistent with these findings, ISCL was preferentially expressed in stationary phase promastigotes and amastigotes. Together, our results indicate that SL degradation by Leishmania is critical for parasites to establish and sustain infection in the mammalian host.
| S-EPMC3267774 | biostudies-literature
Project description:Phlebotomine sand flies that transmit the protozoan parasite Leishmania differ greatly in their ability to support different parasite species or strains in the laboratory: while some show considerable selectivity, others are more permissive. In "selective" sand flies, Leishmania binding and survival in the fly midgut typically depends upon the abundant promastigote surface adhesin lipophosphoglycan (LPG), which exhibits species- and strain-specific modifications of the dominant phosphoglycan (PG) repeat units. For the "selective" fly Phlebotomus papatasi PpapJ, side chain galactosyl-modifications (scGal) of PG repeats play key roles in parasite binding. We probed the specificity and properties of this scGal-LPG PAMP (Pathogen Associated Molecular Pattern) through studies of natural isolates exhibiting a wide range of galactosylation patterns, and of a panel of isogenic L. major engineered to express similar scGal-LPG diversity by transfection of SCG-encoded β1,3-galactosyltransferases with different activities. Surprisingly, both 'poly-scGal' and 'null-scGal' lines survived poorly relative to PpapJ-sympatric L. major FV1 and other 'mono-scGal' lines. However, survival of all lines was equivalent in P. duboscqi, which naturally transmit L. major strains bearing 'null-scGal'-LPG PAMPs. We then asked whether scGal-LPG-mediated interactions were sufficient for PpapJ midgut survival by engineering Leishmania donovani, which normally express unsubstituted LPG, to express a 'PpapJ-optimal' scGal-LPG PAMP. Unexpectedly, these "L. major FV1-cloaked" L. donovani-SCG lines remained unable to survive within PpapJ flies. These studies establish that midgut survival of L. major in PpapJ flies is exquisitely sensitive to the scGal-LPG PAMP, requiring a specific 'mono-scGal' pattern. However, failure of 'mono-scGal' L. donovani-SCG lines to survive in selective PpapJ flies suggests a requirement for an additional, as yet unidentified L. major-specific parasite factor(s). The interplay of the LPG PAMP and additional factor(s) with sand fly midgut receptors may determine whether a given sand fly host is "selective" or "permissive", with important consequences to both disease transmission and the natural co-evolution of sand flies and Leishmania.
| S-EPMC2978724 | biostudies-literature
Project description:BackgroundSand fly species able to support the survival of the protozoan parasite Leishmania have been classified as permissive or specific, based upon their ability to support a wide or limited range of strains and/or species. Studies of a limited number of fly/parasite species combinations have implicated parasite surface molecules in this process and here we provide further evidence in support of this proposal. We investigated the role of lipophosphoglycan (LPG) and other phosphoglycans (PGs) in sand fly survival, using Leishmania major mutants deficient in LPG (lpg1(-)), and the phosphoglycan (PG)-deficient mutant lpg2(-). The sand fly species used were the permissive species Phlebotomus perniciosus and P. argentipes, and the specific vector P. duboscqi, a species resistant to L. infantum development.Principal findingsThe lpg2(-) mutants did not survive well in any of the three sand fly species, suggesting that phosphoglycans and/or other LPG2-dependent molecules are required for parasite development. In vitro, all three L. major lines were equally resistant to proteolytic activity of bovine trypsin, suggesting that sand fly-specific hydrolytic proteases or other factors are the reason for the early lpg2(-) parasite killing. The lpg1(-) mutants developed late-stage infections in two permissive species, P. perniciosus and P. argentipes, where their infection rates and intensities of infections were comparable to the wild type (WT) parasites. In contrast, in P. duboscqi the lpg1(-) mutants developed significantly worse than the WT parasites.ConclusionsIn combination with previous studies, the data establish clearly that LPG is not required for Leishmania survival in permissive species P. perniciosus and P. argentipes but plays an important role in the specific vector P. duboscqi. With regard to PGs other than LPG, the data prove the importance of LPG2-related molecules for survival of L. major in the three sand fly species tested.
| S-EPMC2797086 | biostudies-literature
Project description:The major macromolecule on the surface of the protozoan parasite Leishmania major is a lipophosphoglycan (LPG) which contains a glycosylphosphatidylinositol glycolipid anchor. This parasite also synthesizes a complex family of abundant low-molecular-mass glycoinositolphospholipids (GIPLs) which are structurally related to the LPG anchor. In this study, L. major promastigotes were metabolically labelled with [3H]GlcN, and the kinetics of incorporation into free glycolipids and the LPG anchor followed to elucidate the pathway of GIPL biosynthesis and possible precursor-product relationships between the GIPLs and LPG. Labelled GIPLs were identified by TLC and by liquid chromatography of the released headgroups, before and after enzymic and chemical cleavage. On the basis of the measured specific radioactivities of the GIPLs, and their kinetics of radiolabelling, we suggest the pathway GlcN-PI-->Man1GlcN-PI (M1)-->Man2GlcN-PI (iM2)-->GalfMan2GlcN-PI (GIPL-1)-->Gal1GalfMan2GlcN-PI (GIPL-2)-->Gal2GalfMan2GlcN-PI (GIPL-3). All of the GIPLs were shown to contain alkylacylglycerol or lyso-alkylglycerol lipid moieties with the exception of the earliest intermediate, glucosaminylphosphatidylinositol (GlcN-PI), which contained both alkylacylglycerol and diacylglycerol. A significant proportion (approx. 50%) of GIPL-3 appeared to be selectively modified by the addition of a Glc-1-PO4 residue to one of the mannose residues (P-GIPL-3). On the basis of the specific radioactivity and kinetics of labelling of GIPL-3 and P-GIPL-3 we suggest that both of these low-abundance species are rapidly utilized as LPG precursors. The turnover of LPG and the GIPLs was also studied by [3H]Gal pulse-chase labelling and cell-surface labelling experiments. Whereas LPG was rapidly shed from the cell surface, consistent with previous studies, the GIPLs (both the total cellular and cell-surface pools) had a much slower turnover. These results suggest that the majority of the GIPLs do not act as LPG precursors and indicate that the cellular levels of these molecules is determined, at least in part, by the rate at which they are shed from the cell surface.
| S-EPMC1136841 | biostudies-other