MARCKS-related protein (MRP) is a substrate for the Leishmania major surface protease leishmanolysin (gp63).

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP; MacMARCKS) are protein kinase C substrates in diverse cell types. Activation of murine macrophages by cytokines increases MRP expression, but infection with Leishmania promastigotes during activation results in MRP depletion. We therefore examined the effect of Leishmania major LV39 on recombinant MRP. Both live promastigotes and a soluble fraction of LV39 lysates degraded MRP to yield lower molecular weight fragments. Degradation was independent of MRP myristoylation and was inhibited by protein kinase C-dependent phosphorylation of MRP. MRP was similarly degraded by purified leishmanolysin (gp63), a Leishmania surface metalloprotease. Degradation was evident at low enzyme/substrate ratios, over a broad pH range, and was inhibited by 1,10-phenanthroline and by a hydroxamate dipeptide inhibitor of leishmanolysin. Using mass spectrometric analysis, cleavage was shown to occur within the effector domain of MRP between Ser(92) and Phe(93), in accordance with the substrate specificity of leishmanolysin. Moreover, an MRP construct in which the effector domain had been deleted was resistant to cleavage. Thus, Leishmania infection may result in leishmanolysin-dependent hydrolysis of MRP, a major protein kinase C substrate in macrophages.     

Targeted gene deletion of Leishmania major UDP-galactopyranose mutase leads to attenuated virulence

Considering the high incidence of galactofuranose (Gal(f)) in pathogens and its absence from higher eukaryotes, the enzymes involved in the biosynthesis of this unusual monosaccharide appear as attractive drug targets. However, although the importance of Gal(f) in bacterial survival or pathogenesis is established, its role in eukaryotic pathogens is still undefined. Recently, we reported the identification and characterization of the first eukaryotic UDP-galactopyranose mutases. This enzyme holds a central role in Gal(f) metabolism by providing UDP-Gal(f) to all galactofuranosyltransferases. In this work, the therapeutical potential of Gal(f) metabolism in Leishmania major was hence evaluated by targeted replacement of the GLF gene encoding UDP-galactopyranose mutase. In L. major, Gal(f) is present in the membrane anchor of the lipophosphoglycan (LPG) and in glycoinositolphospholipids. Accordingly, the generated glf(-) mutant is deficient in LPG backbone and expresses truncated glycoinositolphospholipids. These structural changes do not influence the in vitro growth of the parasite but lead to an attenuation of virulence comparable with that observed with a mutant exclusively deficient in LPG.     

Expression and function of the Trypanosoma brucei major surface protease (GP63) genes

The genome of the African trypanosome Trypanosoma brucei (Tb) contains at least three gene families (TbMSP-A, -B, and -C) encoding homologues of the abundant major surface protease (MSP, previously called GP63), which is found in all Leishmania species. TbMSP-B mRNA occurs in both procyclic and bloodstream trypanosomes, whereas TbMSP-A and -C mRNAs are detected only in bloodstream organisms. RNA interference (RNAi)-mediated gene silencing was used to investigate the function of TbMSP-B protein. RNAi directed against TbMSP-B but not TbMSP-A ablated the steady state TbMSP-B mRNA levels in both procyclic and bloodstream cells but had no effect on the kinetics of cultured trypanosome growth in either stage. Procyclic trypanosomes have been shown previously to have an uncharacterized cell surface metalloprotease activity that can release ectopically expressed surface proteins. To determine whether TbMSP-B is responsible for this release, transgenic variant surface glycoprotein 117 (VSG117) was expressed constitutively in T. brucei procyclic TbMSP-RNAi cell lines, and the amount of surface VSG117 was determined using a surface biotinylation assay. Ablation of TbMSP-B but not TbMSP-A mRNA resulted in a marked decrease in VSG release with a concomitant increase in steady state cell-associated VSG117, indicating that TbMSP-B mediates the surface protease activity of procyclic trypanosomes. This finding is consistent with previous pharmacological studies showing that peptidomimetic collagenase inhibitors block release of transgenic VSG from procyclic trypanosomes and are toxic for bloodstream but not procyclic organisms.     

Glycoprotein 63 (gp63) genes show gene conversion and reveal the evolution of Old World Leishmania

Species of the subgenus Leishmania (Leishmania) cause the debilitating disease leishmaniasis on four continents. Species grouped within the Leishmania donovani complex cause visceral leishmaniasis, a life-threatening disease, often associated with poverty, and affecting some 0.5 million people each year. The Leishmania glycoprotein GP63, or major surface protease, is a metalloprotease involved in parasite survival, infectivity and virulence. Here, we show that evolution of the gp63 multigene family is influenced by mosaic or fragmental gene conversion. This is a major evolutionary force for both homogenisation and for generating diversity, even in the absence of sexual reproduction. We propose here that the high GC content at the third codon position in the gp63 family of Old World Leishmania may be higher in multicopy regions, under the biased gene conversion model, because increased copy numbers may lead to increased rates of recombination. We confirm that one class of gp63 genes with an extended 3'end signal, gp63(EXT), reveals genetic groups within the complex and gives insights into evolution and host associations. Gp63(EXT) genes can also provide the basis for rapid and reliable genotyping of strains in the L. donovani complex. Our results confirmed that a more stringent definition of Leishmania infantum is required and that the species Leishmania archibaldi should be suppressed.     

The effect of tunicamycin on the protease activity of GP63 from Leishmania major

The protease activity of gp63 from L. major was studied in relation to tunicamycin induced N-deglycosylation. It was found that after tunicamycin treatment, a N-deglycosylated product of gp63 with protease activity is present at the cell surface of Leishmania promastigote.     

Nonrandom spatial distribution of synonymous substitutions in the GP63 gene from Leishmania

In this work we analyze the variability in substitution rates in the GP63 gene from Leishmania. By using a sliding window to estimate substitution rates along the gene, we found that the rate of synonymous substitutions along the GP63 gene is highly correlated with both the rate of amino acid substitution and codon bias. Furthermore, we show that comparisons involving genes that represent independent phylogenetic lines yield very similar divergence/conservation patterns, thus suggesting that deterministic forces (i.e., nonstochastic forces such as selection) generated these patterns. We present evidence indicating that the variability in substitution rates is unambiguously related to functionally relevant features. In particular, there is a clear relationship between rates and the tertiary structure of the encoded protein since all divergent segments are located on the surface of the molecule and facing one side (almost parallel to the cell membrane) on the exposed surface of the organism. Remarkably, the protein segments encoded by these variable regions encircle the active site in a funnel-like distribution. These results strongly suggest that the pattern of nucleotide divergence and, notably, of synonymous divergence is affected by functional constraints.     

Crystallization and preliminary X-ray diffraction studies of leishmanolysin,the major surface metalloproteinase from Leishmania major

The membrane-bound GPI-anchored zinc metalloproteinase leishmanolysin purified from Leishmania major promastigotes has been crystallized in its mature form. Two crystal forms of leishmanolysin have been grown by the vapor diffusion method using 2-methyl-2,4-pentanediol as the precipitant. Macroseeding techniques were employed to produce large single crystals. Protein microhet-erogeneity in molecular size and charge was incorporated into both crystal forms. The tetragonal crystal form belongs to the space group P4₁2₁2 or the enantiomorph P4₃2₁2, has unit cell parameters of a = b = 63.6 Å, c = 251.4 Å, and contains one molecule per asymmetric unit. The second crystal form is monoclinic, space group C2, with unit cell dimensions a = 107.2 Å, b = 90.6 Å, c = 70.6 Å, β = 110.6°, and also contains one molecule per asymmetric unit. Both crystal forms diffract X-rays beyond 2.6 Å resolution and are suitable for X-ray analysis. Native diffraction data sets have been collected and the structure determination of leishmanolysin using a combination of the isomorphous replacement and the molecular replacement methods is in progress. © 1995 Wiley-Liss, Inc.     

Characterization of a multi-copy gene for a major stage-specific cysteine proteinase of Leishmania mexicana.

lmcpb, a gene from Leishmania mexicana that encodes a major cysteine proteinase in the parasite, has been cloned and sequenced. LmCPb is related more to cysteine proteinases from Trypanosoma brucei and Trypanosoma cruzi than to a previously characterized cysteine proteinase, LmCPa, of L. mexicana. It contains a long C-terminal extension characteristic of similar enzymes of T. brucei and T. cruzi. The gene is multi-copy and tandemly arranged. lmcpb RNA levels are developmentally regulated with steady state levels being high in amastigotes, low in metacyclic promastigotes and undetectable in multiplicative promastigotes. This variation correlates with and may account for the stage-specific expression of LmCPb enzyme activity.     

Dok proteins are recruited to the phagosome and degraded in a GP63-dependent manner during Leishmania major infection

Three adaptor molecules of the Dok family, Dok-1, Dok-2 and Dok-3 are expressed in macrophages and are involved in the negative regulation of signaling in response to lipopolysaccharide and various cytokines and growth factors. We investigated the role and the fate of these proteins following infection with Leishmania major promastigotes in macrophages. The protozoan parasite L. major causes cutaneous leishmaniasis and is known for its capacity to alter host-cell signaling and function. Dok-1/Dok-2^−/− bone marrow-derived macrophages displayed normal uptake of L. major promastigotes. Following Leishmania infection, Dok-1 was barely detectable by confocal microscopy. By contrast, phagocytosis of latex beads or zymosan led to the recruitment of Dok-1 to phagosomes. In the absence of the Leishmania pathogenesis-associated metalloprotease GP63, Dok-1 was also, partially, recruited to phagosomes containing L. major promastigotes. Further biochemical analyses revealed that similar to Dok-1, Dok-2 and Dok-3 were targets of GP63. Moreover, we showed that upon infection with wild-type or Δgp63 L. major promastigotes, production of nitric oxide and tumor necrosis factor by interferon-γ-primed Dok-1/Dok-2^−/− macrophages was reduced compared to WT macrophages. These results suggest that Dok proteins may be important regulators of macrophage responses to Leishmania infection.     

Over-expression of Leishmania major MAP kinases reveals stage-specific induction of phosphotransferase activity

During the infectious cycle, protozoan parasites undergo various developmental transitions and switch virulence factors in response to extracellular signals in insect vectors and human hosts. Despite the importance of environmental sensing in parasite pathogenicity, little is known about the pathways that transduce extracellular signals into stage-specific gene expression. Here, we used a transgenic approach to gain insight into localisation and activity of three green fluorescence protein (GFP)-tagged Leishmania major mitogen-activated protein kinases, LmaMPK4, 7 and 10. The GFP-LmaMPKs in both L. major and Leishmania donovani transgenic lines showed predominant cytoplasmic localisation and the over-expression had no effect on promastigote morphology, growth and the ability to differentiate into stationary-phase metacyclics for L. major and axenic amastigotes for L. donovani. We isolated the GFP-tagged MPKs from parasite extracts and tested their phosphotransferase activity across various culture conditions. For all three GFP-LmaMPKs, kinase activity was low or absent in promastigote extracts but significantly increased in L. major promastigotes after exposure to pH 5.5 and 34 degrees C, and in axenic L. donovani amastigotes. Enhanced activity correlated with increased GFP-LmaMPK phosphorylation as judged by phospho-specific fluorescent staining of the immuno-precipitated kinases. We could extend these findings to the endogenous LmaMPK10, which accumulated in the phospho-protein fraction of axenic amastigotes but not promastigotes, and thus follows the stage-specific phosphorylation profile of episomally expressed GFP-LmaMPK10. These results provide evidence for the functional conservation of Leishmania MAP kinases in parasite environmental sensing and underscore the potential of transgenic approaches to gain insight into signaling events during the Leishmania life cycle.     

Identification of genes encoding arabinosyltransferases (SCA) mediating developmental modifications of lipophosphoglycan required for sand fly transmission of leishmania major

At key steps in the infectious cycle pathogens must adhere to target cells, but at other times detachment is required for transmission. During sand fly infections by the protozoan parasite Leishmania major, binding of replicating promastigotes is mediated by galactosyl side chain (scGal) modifications of phosphoglycan repeats of the major surface adhesin, lipophosphoglycan (LPG). Release is mediated by arabinosyl (Ara) capping of LPG scbetaGal residues upon differentiation to the infective metacyclic stage. We used intraspecific polymorphisms of LPG structure to develop a genetic strategy leading to the identification of two genes (SCA1/2) mediating scAra capping. These LPG side chain beta1,2-arabinosyltransferases (scbetaAraTs) exhibit canonical glycosyltransferase motifs, and their overexpression leads to elevated microsomal scbetaAraT activity. Although the level of scAra caps is maximal in metacyclic parasites, scbetaAraT activity is maximal in log phase cells. Because quantitative immunolocalization studies suggest this is not mediated by sequestration of SCA scbetaAraTs away from the Golgi apparatus during log phase, regulation of activated Ara precursors may control LPG arabinosylation in vivo. The SCA genes define a new family of eukaryotic betaAraTs and represent novel developmentally regulated LPG-modifying activities identified in Leishmania.     

Targeted deletion of genes encoding extracellular enzymes in Bacillus licheniformis and the impact on the secretion capability

The general secretory pathway is routinely concerned with a multitude of extracellular enzymes. By eliminating obstructive competitors the export machinery may transport larger quantities of remaining proteins under circumstances in which the secretion machinery is fully loaded. Hence, in this study, genes encoding efficiently expressed but dispensable exoenzymes were knocked out in Bacillus licheniformis MD1. Single, double, and triple mutants with deletions of celA, chiA, and amyB, respectively, were generated via in vivo recombination by making use of a vector with a temperature sensitive origin of replication. Overexpression of a heterologous amylase gene on a multi-copy plasmid, a common scenario in biotechnological processes, resulted in an articulate reduction of chromosomally encoded extracellular enzyme activities indicating that the secretion machinery works to capacity in such transformants. Deletion mutants with the expression plasmid displayed enhanced amylase activities compared to the strain with the wild type genetic background. In addition, the chromosomally encoded protease activity was clearly higher in transformants with deletions.     

Genetic variation in RPOIILS gene encoding RNA polymerase II largest subunit from Leishmania major

Leishmaniasis is a geographically widespread severe disease which includes visceral leishmaniasis, cutaneous leishmaniasis (CL). There are 350 million people at risk in over 80 countries. In the Old World, CL is usually caused by Leishmania major, Leishmania tropica, and Leishmania aethiopica complex which 90 % of cases occurring in Afghanistan, Algeria, Iran, Iraq, Saudi Arabia, Syria, Brazil, and Peru. Recently, some reports showed that some strains of L. major have internal transcribed space (ITS-1) with differential size exhibiting homology with the related gene in a divergent genus of kinetoplastida, the Crithidia. This prompted us to analyze the mentioned gene in 100 isolates obtained from patients with suspected CL. After obtaining samples from 100 patients, DNA extraction was performed and ITS-1 was analyzed using PCR–RFLP. These samples were sequenced for verifying their homology. Then, RPOIILS gene was analyzed in the samples that their ITS-1 gene exhibiting homology with the related gene in Crithidia. Results showed that 10 % of the isolates have ITS-1 exhibiting different size with the routine ones. Sequencing of them showed their similarity to the one from Crithidia fasciculata. RPOIILS gene encoding RNA polymerase II largest subunit analysis showed genetic diversity. This study might also help in solving the problems concerning Leishmaniasis outbreak currently facing in Iran and some other endemic regions of the world.     

The tissue- and developmental stage-specific involvement of autophagy genes in aggrephagy

ABSTRACT

Genetic screens have identified two sets of genes that act at distinct steps of basal autophagy in higher eukaryotes: the pan-eukaryotic ATG genes and the metazoan-specific EPG genes. Very little is known about whether these core macroautophagy/autophagy genes are differentially employed during multicellular organism development. Here we analyzed the function of core autophagy genes in autophagic removal of SQST-1/SQSTM1 during C. elegans development. We found that loss of function of genes acting at distinct steps in the autophagy pathway causes different patterns of SQST-1 accumulation in different tissues and developmental stages. We also identified that the calpain protease clp-2 acts in a cell context-specific manner in SQST-1 degradation. clp-2 is required for degradation of SQST-1 in the hypodermis and neurons, but is dispensable in the body wall muscle and intestine. Our results indicate that autophagy genes are differentially employed in a tissue- and stage-specific manner during the development of multicellular organisms.

Abbreviations: ATG: autophagy related; CLP: calpain family; EPG: ectopic PGL granules; ER: endoplasmic reticulum; ESCRT: endosomal sorting complex required for transport; GFP: green fluorescent protein; LGG-1/LC3: LC3, GABARAP and GATE-16 family; MIT: microtubule interacting and transport; PGL: P granule abnormality protein; SQST-1: sequestosome-related; UPS: ubiquitin-proteasome system     

Widespread differentiation stage-specific expression of the gene encoding phosphoprotein p19 (metablastin) in mammalian cells

p19 is a highly conserved 19 kD cytosolic protein that undergoes phosphorylation in response to diverse extracellular factors in mammalian cells. Its expression is abundant in brain and testis and is developmentally regulated. To gain insights regarding its function, we analyzed the expression of p19 mRNA in a variety of cell types during induction of differentiation. Murine erythroleukemia cells showed a moderate increase followed by a marked decrease in the abundance of p19 mRNA during induction of differentiation. In murine C2 myoblasts and primary fetal rat osteoblasts, p19 mRNA was abundant in replicating cells and decreased to undetectable levels during differentiation. In resting human peripheral blood lymphocytes, p19 mRNA was virtually undetectable but was strongly induced during blast transformation of both B and T cells. In rat liver, p19 mRNA was abundant on embryonic day 17 and decreased during early postnatal development. Upon fractionation of adult rat liver cells by centrifugal elutriation, p19 mRNA was not detected in hepatocytes while a low level was observed in a fraction enriched in non-parenchymal epithelial cells. CCl4-induced liver regeneration resulted in induction of p19 mRNA in hepatocytes. Primary cultures of embryonic and neonatal rat brain were analyzed by indirect immunofluorescence using co-staining with stage-specific markers. p19 expression was restricted to immature neurons and oligodendrocyte precursors. In contrast to the other cell types examined, the neuronal and glial precursors that express p19 were shown, using BrdU labeling, to be postmitotic both in primary culture and in vivo. The data demonstrate widespread, stage-specific expression of p19 and suggest that the protein exerts a general, lineage-independent function during induction of differentiation of mammalian cells. In view of the available evidence on the stimulation of serine phosphorylation of p19 by several growth factors, our working hypothesis is that phosphorylation of p19 may be involved in the mechanism by which growth factors control cell differentiation.     

The involvement of the major surface glycoprotein (gp63) of Leishmania promastigotes in attachment to macrophages

The interaction between the macrophage and the parasite plays a central role in the continued success of Leishmania infection. The promastigote surface ligand, and its complementary macrophage membrane receptor, involved in attachment and phagocytosis are likely to exert considerable influence over the outcome of a new infection. In this study, we report experiments pertaining to one such parasite membrane protein. Initial examination of promastigote surface proteins by radiolabeling and two-dimensional-polyacrylamide gel electrophoresis revealed an abundant polypeptide with an apparent m.w. of 63,000. Lectin-binding studies indicated that it was a glycoprotein containing mannose, N-acetyl glucosamine, and N-acetyl galactosamine residues. Monospecific antiserum raised against this glycoprotein, gp63, decorated the entire promastigote plasmalemma. Univalent antibody fragments from this antiserum blocked the interaction between promastigotes and macrophages by inhibiting attachment. Anti-gp63-inhibition reduced parasite/macrophage binding to 30 to 35% of the control binding level. Additional evidence of the involvement of gp63 in attachment to macrophages was provided by studies that made use of gp63-containing proteoliposomes. These vesicles were avidly phagocytosed by macrophages. Uptake of the gp63-containing liposomes was suppressed by greater than 90% by both anti-gp63 F(ab) fragments and the oligosaccharide mannan, indicating that their phagocytosis was receptor dependent. These results demonstrate that the abundant glycoprotein gp63 plays an important role in attachment of promastigotes to macrophages, and attachment via this parasite ligand is sufficient to trigger phagocytosis.     

Variation in Leishmania chemokine suppression driven by diversification of the GP63 virulence factor

Leishmaniasis is a neglected tropical disease with diverse outcomes ranging from self-healing lesions, to progressive non-healing lesions, to metastatic spread and destruction of mucous membranes. Although resolution of cutaneous leishmaniasis is a classic example of type-1 immunity leading to self-healing lesions, an excess of type-1 related inflammation can contribute to immunopathology and metastatic spread. Leishmania genetic diversity can contribute to variation in polarization and robustness of the immune response through differences in both pathogen sensing by the host and immune evasion by the parasite. In this study, we observed a difference in parasite chemokine suppression between the Leishmania (L.) subgenus and the Viannia (V.) subgenus, which is associated with severe immune-mediated pathology such as mucocutaneous leishmaniasis. While Leishmania (L.) subgenus parasites utilize the virulence factor and metalloprotease glycoprotein-63 (gp63) to suppress the type-1 associated host chemokine CXCL10, L. (V.) panamensis did not suppress CXCL10. To understand the molecular basis for the inter-species variation in chemokine suppression, we used in silico modeling to identify a putative CXCL10-binding site on GP63. The putative CXCL10 binding site is in a region of gp63 under significant positive selection, and it varies from the L. major wild-type sequence in all gp63 alleles identified in the L. (V.) panamensis reference genome. Mutating wild-type L. (L.) major gp63 to the L. (V.) panamensis sequence at the putative binding site impaired cleavage of CXCL10 but not a non-specific protease substrate. Notably, Viannia clinical isolates confirmed that L. (V.) panamensis primarily encodes non-CXCL10-cleaving gp63 alleles. In contrast, L. (V.) braziliensis has an intermediate level of activity, consistent with this species having more equal proportions of both alleles. Our results demonstrate how parasite genetic diversity can contribute to variation in immune responses to Leishmania spp. infection that may play critical roles in the outcome of infection.