Identification of macrosialin (CD68) on the surface of host macrophages as the receptor for the intercellular adhesive molecule (ICAM-L) of Leishmania amazonensis

The intercellular adhesive molecule, ICAM-L, of Leishmania amazonensis is known to block the attachment as well as internalisation of Leishmania for infection in host macrophages. We employed monoclonal antibodies (mAb) to the surface molecules of a macrophage to block the attachment of ICAM-L to the macrophage surface and identified that CD68 macrosialin is likely the receptor molecule on the macrophage for ICAM-L. We then demonstrated physical interaction between ICAM-L and macrosialin by co-immunoprecipitation of macrosialin with ICAM-L or vice versa. Finally, macrosialin is expressed in macrosialin-negative murine fibroblast cell line NCTC clone 2555 and demonstrates that both ICAM-L and promastigotes of L. amazonensis can bind to the CD68 transfectant. We thus conclude that CD68 macrosialin is the receptor on host macrophages for ICAM-L. Also, involvement of ICAM-L-macrosialin interaction in other Leishmania species and other mammalian macrophages were demonstrated, indicating the biological relevance of this ligand–receptor interaction.     

Leishmania cell surface prohibitin: role in host–parasite interaction

Proteins selectively upregulated in infective parasitic forms could be critical for disease pathogenesis. A mammalian prohibitin orthologue is upregulated in infective metacyclic promastigotes of Leishmania donovani, a parasite that causes visceral leishmaniasis. Leishmania donovani prohibitin shares 41% similarity with mammalian prohibitin and 95–100% within the genus. Prohibitin is concentrated at the surface of the flagellar and the aflagellar pole, the aflagellar pole being a region through which host–parasite interactions occur. Prohibitin is attached to the membrane through a GPI anchor. Overexpression of wild‐type prohibitin increases protein surface density resulting in parasites with higher infectivity. However, parasites overexpressing a mutant prohibitin with an amino acid substitution at the GPI anchor site to prevent surface expression through GPI‐link show lesser surface expression and lower infective abilities. Furthermore, the presence of anti‐prohibitin antibodies during macrophage–Leishmania interaction in vitro reduces infection. The cognate binding partner for Leishmania prohibitin on the host cell appears to be macrophage surface HSP70, siRNA mediated downregulation of which abrogates the capability of the macrophage to bind to parasites. Leishmania prohibitin is able to generate a strong humoral response in visceral leishmaniasis patients. The above observations suggest that prohibitin plays an important role in events leading to Leishmania–host interaction.     

Experimental study of the function of the excreted/secreted <Emphasis Type="Italic">Leishmania</Emphasis>LmSIR2 protein by heterologous expression in eukaryotic cell line

Background  

In yeast and Caenorhabditis elegans, Silent Information Regulator (SIR2) proteins have been shown to be involved in ageing regulation. In Leishmania, the LmSIR2rp was originally isolated from the excreted/secreted material of the Leishmania parasites. Among the function(s) of this protein in Leishmania biology, we have documented its implication in parasite survival, and in particular in Leishmania amastigotes. In this paper we question the role of the excreted/secreted form of the protein. In particular we wonder if the Leishmania Sir2 homologue is involved in some aspect of its biological function(s), in various components and pathways, which could promote the host cell survival. To test this hypothesis we have mimicked an intracellular release of the protein through constitutive expression in mouse L929 fibrosarcoma cells.     

Lutzomyia longipalpis:pH in the Gut, Digestive Glycosidases, and Some Speculations uponLeishmaniaDevelopment

Gontijo, N. F., Almeida-Silva, S., Costa, F. F., Mares-Guia, M. L., Williams, P., and Melo, M. N. 1998.Lutzomyia longipalpis:pH in the gut, digestive glycosidases, and some speculations uponLeishmaniadevelopment.Experimental Parasitology90, 212–219. Screening for digestive glycosidases in different parts of the gut and associated organs ofLutzomyia longipalpisis reported. Searches for the enzymes were made in blood-fed and non-blood-fed females and the enzymes were characterized as soluble or membrane-bound molecules. A total of four different activities were detected, corresponding to the following specificities: an α-glucosidase, anN-acetyl-β-d-glucosaminidase, anN-acetyl-β-d-galactosaminidase, and an α-l-fucosidase. Their possible role and importance forLeishmaniadevelopment are discussed and the α-glucosidase enzyme was partially characterized. The pH inside the gut of non-blood-fed phlebotomines was measured with pH indicator dyes. The pH ranges obtained for crop, midgut, and hindgut were, respectively, higher than pH 6, pH 6, and lower than pH 6. A hypothesis concerning these data andLeishmaniadevelopment is proposed.     

The preparation of neoglycoconjugates containing inter-saccharide phosphodiester linkages as potential anti-Leishmania vaccines

The Leishmaniaexpress complex glycoconjugates containing phosphosaccharide repeat units at all stages of their life-cycle. One of these molecules, lipophosphoglycan (LPG) has been suggested to be a vaccine candidate. To assess the immunological properties of Leishmaniaphosphosaccharides, we have prepared neoglycoproteins and neoglycolipids containing synthetic Leishmaniaphosphosaccharide repeats. The coupling procedure uses the dec-9-enyl spacer of previously synthesised phosphosaccharides for linkage to protein and phospholipid. This alkene moiety is converted by ozonolysis to an aldehyde which is then attached to protein and phospholipid amino groups by reductive amination. The procedure produces neoglycoconjugates in good yield and without compromising the labile phosphodiester linkages within the phosphosaccharide chains.     

Plasmenylethanolamine synthesis in Leishmania major

Ethanolamine glycerophospholipids are ubiquitous cell membrane components. Trypanosomatid parasites of the genus Leishmania synthesize the majority of their ethanolamine glycerophospholipids as 1‐O‐alk‐1′‐enyl‐2‐acyl‐sn‐glycero‐3‐phosphoethanolamine or plasmenylethanolamine (PME) through the Kennedy pathway. PME is a subtype of ether phospholipids also known as ethanolamine plasmalogen whose functions are not well characterized. In this study, we investigated the role of PME synthesis in Leishmania major through the characterization of an ethanolamine phosphotransferase (EPT) mutant. EPT‐null parasites are largely devoid of PME and fully viable in regular medium but fail to proliferate in the absence of fetal bovine serum. They exhibit significant abnormalities in the synthesis and localization of GPI‐anchored surface molecules. EPT‐null mutants also show attenuated virulence in BALB/c mice. Furthermore, in addition to PME synthesis, ethanolamine also contributes to the production of phosphatidylcholine, the most abundant class of lipids in Leishmania. Together, these findings suggest that ethanolamine production is likely required for Leishmania promastigotes to generate bulk phospholipids, to handle stress, and to control the expression of membrane bound virulence factors.     

Leishmania parasitophorous vacuoles interact continuously with the host cell's endoplasmic reticulum; parasitophorous vacuoles are hybrid compartments

Macrophages that express representative endoplasmic reticulum (ER) molecules tagged with green fluorescence protein were generated to assess the recruitment of ER molecules to Leishmania parasitophorous vacuoles (PVs). More than 90% of PVs harbouring Leishmania pifanoi or Leishmania donovani parasites recruited calnexin, to their PV membrane. An equivalent proportion of PVs also recruited the membrane‐associated soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs), Sec22b. Both ER molecules appeared to be recruited very early in the formation of nascent PVs. Electron microscopy analysis of infected Sec22b/YFP expressing cells confirmed that Sec22b was recruited to Leishmania PVs. In contrast to PVs, it was found that no more than 20% of phagosomes that harboured Zymosan particles recruited calnexin or Sec22b to their limiting phagosomal membrane. The retrograde pathway that ricin employs to access the cell cytosol was exploited to gain further insight into ER–PV interactions. Ricin was delivered to PVs in infected cells incubated with ricin. Incubation of cells with brefeldin A blocked the transfer of ricin to PVs. This implied that molecules that traffic to the ER are transferred to PVs. Moreover the results show that PVs are hybrid compartments that are composed of both host ER and endocytic pathway components.     

Cloning,expression and dynamic simulation of TRYP6 from <Emphasis Type="Italic">Leishmania major</Emphasis> (MRHO/IR/75/ER)

Leishmania, a digenetic protozoan parasite causes severe diseases in human and animals. Efficient evasion of toxic microbicidal molecules, such as reactive oxygen species and reactive nitrogen species is crucial for Leishmania to survive and replicate in the host cells. Tryparedoxin peroxidase, a member of peroxiredoxins family, is vital for parasite survival in the presence of antioxidant, hence it is one of the most important molecules in Leishmania viability and then, it may be an appropriate goal for challenging against leishmaniasis. After cloning and sub-cloning of TRYP6 from Leishmania major (MRHO/IR/75/ER), homology modeling of the LmTRYP6 was proposed to predict some functional property of this protein. The refined model showed that the core structure consists of a seven β stranded β-sheet and five α helices which are organized as a central 7-stranded β2-β1-β5-β4-β3-β6-β7 surrounded by 2-stranded β-hairpin, α helices A and D on one side, and α helices B, C and E on the other side. The peroxidatic active site is located in a pocket formed by the residue Pro45, Met46, Thr49, Val51, Cys52, Arg128, Met147 and Pro 148. The catalytic Cys52, located in the first turn of helix αB, is in van der Waals with a Pro45, a Thr49 and an Arg128 that are absolutely conserved in all known Prx sequences. In this study, an attractive molecular target was studied. These results might be used in designing of drugs to fight an important human pathogen.     

Cystathionine γ‐lyase,an Enzyme Related to the Reverse Transsulfuration Pathway,is Functional in Leishmania spp.

Leishmania parasites seem capable of producing cysteine by de novo biosynthesis, similarly to bacteria, some pathogenic protists, and plants. In Leishmania spp., cysteine synthase (CS) and cystathionine β‐synthase (CBS) are expected to participate in this metabolic process. Moreover, the reverse transsulfuration pathway (RTP) is also predicted to be operative in this trypanosomatid because CBS also catalyzes the condensation of serine with homocysteine, and a gene encoding a putative cystathionine γ‐lyase (CGL) is present in all the sequenced genomes. Our results show that indeed, Leishmania major CGL is able to rescue the wild‐type phenotype of a Saccharomyces cerevisiae CGL‐null mutant and is susceptible to inhibition by an irreversible CGL inhibitor, DL‐propargylglycine (PAG). In Leishmania promastigotes, CGL and CS are cytosolic enzymes. The coexistence of de novo synthesis with the RTP is extremely rare in most living organisms; however, despite this potentially high redundancy in cysteine production, PAG arrests the proliferation of L. major promastigotes with an IC₅₀ of approximately 65 μM. These findings raise new questions regarding the biological role of CGL in these pathogens and indicate the need for understanding the molecular mechanism of PAG action in vivo to identify the potential targets affected by this drug.     

Homology modeling and atomic level binding study of Leishmania MAPK with inhibitors

The current therapy for leishmaniasis is not sufficient and it has two severe drawbacks, host-toxicity and drug resistance. The substantial knowledge of parasite biology is not yet translating into novel drugs for leishmaniasis. Based on this observation, a 3D structural model of Leishmania mitogen-activated protein kinase (MAPK) homologue has been developed, for the first time, by homology modeling and molecular dynamics simulation techniques. The model provided clear insight in its structure features, i.e. ATP binding pocket, phosphorylation lip, and common docking site. Sequence-structure homology recognition identified Leishmania CRK3 (LCRK3) as a distant member of the MAPK superfamily. Multiple sequence alignment and 3D structure model provided the putative ATP binding pocket of Leishmania with respect to human ERK2 and LCRK3. This analysis was helpful in identifying the binding sites and molecular function of the Leishmania specific MAPK homologue. Molecular docking study was performed on this 3D structural model, using different classes of competitive ATP inhibitors of LCRK3, to check whether they exhibit affinity and could be identified as Leishmania MAPK specific inhibitors. It is well known that MAP kinases are extracellular signal regulated kinases ERK1 and ERK2, which are components of the Ras-MAPK signal transduction pathway which is complexed with HDAC4 protein, and their inhibition is of significant therapeutic interest in cancer biology. In order to understand the mechanism of action, docking of indirubin class of molecules to the active site of histone deacetylase 4 (HDAC4) protein is performed, and the binding affinity of the protein-ligand interaction was computed. The new structural insights obtained from this study are all consistent with the available experimental data, suggesting that the homology model of the Leishmania MAPK and its ligand interaction modes are reasonable. Further the comparative molecular electrostatic potential and cavity depth analysis of Leishmania MAPK and human ERK2 suggested several important differences in its ATP binding pocket. Such differences could be exploited in the future for designing Leishmania specific MAPK inhibitors.     

Sialoglycans in protozoal diseases: Their detection, modes of acquisition and emerging biological roles

Protozoan parasites including Plasmodia, Leishmania, Trypanosoma, Entamoeba, Trichomonas and others cause diseases in humans and domestic livestock having far-reaching socio-economic implications. They show remarkable propensity to survive within hostile environments encountered during their life cycle, and the identification of molecules that enable them to survive in such milieu is a subject of intense research. Currently available knowledge of the parasite cell surface architecture and biochemistry indicates that sialic acid and its principle derivatives are major components of the glycocalyx and assist the parasite to interact with its external environment through functions ranging from parasite survival, infectivity and host-cell recognition. This review highlights the present state of knowledge with regard to parasite sialobiology with an emphasis on its mode(s) of acquisition and their emerging biological roles, notably as an anti-recognition molecule thereby aiding the pathogen to evade host defense mechanisms. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

Developmental Life Cycle of Leishmania—Cultivation and Characterization of Cultured Extracellular Amastigotes1

ABSTRACT. The biochemistry and immunology of Leishmania promastigotes has been extensively studied; this is due primarily to the facility with which this stage, in contrast to the amastigotes stage, can be maintained in axenic culture. Several attempts to axenically culture lines of Leishmania amastigotes have been reported in the literature. This paper summarizes methods of adaptation (low pH, elevated temperature and culture medium) and characterization of several axenic lines of Leishmania amastigotes. Based on morphological, biological, immunological and biochemical evidence, these organisms appear to resemble amastigotes from infected macrophages or tissue. The axenically cultured amastigotes appear to be distinct from shocked (heat, serum deprivation, stressed) Leishmania promastigotes in the plethora of proteins synthesized, growth (multiplication) in culture, and developmental regulation observed. These data suggest that Leishmania organisms have a significant developmental response to certain signals (pH, temperature) mimicking their in vivo macrophage milieu. The response to other environmental parameters characteristic of the host-macrophage remain to be determined. These axenically cultured amastigotes should be of interest for further immunological, biochemical and developmental investigations of the disease-maintaining stage of this parasite.     

Biology of theLeishmania surface: With particular reference to the surface proteinase,gp 63

Summary The protistan parasiteLeishmania is a dimorphic cell that survives as a motile promastigote in the insect digestive tract, and a non-motile, amastigote from within the phagolysosomal compartment of the vertebrate host's phagocytes. The surface ofLeishmania must interface with a range of differing environments and facilitate uptake of nutrients, whilst protecting the parasite from various host defence mechanisms. This review discusses the organization of the leishmanial cell, and the biology of its major surface constituents, the lipophosphoglycan and the surface proteinase, gp 63.     

Joining forces: first application of a rapamycin‐induced dimerizable Cre system for conditional null mutant analysis in Leishmania

Reverse genetics in Leishmania spp has gained importance beyond basic research as efforts increase to discover and validate new drug targets. Often, the most promising targets are essential for viability of the parasites, defying a genetic analysis by current gene replacement strategies. Duncan et al. demonstrate the applicability of DiCre recombination in Leishmania for induced replacement of the kinase CRK3 gene in promastigotes. DiCre gene replacement leads to the rapid loss of the gene and allows monitoring the phenotypic effects of the loss of function, eliminating the need for prolonged cultivation and selection. Implementation of the DiCre approach will allow functional genetics of the most important of Leishmania genes and is likely to boost genetic research and drug target discovery.     

Isolation and detection of Leishmania species among naturally infected Rhombomis opimus, a reservoir host of zoonotic cutaneous leishmaniasis in Turkemen Sahara, North East of Iran

In Iran, three species of Leishmania have been incriminated as the causative agents of human leishmaniasis, Leishmania (L.) major, Leishmania tropica, and Leishmania infantum.Rhombomis opimus have been incriminated as a principal reservoirs of the parasitic protozoan Leishmania major, the causative agent of rural zoonotic cutaneous leishmaniasis (ZCL) in Iran. Rodents captured and examined to find Leishmania species using conventional methods including direct impression smear and microscopic observation inoculation samples to Balb/c and culture in NNN medium. Also molecular method was employed to detect Leishmania in rodents by amplifying a region of the ribosomal RNA amplicon of Leishmania (ITS1-5.8S rRNA–ITS2) using Nested PCR. Leshmania species were specified by DNA sequences. 36 (38.3%) of R. opimus were Leishmania positive using at least one conventional methods. Many more ITS-rDNA fragments were amplified from R. opimus but only 65 out of 74 PCR products contained enough DNA for direct sequencing or readable sequences. The PCR assays detected in Iranian R. opimus not only Leishmania major in 59 (79.7%) rodents but also Leishmania turanica in 6 (8.1%) rodents, another parasite of the great gerbil. These parasites were found in Turkemen Sahara, North East of Iran, in a focus of rural (ZCL). L. major and L. turanica in R. opimus firmly identified from Turkemen Sahara. Nine rodents with Leishmania infections unidentified which some were unreadable sequences, these could be mixed infections of L. major, L. turanica, Leishmania gerbillisensu lato and Leishmania close to L. gerbilli or a related species reported in sandflies previously from this location. The haplotypes of L. major and L. turanica were found to be identical to that of isolates of L. major and L. turanica from Iran and in GenBank elsewhere. R. opimus is probably the key reservoir in this ZCL focus because of its abundance and its infection rates with both L. major and L. turanica.     

Evidence for Rosettes as an Unrecognized Stage in the Life Cycle of Leishmania Parasites

ABSTRACT. Leishmania parasites, which afflict 12 million people in 88 countries, exist as promastigotes transmitted by insect vectors and as amastigotes residing in mammalian macrophages. Promastigote cells arranged in rosettes have also been described but universally disregarded as a distinct stage in the life cycle. We present evidence that only rosettes of Leishmania major promastigotes express cell surface poly‐α2,8 N‐acetyl neuraminic acid (PSA) and PSA containing de‐N‐acetyl neuraminic acid (NeuPSA). Expression of rosette‐specific PSA antigens was mosaic, with individual promastigotes expressing PSA, NeuPSA or both. A 50 kDa protein was detected by Western blot analysis of a detergent‐insoluble cell fraction with both PSA and NeuPSA‐reactive antibodies. Frequencies of rosette formation as well as cell surface PSA/NeuPSA expression were temperature dependent. Rosettes also engaged in an unusual swarming behavior, congregating into extended clusters. Distinct structures resembling cellular fusion bodies were formed in and released from rosettes. The results indicate that rosettes are an unrecognized stage in the life cycle of Leishmania. We hypothesize that rosettes initiate mating in Leishmania during which PSA/NeuPSA expression plays an important role. Recognizing rosettes as a distinct form of the Leishmania life cycle opens new possibilities for treatment or prevention of disease and, possibly, in vitro genetic recombination without passage of cells through insect vectors.     

The effect of tunicamycin on Leishmania brasiliensis. Glycosylation and the cell surface components

Culture conditions of Leishmania cells were developed to allow the study of the effect of tunicamycin (TM) on glycosylation and on the cell surface components. Leishmania incorporate [¹⁴C]-mannose and [³⁵S]-methionine in vitro. The incorporation of [¹⁴C]-mannose is linear for 150 min and is inhibited by TM (2 g/ml) in a time dependent effect which reaches a plateau of 45% inhibition at 36 h. Under the same experimental conditions [³⁵S]-methionine incorporation into protein is slightly affected. This is reflected by an almost identical polypeptide pattern for TM treated and non-treated cells when analyzed on SDS-PAGE. On the contrary, strong differences were detected on the labeled compounds analyzed on SDS-PAGE followed by autoradiography when the precursor used was [¹⁴C]-mannose. A shift in the electrophoretic mobility of most of the glycopeptides synthesized in the presence of TM was observed, which is also reflected in the structure of the main Leishmania cell surface components.The findings are discussed in the light of biological implications.     

Influence of long-term treatment with pravastatin on the survival, evolution of cutaneous lesion and weight of animals infected by Leishmania amazonensis

The high toxicity of current drugs for treatment of leishmaniasis is a major hindrance for controlling the disease. Pravastatin is a well-known drug with anti-inflammatory and immunomodulatory properties that may modulate host defense mechanisms against Leishmania. We evaluated the influence of prolonged pravastatin treatment on the survival of Leishmania amazonensis-infected animals (BALB/c, C57BL6 mice and Syrian hamsters), including weekly measurement of cutaneous lesions (footpad thickness) and weight. Pravastatin improved survival of Leishmania-infected BALB/c mice but not of infected C57BL6 mice or hamsters. On the 50th week of follow-up, 71% of pravastatin-treated Leishmania-infected BALB/c mice were alive against 29% of control group (p < 0.01). Low footpad thickness was found on BALB/c pravastatin treated mice from the 14th week (p < 0.05), and 20th week onward for C57BL6 treated mice. Pravastatin treatment decreased weight loss in Leishmania-infected C57BL6 mice and Syrian hamsters, but not infected BALB/c mice. Our results points to beneficial effects of pravastatin on the evolution of the disease in the murine leishmaniasis model.     

Localization by scanning immunoelectron microscopy of triosephosphate isomerase,the molecules responsible for contact‐mediated killing of Cryptococcus,on the surface of Staphylococcus

In our previous studies, TPI were found to be the molecules responsible for contact‐killing of C. neoformans by S. aureus cells. Since TPI is a glycolytic protein that functions in the cytoplasm, evidence that TPI is present on the surface of S. aureus was required. In the present study, the presence of TPI on the cell surface of S. aureus was demonstrated by agglutination test and scanning immunoelectron microscopy. Furthermore, TPI was found to be present at a lower density than protein A/G molecules on the surface of S. aureus.