Genomic and proteomic expression analysis of Leishmania promastigote and amastigote life stages: the Leishmania genome is constitutively expressed

Leishmania are protozoan parasites that cause a wide spectrum of clinical diseases in humans and are a major public health risk in several countries. Leishmania life cycle consists of an extracellular flagellated promastigote stage within the midgut of a sandfly vector, and a morphological distinct intracellular amastigote stage within macrophages of a mammalian host. This study reports the use of DNA oligonucleotide genome microarrays representing 8160 genes to analyze the mRNA expression profiles of L. major promastigotes and lesion derived amastigotes. Over 94% of the genes were expressed in both life stages. Advanced statistical analysis identified a surprisingly low degree of differential mRNA expression: 1.4% of the total genes in amastigotes and 1.5% in promastigotes. These microarray results demonstrate that the L. major genome is essentially constitutively expressed in both life stages and suggest that Leishmania is constitutively adapted for survival and replication in either the sandfly vector or macrophage host utilizing an appropriate set of genes for each vastly different environment. Quantitative proteomics, using the isotope coded affinity tag (ICAT) technology and mass spectrometry, was used to identify L. infantum promastigote and axenic amastigote differentially expressed proteins. Of the 91 distinct proteins identified, 8% were differentially expressed in the amastigote stage, 20% were differentially expressed in the promastigote stage, and the remaining 72% were considered constitutively expressed. The differential expression was validated by the identification of previously reported stage specific proteins and identified several amastigote and promastigote novel stage specific proteins.     

Culture systems for production of promastigote and amastigote forms of Leishmania. Application to serological diagnosis and therapeutic trials

Several species of leishmania and three methods of cultivation: monophasic, biphasic and co-cultivation were used in a compared study bearing on the intensive production of leishmania. In addition by applying, a new in vivo model, comprising an injection of sarcomatous cells and promastigotes into BALB/c mice and also an extraction on a discontinuous gradient (Radioselectan 60%), it was possible to obtain highly purified isolates of amastigote forms. The use of two antigens: promastigotes and amastigotes, is to be recommended for the serological diagnosis, by indirect immunofluorescence, of kala-azar. The new in vivo model merits further consideration for research concerning new molecules active against leishmania.     

Intracellular replication of Leishmania tropica in mouse peritoneal macrophages: comparison of amastigote replication in adherent and nonadherent macrophages.

Intracellular replication of Leishmania tropica was assessed in mouse peritoneal macrophage cultures. L. tropica replicated poorly in macrophage monolayers: both the percentage of cells infected and the number of intracellular amastigotes decreased with time in culture. In contrast, nonadherent macrophages supported continuous replication of the parasite, and intracellular amastigotes increased more than 10-fold in these cultures over 8 days.     

Intracellular replication of Leishmania tropica in mouse peritoneal macrophages: amastigote infection of resident cells and inflammatory exudate macrophages.

C3HeB/FeJ peritoneal exudate cells elicited by a variety of sterile inflammatory agents were exposed to Leishmania tropica amastigotes in vitro. Cytochemical characterization of cells that contained intracellular parasites suggested that young, peroxidase-positive macrophages were more susceptible to infection by amastigotes than more mature cells. Replication of the parasite in these younger cells, however, was similar to that observed in resident peritoneal macrophages.     

The surface membrane ofLeishmania mexicana mexicana: Comparison of amastigote and promastigote using freeze-fracture cytochemistry

The freeze fracture replica technique has been used to compare the plasma membranes of amastigote and promastigote stages ofLeishmania mexicana mexicana with respect to intramembranous particle (integral protein) distribution and to -hydroxysterol content as revealed by the distribution of lesions induced by the polyene antibiotic filipin. Intramembranous particle (IMP) density was greater in promastigote than in amastigote plasma membranes. Intramembranous particles were more abundant in the protoplasmic face (PF) than in the exoplasmic face (EF) of promastigotes, but this situation was found to be reversed in amastigotes. Filipin-induced lesions in glutaraldehyde-fixed parasites indicated higher levels of -hydroxysterols in the amastigote than in the promastigote plasma membrane, and in the promastigote flagellar membrane than in the body membrane. Amphotericin B (a related polyene antibiotic used in chemotherapy of leishmaniasis) induced IMP aggregation in the PF of unfixed amastigotes but did not appear to influence sterol distribution as demonstrated by freeze-fracture of subsequently-fixed and filipin-treated organisms.     

Antileishmanial activity of a naphthoquinone derivate against promastigote and amastigote stages of Leishmania infantum and Leishmania amazonensis and its mechanism of action against L. amazonensis species

Parasitology Research - Leishmaniasis has become a significant public health issue in several countries in the world. New products have been identified to treat against the disease; however,...     

Radioiodination and identification of externally disposed membrane components of Leishmania tropica

Two methods, one involving a lactoperoxidase-glucose oxidase coupled reaction and the other employing the insoluble catalyst 1,3,4,6-tetrachloro-3 alpha, 6-alpha-diphenyl-glycoluril (Iodo-Gen), were used to label the surface membrane of promastigotes of Leishmania tropica major. Both methods labelled approximately 20 proteins or glycoproteins (apparent size range 10 to 110 kDa) in a qualitatively similar manner, however the lactoperoxidase method labelled one additional constituent (260 kDa). By omission of both enzymes, or of Iodo-Gen; by comparison of radioactivity incorporated by particulate and soluble cell fractions; and through the action of proteases on live, labelled promastigotes, the surface-labelling specificity of both procedures was confirmed. Immunoprecipitation of Triton X-100 extracts of labelled cells with rabbit antisera revealed a minimum of twelve (seven major) protein antigens in the homologous system and different but cross-reactive protein species from two other isolates of L. tropica. Lectin precipitation of radiolabelled surface components was possible with concanavalin A (but not with other lectins tested) identifying a minimum of 12 glycoproteins. Two of these glycoproteins (210 and 88 kDa) were not recognized by rabbit antiserum.     

The promastigote surface protease (gp63) of Leishmania is expressed but differentially processed and localized in the amastigote stage

The expression, processing and localization of the promastigote surface glycoprotein, gp63, in the amastigote form of Leishmania mexicana was examined. Metabolically labeled protein was immunoprecipitated from promastigotes and amastigotes. The isolated proteins were subjected to deglycosylation and partial peptide mapping. The cleavage products generated migrated similarly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the proteins were closely related. The majority of gp63 in amastigotes was inaccessible to surface-labeling procedures, and lacked the phosphatidylinositol membrane anchor. Immunolocalization of this subpopulation of gp63 revealed it to be present within the parasite's flagellar pocket. Despite the relative paucity of 'membrane-form' gp63, isolation and analysis of surface proteins from lesion amastigotes indicated that gp63 was the most abundant protein on the amastigote surface.     

Characterization of RNA from Leishmania tropica and Leishmania d.donovani promastigotes

RNA has been prepared from promastigotes of Leishmania tropica and Leishmania d.donovani using three different methods. Extraction by hot phenol/isothiocyanate gave the best quantitative and qualitative results. The analysis of total RNA on methyl mercuric agarose gels shows that the large rRNA species is nicked: it is composed of a 630 and a 560 kDa molecule. The small rRNA species has a molecular weight of 800,000. Poly(A+) RNA can be translated in a rabbit reticulocyte lysate system. The newly synthesized products comprise high molecular weight proteins and show different patterns using RNA from L. tropica or from L. d. donovani promastigotes.     

Leishmania tropica in Morocco. IV--Intrafocal enzyme diversity]

Ecoepidemiological analysis of a Moroccan focus of leishmaniasis caused by Leishmania tropica revealed considerable enzymatic diversity. Seven zymodemes belonging to the complex were identified in 149 strains isolated from humans, dogs, and the vector Phlebotomus sergenti. Three distinct subgroups were identifiable, two of which were in turn, composed of three "small variant" zymodemes. The diversity appears to be related to the age of the focus, which may have allowed colonization by zymodemes of different geographic origins. Diversification into "small variants" is apparently the result of recent mutation, possibly associated with genetic exchange.     

Protein methylation and protein methylases in Leishmania donovani and Leishmania tropica promastigotes

We studied the content of acid-stable methylated amino acids of soluble proteins in promastigotes of Leishmania donovani and L. tropica. epsilon-N-Trimethyllysine and NG,NG-dimethylarginine were found in both Leishmania species after culture in the presence of [methyl-14C]methionine. In addition, 3-N-methylhistidine was found only in L. tropica and epsilon-N-dimethyllysine only in proteins of L. donovani. As sinefungin, an antileishmanial nucleoside antibiotic, is a known transmethylase inhibitor, its effect on protein methylation was studied, in whole cells and in vitro. In the first case the drug had no effect on the content of methylated amino acid residues of soluble proteins. In vitro, histone methylation by crude extracts was studied at pH 7.2 and 9.0, known in other organisms as optimum pH values for arginine and lysine methylation, respectively. Surprisingly, arginine methylation by extracts of L. donovani was the same at both pH values while lysine residues were more efficiently methylated at pH 7.2 than at pH 9 by the extracts of the two species. These results indicate that the properties of protein methylases I and III of these parasites are different from those of other organisms hitherto studied. The inhibition constants of sinefungin for the leishmanial protein methylases were weak in comparison with those for enzymes from other sources, with the exception of the constant of L. donovani enzyme at pH 9.     

Metacyclogenesis is a major determinant of Leishmania promastigote virulence and attenuation.

The in vivo virulence patterns of promastigote populations defined on the basis of agglutination by the lectin peanut agglutinin (PNA) were studied for various cloned lines of Leishmania major. Promastigotes derived from logarithmic-phase cultures, which were routinely 100% agglutinated at 100 micrograms of PNA per ml, were relatively avirulent for BALB/c mice. The relative virulence of stationary-phase promastigotes appeared to be attributable to the proportion of nonagglutinable (PNA-) promastigotes contained within these populations. Purification of PNA- organisms from stationary cultures provided for each clone the most virulent inoculum, supporting the view that this change in lectin binding accurately reflects the development of infective metacyclic stage promastigotes. By studying this marker, we found that there was considerable variation in the degree to which different strains and clones underwent metacyclogenesis during growth. Examination of a reportedly avirulent L. major clone revealed that metacyclogenesis was unusually delayed and inefficient for this clone, but that those PNA- promastigotes which could be recovered from late-stationary-phase cultures were virulent for BALB/c mice. The loss of virulence associated with frequent subculture could also be attributed to a drastic diminution in metacyclogenesis potential over time. A clone which yielded over 90% PNA- promastigotes during growth within passage 1 generated fewer than 10% PNA- promastigotes during growth by passage 94. Subcloning of late-passage attenuated promastigotes yielded a clone for which no PNA- promastigotes could be generated during growth, and an infective population could not be derived from this clone. Thus, metacyclogenesis does not appear to be stable for even cloned lines of Leishmania promastigotes, and virulence comparisons between different strains and clones can be meaningfully made only if the metacyclic populations contained within the respective inocula are determined.     

Intracellular protein degradation in Leishmania tropica promastigotes

A labile class of proteins in the range of M_r = 30 000–60 000 which turn over rapidly have been demonstrated in Leishmania tropica promastigotes. The rate of protein degradation is increased by exhaustion of nutrients or inhibition of energy metabolism. Proteolysis is reduced when the parasites are provided with a readily utilizable carbon and energy source such as glucose, glutamate or proline. The breakdown of proteins in L. tropica is dependent on continuous protein synthesis probably for protease synthesis. It is suggested that the relatively high rate of intracellular protein degradation is an auxiliary means for generating carbon and energy sources during nutritional stress.     

Leishmania pifanoi amastigote antigens protect mice against cutaneous leishmaniasis.

In the search for a leishmaniasis vaccine, extensive studies have been carried out with promastigote (insect stage) molecules. Information in this regard on amastigote (mammalian host stage) molecules is limited. To investigate host immune responses to Leishmania amastigote antigens, we purified three stage-specific antigens (A2, P4, and P8) from in vitro-cultivated amastigotes of Leishmania pifanoi by using immunoaffinity chromatography. We found that with Corynebacterium parvum as an adjuvant, three intraperitoneal injections of 5 micrograms of P4 or P8 antigen provided partial to complete protection of BALB/c mice challenged with 10(5) to 10(7) L. pifanoi promastigotes. These immunized mice developed significantly smaller or no lesions and exhibited a 39- to 1.6 x 10(5)-fold reduction of lesion parasite burden after 15 to 20 weeks of infection. In addition, P8 immunization resulted in complete protection against L. amazonensis infection of CBA/J mice and partial protection of BALB/c mice, suggesting that this antigen provided cross-species protection of mice with different H-2 haplotypes. At different stages during infection, vaccinated mice exhibited profound proliferative responses to parasite antigens and increased levels of gamma interferon production, suggesting that a Th1 cell-mediated immune response is associated with the resistance in these mice. Taken together, the data in this report indicate the vaccine potential of amastigote-derived antigens.     

Comparisons of mutants lacking the Golgi UDP-galactose or GDP-mannose transporters establish that phosphoglycans are important for promastigote but not amastigote virulence in Leishmania major

Abundant surface Leishmania phosphoglycans (PGs) containing [Gal(beta1,4)Man(alpha1-PO(4))]-derived repeating units are important at several points in the infectious cycle of this protozoan parasite. PG synthesis requires transport of activated nucleotide-sugar precursors from the cytoplasm to the Golgi apparatus. Correspondingly, null mutants of the L. major GDP-mannose transporter LPG2 lack PGs and are severely compromised in macrophage survival and induction of acute pathology in susceptible mice, yet they are able to persist indefinitely and induce protective immunity. However, lpg2(-) L. mexicana amastigotes similarly lacking PGs but otherwise normal in known glycoconjugates remain able to induce acute pathology. To explore this further, we tested the infectivity of a new PG-null L. major mutant, which is inactivated in the two UDP-galactose transporter genes LPG5A and LPG5B. Surprisingly this mutant did not recapitulate the phenotype of L. major lpg2(-), instead resembling the L. major lipophosphoglycan-deficient lpg1(-) mutant. Metacyclic lpg5A(-)/lpg5B(-) promastigotes showed strong defects in the initial steps of macrophage infection and survival. However, after a modest delay, the lpg5A(-)/lpg5B(-) mutant induced lesion pathology in infected mice, which thereafter progressed normally. Amastigotes recovered from these lesions were fully infective in mice and in macrophages despite the continued absence of PGs. This suggests that another LPG2-dependent metabolite is responsible for the L. major amastigote virulence defect, although further studies ruled out cytoplasmic mannans. These data thus resolve the distinct phenotypes seen among lpg2(-) Leishmania species by emphasizing the role of glycoconjugates other than PGs in amastigote virulence, while providing further support for the role of PGs in metacyclic promastigote virulence.     

Mixed Mucosal Leishmaniasis Infection Caused by Leishmania tropica and Leishmania major

Mixed infections with different Leishmania species could explain differences in the clinical courses of these infections. On identification of Leishmania parasites from Iranian patients with mucosal leishmaniasis (ML), a patient with both oral and nasal lesions was found to be concomitantly infected with Leishmania tropica and L. major. Mixed infection was identified by PCR amplification of Leishmania kinetoplast DNA on scraping of cytological smears and histopathological sections. L. major and L. tropica were isolated from the nasal and oral lesions, respectively. These species were also confirmed by immunohistochemistry. This seems to be the first reported case of concurrent ML infection with two Leishmania species. It indicates that, at least in this patient, previous infection with one of these Leishmania species did not protect against infection with the other. This result has important implications for the development of vaccines against leishmaniases and implies careful attention in the treatment of this infectious disease.     

Generation of adenosine tri-phosphate in Leishmania donovani amastigote forms

Leishmania, the causative agent of various forms of leishmaniasis, is the significant cause of morbidity and mortality. Regarding energy metabolism, which is an essential factor for the survival, parasites adapt to the environment under low oxygen tension in the host using metabolic systems which are very different from that of the host mammals. We carried out the study of susceptibilities to different inhibitors of mitochondrial electron transport chain and studies on substrate level phosphorylation in wild-type L. donovani. The amastigote forms of L. donovani are independent on oxidative phosphorylation for ATP production. Indeed, its cell growth was not inhibited by excess oligomycin and dicyclohexylcarbodiimide, which are the most specific inhibitors of the mitochondrial Fo/F1-ATP synthase. In contrast, mitochondrial complex I inhibitor rotenone and complex III inhibitor antimycin A inhibited amastigote cell growth, suggesting the role of complex I and complex III in cell survival. Complex II appeared to have no role in cell survival. To further investigate the site of ATP production, we studied the substrate level phosphorylation, which was involved in the synthesis of ATP. Succinate-pyruvate couple showed the highest substrate level phosphorylation in amastigotes whereas NADH-fumarate and NADH-pyruvate couples failed to produce ATP. In contrast, NADPH-fumarate showed the highest rate of ATP formation in promastigotes. Therefore, we can conclude that substrate level phosphorylation is essential for the survival of amastigote forms of Leishmania donovani.     

Leishmania promastigote membrane antigen-based enzyme-linked immunosorbent assay and immunoblotting for differential diagnosis of Indian post-kala-azar dermal leishmaniasis

Diagnosis of post-kala-azar dermal leishmaniasis (PKDL), caused by Leishmania donovani, is difficult, as the dermal lesions are of several types and resemble those caused by other skin diseases, especially leprosy. Since the disease generally appears very late after the clinical cure of kala-azar in India, it is also difficult to correlate PKDL with a previous exposure to L. donovani. Very few attempts have been made so far to diagnose PKDL serologically, and the diagnostic methods vary in their sensitivities and specificities. Diagnosis of PKDL through sophisticated PCR methods, although highly sensitive, has limited practical use. We have developed a serodiagnostic method using an enzyme-linked immunosorbent assay to detect specific immunoglobulin (Ig) isotypes and IgG subclass antibodies in the sera of Indian PKDL patients. Our assay, which uses L. donovani promastigote membrane antigens, was 100% sensitive for the detection of IgG and 96.7% specific for the detection of IgG and IgG1. Optical density values for individual patients, however, demonstrated wide variations. Western blot analysis based on IgG reactivity could differentiate patients with PKDL from control subjects, which included patients with leprosy, patients from areas where kala-azar is endemic, and healthy subjects, by the detection of polypeptides of 67, 72, and 120 kDa. The recognition patterns of the majority of serum samples from patients with PKDL were also distinct from those of the serum samples from patients with visceral leishmaniasis (VL), at least for a 31-kDa polypeptide. To further differentiate patients with PKDL from those with active and cured VL, we analyzed the specific titers of the Ig isotypes and IgG subclasses. High levels of IgG, IgG1, IgG2, and IgG3 antibodies significantly differentiated patients with PKDL from patients cured of VL. The absence of antileishmanial IgE and IgG4 in patients with PKDL differentiated these patients from those with active VL. These results imply intrinsic differences in the antibodies generated in the sera from patients with PKDL and VL.     

Lipophosphoglycan and secreted acid phosphatase of Leishmania tropica share species-specific epitopes.

Several species-specific monoclonal antibodies (T11, T13-T15) which only react with Leishmania tropica, recognize phosphorlated carbohydrate epitopes on lipophosphoglycan and the structurally related molecule, phosphoglycan, which is shed by promastigotes into spent culture medium. During immunoaffinity isolation of [32P]orthophosphate-labeled phosphoglycan on monoclonal antibody T15 conjugated to Sepharose 4B, a high-Mr component (approx. 200,000) was co-purified. The latter material is metabolically labeled with [35S]methionine and [3H]glucosamine. This glycoprotein was separated from phosphoglycan by chromatography on lentil lectin resin. The glycoprotein exhibited a L-tatrate-sensitive acid phosphatase activity, typical of secreted acid phosphatase (EC 3.1.3.2) from Leishmania. Monospecific antibodies to Leishmania donovani-secreted acid phosphatase selectively precipitated the L. tropica enzyme from immunoaffinity purified mixtures of the two antigens, and monoclonal antibodies to lipophosphoglycan precipitate the pure enzyme. Species-specific monoclonal antibodies to L. major lipophosphoglycan also recognized both L. tropica antigens. Treatment of the acid phosphatase with periodate or phosphodiesterase I abolished binding by the monoclonal antibodies to the pure enzyme. These results demonstrate that the two major secreted glycoconjugates of Leishmania tropica, the lipophosphoglycan and the acid phosphatase, share species-specific phosphorylated carbohydrate epitope(s).