In vitro parasite-monocyte interactions in human leishmaniasis: possible role of fibronectin in parasite attachment.

Leishmania spp. must attach to mononuclear phagocyte surfaces before entering this host cell. We investigated the potential role of fibronectin in facilitating parasite attachment. Human plasma fibronectin bound to axenically cultured promastigotes, and promastigotes and amastigotes preferentially bound to fibronectin-coated cover slips. Promastigotes grown in the absence of fibronectin were strikingly deficient in their ability to attach to human monocytes compared with promastigotes grown in the presence of fibronectin. Rabbit anti-human plasma fibronectin antiserum decreased promastigote and amastigote attachment to monocytes. Immunoglobulin G F(ab')2 and Fab fragments also reduced the ability of amastigotes to bind to monocytes. Antiserum pretreatment of amastigotes followed by washing resulted in reduced parasite binding, whereas antibody pretreatment of monocytes did not. Addition of exogenous fibronectin did not enhance parasite attachment to monocytes. These findings suggest that Leishmania spp. can bind fibronectin and may utilize this glycoprotein to facilitate attachment to the mononuclear phagocytes that they infect.     

Monoclonal antibodies recognizing determinants specific for the promastigote stage of Leishmania mexicana

Two monoclonal antibodies (IX-IF9-D8 and IX-5H9-CI) produced to a membrane enriched fraction of Leishmania mexicana amazonensis promastigotes have been demonstrated to be specific for the promastigote (insect) form and not the amastigote (mammalian host) form of the parasite. The antigens recognized by these monoclonal antibodies are not found on amastigotes isolated from infected animals or on amastigotes isolated from a macrophage cell line J774 infected initially with promastigotes. The antigens are not re-expressed by amastigotes cultured at 34°C; however, amastigotes cultured at 24°C that have begun transformation into promastigotes do express these antigens. The level of expression of these antigens in cultures of amastigotes undergoing transformation into promastigotes, increases with time from 16 to 36 h and appears to correlate with the percentage of promastigotes. Two protein molecules with apparent molecular weights of 40 000 and 92 000 have been identified by radioimmune precipitation as associated with L. mexicana promastigote stage specific determinants.     

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.     

Expression of Leishmania antigen on the surface membrane of infected human macrophages in vitro.

Resolution of leishmaniasis is associated with host immunological responsiveness to parasite antigens. In clinical disease, leishmania are found as amastigotes contained with macrophages. We investigated the possibility that Leishmania antigens are expressed on the infected macrophage surface by reacting infected macrophages with antibody to Leishmania. In vitro-infected human monocyte-derived macrophages were labelled with antibody to amastigotes when examined with immunofluorescent or immunoelectron microscopic techniques. Infected macrophages were poorly labelled by antibody to promastigotes (insect forms of Leishmania). Certain antisera that reacted with the surface membranes of amastigotes did not label the infected macrophage surface. These results indicate that human macrophages infected in vitro express Leishmania amastigote antigen(s) on their surface membranes, that such antigen(s) may not be present in large quantities in promastigotes, and that certain antigen(s) on the amastigote surface are not expressed on the surface membranes of infected macrophages.     

Internal and surface subpopulations of the major surface protease (MSP) of Leishmania chagasi

Major surface protease (MSP) facilitates Leishmania promastigote evasion of complement-mediated lysis in the mammalian host and enhances host macrophage phagocytosis of the promastigotes. We previously showed that the steady-state abundance of MSP protein increases 14-fold during in vitro cultivation of L. chagasi promastigotes from logarithmic to stationary phase, despite the fact that the total amount of MSP mRNA does not increase. Furthermore, 10 major MSP isoforms are differentially expressed in different promastigote growth phases, and attenuation of parasites by long-term in vitro cultivation influences MSP isoform expression. Herein, we report that although about two-thirds of newly synthesized MSP becomes surface localized, the rest of the MSP does not reach the promastigote surface. This internal MSP is stable without detectable decrease in abundance up to 6 days after biosynthesis. Furthermore, surface-localized MSP is released at different rates from logarithmic and stationary phase virulent Leishmania promastigotes. These data are consistent with the hypothesis that the major mechanism regulating MSP abundance is the rate of loss of surface-localized MSP from the promastigote surface, and that internally localized MSP is very stable.     

Differences in expression and exposure of promastigote and amastigote membrane molecules in Leishmania tropica.

The accessibility of particular Leishmania tropica promastigote (extracellular) and amastigote (intracellular) membrane molecules might be related to the relative abilities of the two stages to induce host immune responses. To examine the exposure of membrane antigens on resident macrophage-susceptible promastigotes and resident macrophage-resistant amastigotes, both stages were analyzed by polyacrylamide gel electrophoresis and immunoblotting after specific labeling and extraction procedures. Protein compositional studies, using metabolic labeling of promastigotes and amastigotes, demonstrated that both forms possessed numerous endogenously synthesized proteins. In addition, a marked difference was revealed in the external exposure of promastigote and amastigote membrane constituents when analyzed by 125I surface labeling or Western blot analysis. Whereas nine promastigote proteins were intensely to moderately iodinated, only one amastigote membrane component was similarly labeled (9.5K band). Western blot analyses with serum from a rabbit immunized with a mixture of both L. tropica stages indicated that the majority of promastigote molecules accessible to 125I may also react with immune serum. However, Western blots of extracted amastigotes identified several bands not seen on radiographs and thus not accessible to 125I. The external exposure of these amastigote molecules was confirmed in that immune serum adsorbed with viable, intact amastigotes was no longer reactive with amastigote extracts. Further, by Western blot analyses of sodium dodecyl sulfate- but not Nonidet P-40-extracted amastigotes, three amastigote-specific membrane antigens not previously observed with nonionic detergent extraction methods were identified. The autofluorographic pattern of amastigotes intrinsically labeled with N-[3H]acetylglucosamine, an amino sugar which is incorporated into membrane carbohydrates, was in excellent agreement with the pattern of antigens reactive with antibody in Western blots. Thus, with these cell surface labeling and extraction methods, promastigote and amastigote membranes were shown to be significantly different. Amastigotes possessed several membrane-associated molecules, but few appeared to be either accessible or reactive with 125I. Moreover, the majority of molecules not reactive with 125I, but reactive with antibodies, may be glycosylated. These observations are discussed relative to the ability of amastigotes both to survive within the degradative milieu of macrophage phagolysosomes and to evade host immune reactivity.     

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.     

Biochemical and molecular characterization of Leishmania pifanoi amastigotes in continuous axenic culture.

Inability to culture the disease-producing amastigote form of Leishmania has greatly hampered its study. We have biochemically characterized an axenically cultured amastigote-like form of Leishmania pifanoi. This form closely resembles amastigotes in proteinase, ribonuclease, adenine deaminase and peroxidase activity. It also exhibits comparable rates of growth, transformation, synthesis of DNA, RNA and protein, and metabolism of glucose and linoleic acid. It is distinct from promastigotes in these characteristics. The expression of the genes for beta-tubulin and the P100/11E reductase is developmentally regulated in this axenic form as in amastigotes. These results, combined with previous demonstrations of amastigote morphology and antigenicity in the culture form, confirm that Leishmania amastigotes have been successfully propagated in axenic media. This strain should serve as an excellent model for the study of amastigote biochemistry, pharmacology and immunology, and the molecular genetics of the transformation between amastigote and promastigote forms.     

Cutaneous host defense in leishmaniasis: interaction of isolated dermal macrophages and epidermal Langerhans cells with the insect-stage promastigote.

Leishmania species are obligate intracellular pathogens of mononuclear phagocytes. Successful infection depends on sequestration of the promastigote (insect form) within host cells, allowing transformation into the relatively hardy amastigote stage. Promastigotes are killed readily by circulating phagocytes and nonimmune serum, suggesting that cutaneous infection is initiated within a permissive cell in the epidermis or dermis. From large sections of primate skin dermal macrophages and epidermal Langerhans cells were isolated, and their interaction with promastigotes of Leishmania major was investigated in vitro. Dermal macrophages were readily infected with promastigotes, and successful transformation to and replication of amastigotes was observed. Ingestion of promastigotes by dermal macrophages was not associated with a significant respiratory burst, in contrast to that by other macrophage populations, and was associated with significantly greater survival of parasites. Stimulation of these cells with phorbol myristate acetate or opsonized zymosan revealed that those cells were generally oxidatively deficient. Langerhans cells could not be successfully infected by promastigotes under similar conditions. Examination of these cells for expression of CR3, which has been identified as a potential Leishmania receptor, revealed that Langerhans cells did not express the alpha M subunit of CR3, whereas dermal macrophages were CR3 positive. These data support the concept that dermal macrophages are the site of initiation of Leishmania infection.     

Changes in the proteome and infectivity of Leishmania infantum induced by in vitro exposure to a nitric oxide donor

Leishmania species are protozoan parasites that exhibit an intracellular amastigote form within mammalian macrophages and an extracellular promastigote form inside the sandfly vector. The generation of nitric oxide (NO) upon activation of macrophages is surely the principal killing effector of intracellular amastigotes but little is known about the potential action of NO against the promastigote phase during its multiplication inside the digestive tract of the sandfly vector. Therefore, we have approached this issue by using an in vitro model to study the effect of an NO donor, 3-morpholinosydnonimine (SIN-1), on the proteome and infectivity of promastigotes of Leishmania infantum. Exposure of promastigotes to SIN-1 during its logarithmic growth phase caused a dramatic effect on parasite protein expression and viability, consequently killing about 60-70% of the promastigotes. The significant changes in the proteome included the over-expression of enolase, peroxidoxin precursors, and heat-shock protein 70 (HSP70), under-expression of 20S proteasome alpha 5 unit, and phosphomannomutase and induced expression of 3-hydroxy-3-methyglutaryl-CoA (HMG-CoA) synthase and prostaglandine f2-alpha (PGD2) synthase. Interestingly, promastigotes that resisted treatment showed enhanced infectivity to J774 macrophages in comparison to the controls. This finding together with the appearance of the PGD2S and an over-expression of HSP70 isoforms in treated promastigotes led us to speculate the existence of NO-mediated programmed cell death (PCD) events as a potential mechanism of population regulation and selection of properly infecting forms that predominantly operate on the promastigote stage.     

Role of beta-D-galactofuranose in Leishmania major macrophage invasion

The role of glycosylinositol phospholipid 1 (GIPL-1) of Leishmania (Leishmania) major in the interaction of promastigotes and amastigotes with macrophages was analyzed. Monoclonal antibody MEST-1, which recognizes glycolipids containing terminal galactofuranose (Galf) residues (E. Suzuki, M. S. Toledo, H. K. Takahashi, and A. H. Straus, Glycobiology 7:463-468, 1997), was used to detect GIPL-1 in Leishmania by indirect immunofluorescence and to analyze its role in macrophage infectivity. L. major promastigotes showed intense fluorescence with MEST-1, and GIPL-1 was detected in both amastigote and promastigote forms by high-performance thin-layer chromatography immunostaining by using MEST-1. Delipidation of L. major promastigotes with isopropanol-hexane-water eliminated the MEST-1 reactivity, confirming that only GIPL-1 is recognized in either amastigotes or promastigotes of this species. The biological role of GIPL-1 in the ability of L. major to invade macrophages was studied by using either Fab fragments of MEST-1 or methylglycosides. Preincubation of parasites with Fab fragments reduced macrophage infectivity in about 80% of the promastigotes and 30% of the amastigotes. Preincubation of peritoneal macrophages with p-nitrophenyl-beta-galactofuranoside (10 mM) led to significant ( approximately 80%) inhibition of promastigote infectivity. These data suggest that a putative new receptor recognizing beta-D-Galf is associated with L. major macrophage infectivity and that GIPL-1 containing a terminal Galf residue is involved in the L. major-macrophage interaction.     

Antigenic changes during intracellular differentiation of Leishmania mexicana in cultured macrophages.

Antigenic changes during the intracellular transformation of Leishmania mexicana subsp. amazonensis from promastigotes to amastigotes in macrophages of J774G8 line were noted mostly among protein bands of 24 to 68 kilodaltons in apparent molecular weight. In this region, six were identified as common antigens of both stages, six to seven were identified as promastigote specific, and three to five were identified as amastigote specific. At the higher-molecular-weight region (greater than 68 kilodalton) were two bands, one being predominant in amastigotes and the other in promastigotes. There may be a transformation-specific band (apparent molecular weight = 20 kilodaltons). The transition of these stage-specific antigens varies considerably with different protein species and may play important roles in intracellular leishmanial differentiation.     

Valeriana wallichii root extracts and fractions with activity against Leishmania spp

Leishmanial diseases, posing a public health problem worldwide, are caused by Leishmania parasites with a dimorphic life cycle alternating between the promastigote and amastigote forms. Promastigotes transmitted by the vector are transformed into amastigotes residing in the host tissue macrophages. Presently, new antiparasitic agents are needed against Leishmania donovani and Leishmania major, the respective organisms causing visceral and cutaneous leishmaniasis, since the available treatments are unsatisfactory due to toxicity, high cost, and emerging drug resistance. Over the years, traditional medicinal flora throughout the world enriched the modern pharmacopeia. Hence, roots of 'Indian Valerian' (Valeriana wallichii DC) were studied for its antileishmanial activity for the first time. The methanol and chloroform extracts showed activity against L. donovani promastigotes and both promastigotes and amastigotes of L. major. The most active fraction, F3, obtained from the chloroform extract, showed IC(50) at ～ 3-7 μg/ml against both the promastigotes and 0.3 μg/ml against L. major amastigotes. On investigation of the mechanism of cytotoxicity in L. donovani promastigotes, the 'hall-mark' events of morphological degeneration, DNA fragmentation, externalization of phosphatidyl serine, and mitochondrial membrane depolarization indicated that F3 could induce apoptotic death in leishmanial cells. Therefore, the present study revealed a novel and unconventional property of V. wallichii root as a prospective source of effective antileishmanial agents.     

Stage-specific development of a novel adenosine transporter in Leishmania donovani amastigotes

Leishmania donovani, like all other kinetoplastida, is a purine auxotroph. Comparative studies of adenosine transport in L. donovani amastigotes and promastigotes revealed that, unlike the promastigote stage, the amastigote possesses two distinct adenosine transporters (T(1) and T(2)) both with high affinities (K(m), 1.14+/-0.05 and 2. 09+/-0.13 microM, respectively). One of these transporters (T(1)) appears to be identical with the adenosine/pyrimidine nucleoside transporter of the promastigote reported earlier. The other transporter (T(2)) is specific for the amastigote stage and transports only purine nucleosides. The biological significance of this stage-specific development of the second adenosine transporter has been briefly discussed.     

Involvement of a Leishmania thymidine kinase in flagellum formation, promastigote shape and growth as well as virulence

Leishmania promastigote cells transmitted by their insect vector get phagocytosed by macrophages and convert into the amastigote form. In a recently performed proteomic study, a thymidine kinase (TK) was found to be preferentially expressed in amastigotes. Western blot analysis showing a marked increase in TK protein synthesis during stage differentiation from promastigotes to amastigotes confirmed this result. After comparison of the amino acid sequence of Leishmania donovani and Leishmania major thymidine kinases with thymidine kinases of other organisms the Leishmania protein has to be classified as a type II TK. Therefore, in accordance with the nomenclature of other thymidine kinases we named the Leishmania enzymes LdTK1 and LmTK1, respectively. The LdTK1 is localised within the cytoplasm of promastigotes. In amastigotes, increased expression and a clustered distribution of the protein can be observed. Lmtk1 single allele gene replacement mutants have significantly elongated flagellum. In contrast, lmtk1 double allele gene replacement mutants show a remarkably reduced flagellar length, diminished overall size and a deformed body shape. In addition, they have a 12-fold reduced growth rate. For both mutant strains, macrophage infectivity is clearly reduced compared to a L. major wildtype infection.     

The effects of carbon dioxide and oxygen upon the growth and in vitro transformation of Leishmania mexicana mexicana

Growth of Leishmania mexicana promastigotes is highly dependent upon O2 tension. There was a strong positive correlation between the level of O2, growth rate and maximum parasite density. Promastigotes under low oxygen tension decreased in size, protein content and motility, and deaths occurred. Changes in the carbon dioxide concentration (0.1-5.0%) had little effect on promastigote growth. Transformation in vivo of L. mexicana amastigotes to promastigotes also required oxygen, but a low level (0.4%) was sufficient for a high percentage of the amastigotes to transform. At high O2 concentrations, transformation was a little speedier but the number of parasites transforming was little affected. A greater effect was found with CO2. At 5%, transformation was much more rapid than at 0.1% and also an even greater percentage of amastigotes transformed within 48 h. The results give some indication that amastigotes are adpated for growth at low oxygen tensions encountered in vivo and that high carbon dioxide levels may act as a trigger for transformation of the amastigote to promastigote after it is taken up by the sandfly.     

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.