Role of intracellular cAMP in differentiation-coupled induction of resistance against oxidative damage in Leishmania donovani

Even though the human parasite Leishmania donovani encounters tremendous oxidative burst during macrophage invasion, a set of parasites survives and proliferates intracellularly, leading to transformation from promastigote to amastigote form and disease manifestation. The striking shifts in temperature (from 22 degrees C in the insect gut to 37 degrees C in the mammalian host) and pH (7.2 in the insect gut to 5.5 in the parasitophorous vacuole of macrophages) are the key environmental triggers for differentiation as these cause an arrest in the G1 stage of the cell cycle and initiate transformation. Using an established in vitro culture and differentiation system our study demonstrates that the differentiation-triggering environment induces resistance to oxidative damage and consequently enhances infectivity. Differentiation conditions caused a three- to fourfold elevation in cAMP level as well as cAMP-dependent protein kinase activity. Similar to stress exposure, positive modulation of intracellular cAMP resulted in blockage of cell cycle progression and induction of resistance against oxidative damage. Resistance against pro-oxidants from either stress or cAMP may be associated with upregulation of the expression of three major antioxidant genes, peroxidoxin 1, trypanothione reductase, and superoxide dismutase A. Positive modulation of the intracellular cAMP response enables cells to resist the cytotoxic effects of pro-oxidants. In contrast, downregulation of intracellular cAMP by overexpression of cAMP phosphodiesterase A resulted in a decrease in resistance against oxidative damage and reduced infectivity toward activated macrophages. This study for the first time reveals the importance of cAMP response in the life cycle and infectivity of the Leishmania parasite.     

Leishmania histone H1 overexpression delays parasite cell-cycle progression, parasite differentiation and reduces Leishmania infectivity in vivo

Episomal expression of Leishmania histone H1 sense mRNAs in Leishmania major promastigotes was found previously to result in overexpression of this molecule and to reduce parasite infectivity in vitro. Herein, we evaluated the in vivo infectivity of these transfectants, in BALB/c mice, and showed that it is dramatically reduced. No lesions were observed in this group of mice and this was associated with an extremely low number of parasites both in the footpad and in the draining lymph nodes. Interestingly, the transfectants-reduced infectivity was associated with a delay in their cell-cycle progression and differentiation to axenic amastigotes, assessed in vitro. Therefore, the dramatic reduction in their infectivity may be attributed to the above-mentioned phenotypic modifications. As the metazoan linker histone H1(0) homologue is known to delay cell-cycle progression in mammalian cells we investigated whether its Leishmania counterpart, which possesses homology to its C-terminal region, when expressed in mammalian cells may also affect their cell-cycle progression. It was thus shown that Leishmania histone H1 expressed in COS7 and NIH 3T3 cells, delays cell-cycle progression in these cells too. The latter strengthens the phenotype observed in Leishmania and provides evidence that critical functions of histone H1 molecules are conserved throughout evolution.     

Developmentally regulated sphingolipid degradation in Leishmania major

Leishmania parasites alternate between extracellular promastigotes in sandflies and intracellular amastigotes in mammals. These protozoans acquire sphingolipids (SLs) through de novo synthesis (to produce inositol phosphorylceramide) and salvage (to obtain sphingomyelin from the host). A single ISCL (Inositol phosphoSphingolipid phospholipase C-Like) enzyme is responsible for the degradation of both inositol phosphorylceramide (the IPC hydrolase or IPCase activity) and sphingomyelin (the SMase activity). Recent studies of a L. major ISCL-null mutant (iscl(-)) indicate that SL degradation is required for promastigote survival in stationary phase, especially under acidic pH. ISCL is also essential for L. major proliferation in mammals. To further understand the role of ISCL in Leishmania growth and virulence, we introduced a sole IPCase or a sole SMase into the iscl(-) mutant. Results showed that restoration of IPCase only complemented the acid resistance defect in iscl(-) promastigotes and improved their survival in macrophages, but failed to recover virulence in mice. In contrast, a sole SMase fully restored parasite infectivity in mice but was unable to reverse the promastigote defects in iscl(-). These findings suggest that SL degradation in Leishmania possesses separate roles in different stages: while the IPCase activity is important for promastigote survival and acid tolerance, the SMase activity is required for amastigote proliferation in mammals. Consistent with these findings, ISCL was preferentially expressed in stationary phase promastigotes and amastigotes. Together, our results indicate that SL degradation by Leishmania is critical for parasites to establish and sustain infection in the mammalian host.     

Growth phase regulation of the main folate transporter of Leishmania infantum and its role in methotrexate resistance

The protozoan parasite Leishmania relies on the uptake of folate and pterin from the environment to meet its nutritional requirements. We show here that a novel gene (folate transporter 1 (FT1)) deleted in a Leishmania infantum methotrexate-resistant mutant corresponds to the main folate transporter (K(m), 410 nM). FT1 was established as the main folate transporter by both gene transfection and by targeted gene deletion. Modulation of the expression of FT1 by these manipulations altered the susceptibility of Leishmania cells to methotrexate. Folate transport was stage-regulated with higher activity in the logarithmic phase and less in the stationary phase. FT1 fused to green fluorescent protein led to the observation that FT1 was located in the plasma membrane in the logarithmic phase but was retargeted to an intracellular organelle followed by a degradation of the protein in stationary phase. Leishmania has several folate transporters with different characteristics, and the growth stage-related activity of at least one transporter is regulated post-translationally.     

Leishmania braziliensis: protein, carbohydrate, and antigen differences between log phase and stationary phase promastigotes in vitro

When Leishmania species are grown in vitro, parasites from the stationary phase differ from those in log phase growth in being more infective and more resistant to complement and macrophage mediated killing. In the present study, log phase and stationary phase promastigotes of Leishmania braziliensis panamensis were compared at the molecular level. Differences in polypeptide and glycoprotein composition and antigenicity between log and stationary phase promastigotes of L. b. panamensis were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting; the former showed that two polypeptides were unique to log phase promastigotes and one was unique to stationary phase promastigotes. There were also differences in surface lectin binding characteristics of log and stationary phase promastigotes. Live stationary phase promastigotes bound more concanavalin and lentil lectin than log phase promastigotes, indicating a greater number of mannose residues on their surfaces.     

Leishmania donovani metacyclic promastigotes: transformation in vitro, lectin agglutination, complement resistance, and infectivity

Freshly transformed Leishmania donovani amastigotes from hamster spleen were used to establish axenic cultures at high density in a modified Grace's medium, which was only partly replenished when cultures were fed. Small, free-swimming, highly active stationary phase promastigotes with a short cell body and long flagellum were induced in this medium. The freshly transformed stationary phase promastigotes so induced were less able to bind peanut agglutinin, had more than 40-fold increased resistance to killing by normal human serum, and 15-fold increased infectivity both in vivo and in vitro when compared to freshly transformed logarithmic phase or long term culture promastigotes. These short form promastigotes may correspond to the metacyclic promastigote forms in the sand fly vector.     

Overexpression of the Leishmania amazonensis Ca2+-ATPase gene lmaa1 enhances virulence

A gene for a Ca2+-transporting ATPase (lmaa1) from the trypanosomatid parasite Leishmania (mexicana) amazonensis was overexpressed in two clones of L. amazonensis differing in their virulence. RNA and protein expression of the gene was increased in transfectants, as was the infectivity of transfectants versus parental types in both mouse and in vitro macrophage infection experiments. The virulence of the almost avirulent clone was enhanced such that it was more virulent than the parental 'virulent' clone. Growth of the parasites in culture as promastigotes, after isolation from mouse lesions, indicated that transfection led to improved survival of promastigotes during the stationary phase of culture. As it is in this culture phase that infective metacyclic forms develop, the key role of the Lmaa1 protein may be in metacyclogenesis. The protein may be important in the synthesis and trafficking of new proteins through the secretory pathway, as we demonstrate, using a green fluorescent protein hybrid and by immunofluorescence, that the Lmaa1 protein is located in the endoplasmic reticulum in promastigotes and amastigotes of L. amazonensis.     

Growth phase and medium ph modulate the expression of proteinase activities and the development of megasomes in axenically cultivated Leishmania (Leishmania) amazonensis amastigote-like organisms

Leishmania (Leishmania) amazonensis LV79 (MPRO/BR/72/M1841) has been adapted to grow at 33 C as amastigote-like (AL) organisms in modified UM-54 medium initially adjusted to a pH of 4.8-5.0. Axenic cultures could be routinely restarted from parasites recovered from footpad lesions obtained by inoculation of BALB/c mice with preadapted culture stages. Morphological features, proteinase activities, and infectivity of AL organisms were examined during the in vitro growth cycle, and differences were found between log- and stationary-phase parasites. Stationary-phase AL organisms were morphologically similar to lesion amastigotes, did not react with a paraflagellar rod-specific monoclonal antibody in western blots, and contained proteinase activities resolving identically to the enzymes of lesion amastigotes in gelatin gels. Whereas typical megasomes could be identified in about a third of the stationary-phase AL population, the organelles were rarely seen in log-phase organisms. Azocaseinolytic activity progressively increased during the exponential growth phase and reached its highest values (approximately 65-70% of those determined in lesion amastigotes) at the stationary phase; the association of total proteinase activity with increased expression of cysteine proteinases was indicated by the strong inhibition of azocasein hydrolysis by E-64, the intensified banding of the 28-, 31-, and 35-kDa proteinases in gelatin gels, and the higher susceptibility of stationary-phase AL organisms to L-leucine methyl ester. Although overall axenic amastigotes were less infective to BALB/c mice than were lesion-derived parasites, stationary-phase AL organisms were more infective than were log-phase parasites. Medium pH increased during the exponential growth phase, but dropped in the stationary phase, when the observed morphological, biochemical, and biological changes became apparent.     

Characterisation of a new Leishmania META gene and genomic analysis of the META cluster

The META1 gene of Leishmania is upregulated in metacyclic promastigotes and encodes a 12 kDa virulence-related protein, conserved in all Leishmania species analysed. In this study, the genomic region adjacent to the Leishmania amazonensis META1 gene was characterised and compared to the Leishmania major META1 locus as well as to syntenic loci identified in Trypanosoma brucei and Trypanosoma cruzi. Three new genes expressed with increased abundance of steady state mRNA in L. amazonensis promastigotes were identified, two of which are upregulated in stationary phase promastigotes, sharing the pattern of expression previously described for the META1 mRNA. One of these new genes, named META2, encodes a polypeptide of 444 amino acid residues with a repetitive structure showing three repeats of the META domain (defined as a small domain family found in the Leishmania META1 protein and in bacterial proteins hypothetically secreted and/or implicated in motility) and a carboxyl-terminal region similar to several putative calpain-like proteins of Trypanosoma and Leishmania.     

Metacyclogenesis: a basic process in the biology of Leishmania

Metacyclogenesis is a process whereby Leishmania transforms from poorly infective procyclic promastigotes into highly infective metacyclic promastigotes. In nature, metacyclogenesis occurs in the insect vector. This transformation is accompanied by an increased ability to infect and survive in the vertebrate host, where the parasite is attacked by the host's immune system. Metacyclogenesis has also been shown to occur in axenic cultures of promastigotes. Morphological changes in size and shape, and length of flagellum were first associated with differentiation in the insect gut and in different phases of growth in culture. Later, the expression of molecules such as LPG and the surface protease gp63 were associated with this process. These two molecules were observed to undergo qualitative and quantitative modifications as the promastigotes differentiated from procyclic to metacyclic forms. Using cDNA subtractive hybridization-based methods or differential amplification, previously unknown genes tightly linked to metacyclogenesis have been identified. Gene products exclusively expressed in metacyclic promastigotes included a gene B product and Mat-1--a gene associated with metacyclogenesis. Other proteins, Meta-1, SHERP and HASP, were up-regulated during the metacyclic stage. The function and stage-regulated expression of these molecules and their relationship with infectivity are now under investigation.     

Leishmania: overexpression and comparative structural analysis of the stage-regulated meta 1 gene.

The meta 1 gene of Leishmania major is upregulated in metacyclic promastigotes and encodes an 11.5-kDa protein with no significant similarities to other proteins in the existing databases. In this paper, we characterize the homologous meta 1 genes in L. amazonensis and L. donovani. Proteins encoded by this gene in all three species present a high degree of identity. The meta 1 gene cannot be replaced by gene targeting in L. major, suggesting an essential role for the protein, at least in promastigotes. Overexpression of the meta 1 protein in L. amazonensis generates parasites that are more virulent than wild-type organisms in vivo.     

Effects of Promastigote Growth Phase,Frequency of Subculture,and Host Age on Promastigote-initiated Infections with Leishmania donovani in the Golden Hamster*†

Promastigotes of Leishmania donovani, 3S strain, were cultured from homogenized infected hamster spleen incubated at 25 C in a particle-free modification of Tanabe's (1923) medium, and were subcultured in this medium from 1 to 4 times. Promastigotes were inoculated intracardially to golden hamsters (Mesocricetus auratus). Promastigotes that were subcultured frequently by transfer from log phase of growth retained their infectivity for hamsters, as assayed by numbers of amastigotes in the liver at 16 days post infection. Promastigotes that were subcultured infrequently by transfer from stationary phase declined in infectivity. The extent of the decline was roughly proportional to the length of the incubation periods of the primary culture plus 1st subculture. Promastigotes harvested from log phase of growth were significantly less infective for hamsters than those harvested from stationary phase of growth, in that numbers of amastigotes found in the liver after 16 days were lower, and times to death longer, when log phase organisms were used to infect hamsters. The age of the hamster at the time of inoculation was found to affect the apparent infectivity of promastigotes from a 1st or 2nd subculture. When weanling (age 4 weeks), juvenile (age 8 weeks) and adult (age 24 to 32 weeks) hamsters received the same numbers of promastigotes, the weanlings had the highest numbers of liver amastigotes at 16 days, and shortest times to death, of the 3 groups; juveniles were intermediate between weanlings and adults; and adults had the lowest numbers of parasites and longest times to death of the host. Differences were statistically significant only between weanlings and adults. Responses of weanling and adult hamsters to infection with promastigotes could be rendered indistinguishable if the promastigotes were inoculated on the basis of 10⁵ promastigotes per g of host body weight.     

Leishmania mexicana: promastigotes migrate through osmotic gradients

During the insect phase of the parasite lifecycle, Leishmania promastigotes move from the midgut to the anterior regions of the alimentary tract of their sandfly vector. Chemotaxis of Leishmania promastigotes towards sugars has been reported, and the putative presence of sugar gradient in the insect foregut has been suggested to play a role in promastigote development in the insect. We have further investigated the potential of Leishmania mexicana promastigotes to respond to chemical stimulii. We find that promastigotes move towards concentrations of all substances tested and that this taxis requires the presence of an osmotic gradient. Our results indicate that behaviour that has previously been interpreted as chemotaxis is better understood as osmotaxis. The implications of this observation are discussed in the context of promastigote development.     

Leishmania major: Reactive oxygen species and interferon gamma induction by soluble lipophosphoglycan of stationary phase promastigotes

Protozoan parasites of the genus Leishmania cause a number of important human diseases. One of the key determinants of parasite infectivity and survival is membrane glycoconjugate lipophosphoglycan (mLPG). In addition, it has been shown that mLPG could be used as a transmission blocking vaccine. Since culture supernatant of parasite promastigotes is a good source of LPG, we attempted to compare the immunological properties of culture supernatant and membrane LPG prepared from stationary phase promastigotes of Leishmania major. The purity of supernatant LPG (sLPG) and membrane LPG (mLPG) was determined by thin layer chromatography. The effect of sLPG and mLPG on the production of reactive oxygen species (ROS) was studied using PBMCs isolated from healthy individuals. In addition, induction of IL-12, IFN-gamma and IL-10 secretion in the presence of sLPG and mLPG was investigated. Reactive oxygen species in addition to IL-10 and IL-12 were induced by both sLPG and mLPG. However, IFN-gamma production was promoted only in response to sLPG suggesting its ability to promote Th1 response and implication in vaccine design.     

Substrate preferences and glucose uptake in glibenclamide-resistant Leishmania parasites

Several drug-resistant mammalian cell types exhibit increased glycolytic rates, preferential synthesis of ATP through oxidative phosphorylation, and altered glucose transport. Herein we analyzed the influence of parasite growth phase on energy substrate uptake and use in a Leishmania strain [NR(Gr)] selected for resistance against glibenclamide. Glibenclamide is an ABC-transporter blocker which modulates the function of glucose transporters in some mammalian cells. Our results demonstrate for the first time that compared to glibenclamide-sensitive Leishmania, exponential phase glibenclamide-resistant parasites exhibit decreased use of glucose as energy substrate, decreased glucose uptake and decreased glucose transporter expression. However, compared to glibenclamide-sensitive cells, stationary phase resistant parasites display an increased use of amino acids as energy substrate and an increased activity of the enzymes hexokinase, phosphoglucose isomerase, and especially NAD(+)-linked glutamate dehydrogenase. These results suggest that drug resistance in Leishmania involves a metabolic adaptation that promotes a stage dependent modulation of energy substrate uptake and use as a physiological response to the challenge imposed by drug pressure.     

A sensitive method for assaying chemotaxic responses of Leishmania promastigotes

This article describes a sensitive, cheap, and easy method for assaying chemotaxic responses of Leishmania promastigotes. A gradient of the substance to be assayed was produced inside a series of commercially available capillary tubes submerged in a promastigote suspension. After an incubation period, the attractiveness of the substance under test was measured by counting the number of parasites in the capillaries in a Neubauer chamber. Different responses were detected in two strains of Leishmania amazonensis and one strain of L. chagasi after standardization of the method to assay attraction to carbohydrates. Very different responses were obtained when the test was performed using promastigotes of the same strain in two different physiological states (log and stationary phase). The stationary phase cells showed an enhanced chemotaxic capability, which can be explained by the fact that the metacyclic forms commonest in stationary phase cultures have greater mobility than other promastigotes. This method will permit studies to be made of the attractive response to different substances in Leishmania species and other trypanosomatids and facilitate characterization of the potential receptors involved in the chemotaxic response. An adaptation of the method to assay the response to repellent substances is also provided.     

Expression of biopterin transporter (BT1) protein in Leishmania

The present work focuses on the growth phase regulated expression of biopterin transporter gene (BT1) from the LD1 locus on chromosome 35 of Leishmania donovani. Antiserum against recombinant BT1 detected a polypeptide of 45 kDa of equal intensity at lag, log and stationary phases of promastigote growth, both in L. donovani strain LSB-7.1 (MHOM/BL/67/ITMAP263), and strain LSB-146.1 (HOM/IR/95/X81), a natural isolate from Isfehan, Iran that caused cutaneous leishmaniasis. However, in both these strains an additional polypeptide of higher molecular mass (50 kDa) was also observed during lag phase only. In addition, polypeptides of 40, 20, 18 and 16 kDa were seen only during the lag and log phases of both strains. Analysis of L. donovani single, double and triple (null) BT1 knockout mutants confirmed that the 45-kDa polypeptide was the BT1 gene product, as it was absent in the null mutant. These results indicate that 45-kDa BT1 protein in Leishmania is consistently and constitutively expressed in all the growth stages of the parasite.