Functional aspects of the Leishmania donovani lipophosphoglycan during macrophage infection

The most abundant surface glycoconjugate of the Leishmania promastigotes is lipophosphoglycan, a glycosylphosphatidyl-inositol-anchored polymer of the repeating disaccharide-phosphate Gal(beta1,4)Manalpha1-PO4 unit. This complex molecule possesses properties that contribute to the ability of Leishmania to modulate macrophage signaling pathways during the initiation of infection.     

Suppression of macrophage lysosomal enzymes after Leishmania donovani infection

In order to have an insight into the role of host lysosomal enzymes in the intracellular survival of Leishmania parasites, the activities of beta-galactosidase, alpha-mannosidase, and N-acetyl-beta-D-glucosaminidase were studied in peritoneal macrophages of hamsters infected with L. donovani. There was a significant decrease of all three lysosomal enzymes after infection. Heat-killed or formalin-treated parasites failed to inhibit the enzymes, instead a slight stimulation was observed. Purified excreted factor from promastigotes had no effect on the enzymes except beta-galactosidase which was inhibited up to 20%. Inhibition of enzymes was not due to increased secretion after infection. The absence of induction of any endogenous macrophage inhibitor was confirmed by mixed experiments. The levels of 5'-nucleotidase and lactate dehydrogenase remained unchanged after infection. Thus, the inhibition of lysosomal enzymes appears to be the effect of infection process and reflects to actua decrease rather than increased secretion or the action of any inhibitors present in Leishmania promastigotes.     

Effects of topoisomerases inhibitors protoberberine on Leishmania donovani growth, macrophage function, and infection

DNA topoisomerases play a pivotal role in the regulation of cell division. Inhibition of Leishmania spp. topoisomerases represents an alternative to control parasite growth. Cancer research led to the development of several potent topoisomerase inhibitors such as topoisomerase I, topoisomerase II, or both (monobenzimidazole, terbenzimidazole, and protoberberine alkaloid-related compounds) that are effective antitumor agents. In the present study, we evaluated the efficacy of these compounds against Leishmania spp. growth in vitro. Some protoberberine compounds showed pronounced antileishmanial activity and were selected for further analysis in macrophages. These compounds did not affect macrophage viability and only slightly reduced macrophage nitric oxide generation in response to interferon-gamma. Moreover, exposure of infected macrophages to these compounds significantly reduced parasite loads. Collectively, our data suggest that protoberberine-related compounds have powerful antileishmania action and that minor structural variations among them can substantially improve their activity to restrict Leishmania spp. infection in vitro.     

Leishmania donovani affects antigen presentation of macrophage by disrupting lipid rafts

Leishmania donovani-infected splenic macrophages and P388D1 (P388D1(I)) failed to activate T cells in response to low dose of exogenous peptide. The membrane fluidity of P388D1(I) was greater than that of the normal counterpart P388D1(N), but could be reduced either by exposing the cell below phase transition point or by loading cholesterol into membrane (L-P388D1(I)), and this was associated with enhanced Ag-presenting ability of P388D1(I). Presentation of endogenous leishmanial Ag, kinetoplastid membrane protein-11, was also defective, but could be corrected by loading cholesterol into membrane. Because membrane rafts are important for Ag presentation at a low peptide dose, raft architecture of P388D1(I) was studied using raft (CD48 and cholera toxin-B) and non-raft (CD71) markers in terms of their colocalization with I-A(d). Binding of anti-CD48 mAb and cholera toxin B subunit decreased significantly in P388D1(I), and consequently, colocalization with I-A(d) was not seen, but this could be restored in L-P388D1(I). Conversely, colocalization between I-A(d) and CD71 remained unaffected regardless of the presence or the absence of intracellular parasites. P388D1(N) and L-P388D1(I), but not P388D1(I), formed peptide-dependent synapse with T cells quite efficiently and this was found to be corroborated with both intracellular Ca2+ mobilization in T cells and IL-2 production. This indicated that intracellular parasites disrupt the membrane rafts, possibly by increasing the membrane fluidity, which could be corrected by making the membrane rigid. This may be a strategy that intracellular L. donovani adopts to evade host immune system.     

Effect of five-membered heterocycles against Leishmania donovani infection.

Impact of change of heteroatom in pentavalent heterocycles, viz., pyrroles, isoxazoles, imidazoles and crotonates on the profile of antileishmanial activity against amastigotes of L. donovani using in vivo test system and macrophage-amastigote culture system has been studied. Sixty-three compounds were tested. Nine imidazoles showed marginal activity in vivo, whereas 3 out of 10 compounds of isoxazolone series and 2 out of 4 substituted aminocrotonates exhibited antileishmanial activity. Of the 30 substituted pyrroles, except 8 all showed antileishmanial activity in vivo on day 7 post treatment.     

Comparison of the A2 gene locus in Leishmania donovani and Leishmania major and its control over cutaneous infection

In Old World Leishmania infections, Leishmania donovani is responsible for fatal visceral leishmaniasis, and L. major is responsible for non-fatal cutaneous leishmaniasis in humans. The genetic differences between these species which govern the pathology or site of infection are not known. We have therefore carried out detailed analysis of the A2 loci in L. major and L. donovani because A2 is expressed in L. donovani but not L. major, and A2 is required for survival in visceral organs by L. donovani. We demonstrate that although L. major contains A2 gene regulatory sequences, the multiple repeats that exist in L. donovani A2 protein coding regions are absent in L. major, and the remaining corresponding A2 sequences appear to represent non-expressed pseudogenes. It was possible to restore amastigote-specific A2 expression to L. major, confirming that A2 regulatory sequences remain functional in L. major. Although L. major is a cutaneous parasite in rodents and humans, restoring A2 expression to L. major inhibited its ability to establish a cutaneous infection in susceptible BALB/c or resistant C57BL6 mice, a phenotype typical of L. donovani. There was no detectable cellular immune response against L. major after cutaneous infection with A2-expressing L. major, suggesting that the lack of growth was not attributable to acquired host resistance but to an A2-mediated suppression of parasite survival in skin macrophages. These observations argue that the lack of A2 expression in L. major contributed to its divergence from L. donovani with respect to the pathology of infection.     

Second-site suppression of a nonfunctional mutation within the Leishmania donovani inosine-guanosine transporter

LdNT2 is a member of the equilibrative nucleoside transporter family, which possesses several conserved residues located mainly within transmembrane domains. One of these residues, Asp(389) within LdNT2, was shown previously to be critical for transporter function without affecting ligand affinity or plasma membrane targeting. To further delineate the role of Asp(389) in LdNT2 function, second-site suppressors of the ldnt2-D389N null mutation were selected in yeast deficient in purine nucleoside transport and incapable of purine biosynthesis. A library of random mutants within the ldnt2-D389N background was screened in yeast for restoration of growth on inosine. Twelve different clones were obtained, each containing secondary mutations enabling inosine transport. One mutation, N175I, occurred in four clones and conferred augmented inosine transport capability compared with LdNT2 in yeast. N175I was subsequently introduced into an ldnt2-D389N construct tagged with green fluorescent protein and transfected into a Deltaldnt1/Deltaldnt2 Leishmania donovani knockout. GFP-N175I/D389N significantly suppressed the D389N phenotype and targeted properly to the plasma membrane and flagellum. Most interestingly, N175I increased the inosine K(m) by 10-fold within the D389N background relative to wild type GFP-LdNT2. Additional substitutions introduced at Asn(175) established that only large, nonpolar amino acids suppressed the D389N phenotype, indicating that suppression by Asn(175) has a specific size and charge requirement. Because multiple suppressor mutations alleviate the constraint imparted by the D389N mutation, these data suggest that Asp(389) is a conformationally sensitive residue. To impart spatial information to the clustering of second-site mutations, a three-dimensional model was constructed based upon members of the major facilitator superfamily using threading analysis. The model indicates that Asn(175) and Asp(389) lie in close proximity and that the second-site suppressor mutations cluster to one region of the transporter.     

Early histopathology of experimental infection with Leishmania donovani in hamsters

The extracellular promastigote stage of Leishmania donovani is inoculated by a phlebotomine sandfly into the skin of a susceptible host, after which visceral dissemination and clinical disease may ensue. Using a hamster model we examined the histopathology of early infection with L. donovani after intradermal inoculation of cultured promastigotes. The initial response was a mixed polymorphonuclear (PMN)-mononuclear phagocyte infiltrate, noted between 1 and 24 hr after inoculation, which became primarily mononuclear by 48 hr. Parasites were initially found intracellularly in both PMN's and mononuclear phagocytes, but by 48 hr they had assumed amastigote-like morphology and were found exclusively in macrophages. The number of parasites per infected macrophage increased during the first week after inoculation, suggesting that intracellular replication of the organism was taking place. This was followed by the formation of granulomas between 4 and 6 wk. By 8 wk intracellular parasites were largely gone. The histologic response was consistent with early destruction of parasites in PMN's, and survival and replication of L. donovani in macrophages. Cutaneous infection with the parasite was eventually controlled locally, coincident with granuloma formation. Despite these local responses, the organism was able to disseminate and eventually produce typical visceral leishmaniasis.     

Post-translational modification of cellular proteins during Leishmania donovani differentiation

The pathogenic intracellular parasites Leishmania donovani cycle between sand fly gut and the human macrophage phagolysosome, differentiating from extracellular promastigotes to intracellular amastigote forms. Using isobaric tagging for relative and absolute quantifications (iTRAQ/LC-MS/MS) proteomic methodology, we recently described the ordered gene expression changes during this process. While protein abundance changes in Leishmania were documented, little is known about their PTMs. Here we used iTRAQ to detect protein phosphorylation, methylation, acetylation, and glycosylation sites throughout differentiation. We found methylation of arginines, aspartic acids, glutamic acids, asparagines, and histidines. Detected acetylation sites included serines and protein N-terminal acetylations on methionines, serines, alanines, and threonines. Phosphorylations were detected on serines and threonines, but not tyrosines. iTRAQ identified novel fucosylation sites as well as hexosylations. We observed quantity changes in some modifications during differentiation, suggesting a role in L. donovani intracellular development. This study is the first high-throughput analysis of PTM sites dynamics during an intracellular parasitic development.     

Leishmania donovani infection differentially regulates small G‐proteins

Leishmania is a protozoan parasite that resides and replicates in macrophages and causes leishmaniasis. The parasite alters the signaling cascade in host macrophages and evades the host machinery. Small G‐proteins are GTPases, grouped in 5 different families that play a crucial role in the regulation of cell proliferation, cell survival, apoptosis, intracellular trafficking, and transport. In particular, the Ras family of small G‐proteins has been identified to play a significant role in the cellular functions mentioned before. Here, we studied the differential expression of the most important small G‐proteins during Leishmania infection. We found major changes in the expression of different isoforms of Ras, mainly in N‐Ras. We observed that Leishmania donovani infection led to enhanced N‐Ras expression, whereas it inhibited K‐Ras and H‐Ras expression. Furthermore, an active N‐Ras pull‐down assay showed enhanced N‐Ras activity. L donovani infection also increased extracellular signal–regulated kinase 1/2 phosphorylation and simultaneously decreased p38 phosphorylation. In contrast, pharmacological inhibition of Ras led to reduction in the phosphorylation of extracellular signal–regulated kinase 1/2 and enhanced the phosphorylation of p38 in Leishmania‐infected cells, which could lead to increased interleukin‐12 expression and decreased interleukin‐10 expression. Indeed, farnesylthiosalicyclic acid (a Ras inhibitor), when used at the effective level in L donovani–infected macrophages, reduced amastigotes in the host macrophages. Thus, upregulated N‐Ras expression during L donovani infection could be a novel immune evasion strategy of Leishmania and would be a potential target for antileishmanial immunotherapy.     

Parasite-accessory cell interactions in murine leishmaniasis. II. Leishmania donovani suppresses macrophage expression of class I and class II major histocompatibility complex gene products

In the present study, we examined the modulation of MHC class II and class I gene products on BALB/c macrophages infected with the obligate intracellular protozoan Leishmania donovani. Our findings indicated that this organism suppressed macrophage expression of both classes of MHC antigens. These effects varied somewhat, depending on whether cells were in the basal state or were stimulated with interferon-gamma. Thus, class II density on interferon-gamma-treated infected macrophages was suppressed by as much as 90%, relative to lymphokine-stimulated control cells. Induction of H-2K and H-2D by lymphokine treatment of infected macrophages was also markedly reduced. In the basal (non-lymphokine-treated) state, infected cells also showed reduced expression of H-2K and H-2D, but not I-A or I-E. The latter result was related to minimal levels of class II molecules on normal, in vitro cultured macrophages. Suppression of MHC gene products correlated with both the duration and intensity of leishmania infection and could not be overcome by increasing doses of interferon-gamma. Culture of cells under conditions of cyclooxygenase inhibition completely abolished elevated synthesis of prostaglandin E2 by infected macrophages and augmented their responsiveness to lymphokine induction of class II antigens by 60 to 80%. These results indicate that L. donovani is capable of subverting a critical macrophage accessory function required for the induction of T lymphocyte immunity. This mechanism could account, at least in part, for defective parasite-specific cell-mediated immunity seen during infections with this protozoan.     

Vaccination with recombinant Leishmania donovani gamma-glutamylcysteine synthetase fusion protein protects against L. donovani infection

Visceral leishmaniasis presents a serious health threat in many parts of the world. There is, therefore, an urgent need for an approved vaccine for clinical use to protect against infection. In this study, the ability of recombinant Leishmania donovani gamma-glutamyl cysteine synthetase protein (LdγGCS) alone or incorporated into a non-ionic surfactant vesicle (NIV) delivery system to protect against L. donovani infection was evaluated in a BALB/c mouse model. Immunization with LdγGCS alone or LdγGCS-NIV induced specific IgG1 and IgG2a antibodies compared to controls, with LdγGCS-NIV inducing significantly higher titers of both antibody classes (P < 0.05). Both formulations induced similar increases in splenocyte IFN-γ production following ex vivo antigen stimulation with LdγGCS compared with cells from control mice (P < 0.05). Similar levels of protection against infection were induced by LdγGCS alone and LdγGCS-NIV, based on their ability to suppress liver parasite burdens compared to control values (P < 0.01), indicating that using a carrier system did not enhance the protective responses induced by the recombinant protein. The results of this study indicate that LdγGCS may be a useful component in a vaccine against L. donovani.     

Selective macrophage suppression during sepsis

Polymicrobial sepsis induces suppression of macrophage function as determined by a reduction of pro-inflammatory cytokine production upon re-exposure to lipopolysaccharide (LPS) in vitro. We examined whether macrophages were refractory to only LPS challenge or if they were immunoparalyzed and unable to respond to other stimuli such as lipoteichoic acid (LTA) or zymosan (ZYM). This study evaluated the capacity of peritoneal macrophages to produce pro-inflammatory and anti-inflammatory cytokines as well as chemokines following mild or severe sepsis induced by cecal ligation and puncture (CLP). Peritoneal macrophages were isolated 29 h after CLP and challenged with different stimuli. LPS was a more potent stimulus for cytokine induction than LTA or ZYM in both mild and severe sepsis. In mild sepsis, the macrophage cytokine response to LPS was selective and less refractory than in severe sepsis. While production of IL-6 and KC was reduced, secretion of TNF-alpha and MIP-1alpha was enhanced in those cells isolated from mice with mild sepsis. Production of IL-10 and the IL-1 receptor antagonist , MIP-2, and MCP-1 in response to LPS stimulation was equivalent to the amount produced by naive macrophages. Our results indicate that macrophages are not immunoparalyzed during sepsis and may still be induced to secrete some inflammatory mediators.     

Characterization of the A2-A2rel gene cluster in Leishmania donovani: involvement of A2 in visceralization during infection

The A2 gene family is present in Leishmania donovani, which causes fatal visceral leishmaniasis in human patients, but is not present in Leishmania major, which causes cutaneous leishmaniasis infections. The A2 genes in L. donovani are stage specific and are expressed at high levels in the amastigote stage in the mammalian host, but are not expressed in the promastigote stage in the insect sandfly vector. The A2 genes are tandem repeated with a distinct gene family termed the A2rel genes. In order to characterize the structure and function of the A2-A2rel gene clusters, the 5' and 3' DNA sequences flanking the A2-A2rel cluster were isolated, sequenced and used to generate mutants through gene targeting. Although it was possible to generate partial A2-A2rel gene clusters knock-out mutants, it was not possible to delete all the A2-A2rel gene clusters completely from the L. donovani genome, suggesting that, within this cluster, there are genes that are essential for survival in culture. Characterization of these mutants revealed that A2 and A2rel gene expression was compensated by amplifying the remaining intact A2 and A2rel genes, and the proliferation of these mutants in culture and their virulence in BALB/c mice were compromised. In order to explore further the biological role of A2, the L. donovani A2 gene was introduced into L. major. In comparison with the control L. major, the A2-expressing L. major parasites demonstrated an increased ability to survive in the spleen of BALB/c mice. These data suggest that A2 plays a role in the visceralization of infection associated with L. donovani.     

Parasite accessory cell interactions in murine leishmaniasis. I. Evasion and stimulus-dependent suppression of the macrophage interleukin 1 response by Leishmania donovani

Interleukin 1 (IL 1) is a principal mediator of the host immune response to microbial challenge. Accessory cells of the monocyte-macrophage series are a major source of this cytokine and are also chronically parasitized by protozoa of the genus Leishmania. This suggests that characterization of the macrophage IL 1 response to Leishmania would increase our understanding of the regulation of host immunity to these organisms. In the present study, the macrophage IL 1 response to Leishmania donovani was examined because infections with this organism have findings consistent with parasite-specific T cell unresponsiveness. Cytokine activity was measured either by direct stimulation or by co-stimulation of thymocytes. Conditioned media from BALB/c resident peritoneal macrophages infected with amastigotes of L. donovani contained no more IL 1 than did supernatant fluids of control cells. In contrast, supernatants from cells stimulated with lipopolysaccharide or heat-killed Listeria monocytogenes had significantly increased cytokine content. Resident cells infected with L. donovani for 4 hr before being stimulated with Listeria demonstrated a suppressed IL 1 response (approximately 40% of Listeria alone) to this secondary particulate stimulus. In contrast, the secondary response of leishmania-preinfected cells to lipopolysaccharide was not affected. To examine whether accessory cell nonresponsiveness to L. donovani (with respect to IL 1) was related to the state of macrophage activation, elicited peritoneal macrophages obtained by injection of proteose peptone were also studied. These cells responded to stimulation with lipopolysaccharide and fixed Staphylococcus aureus with increases in intracellular, membrane, and secreted cytokine activities. In contrast, L. donovani failed to induce any of these activities. This was found to be the case irrespective of whether amastigotes were alive or killed or opsonized with specific antibodies. Elicited cells preinfected with Leishmania responded normally to secondary stimulation with lipopolysaccharide, but not S. aureus (64% of Staphylococcus alone). In addition, attachment and penetration of L. donovani promastigotes and their subsequent conversion to amastigotes within macrophages failed to induce IL 1 synthesis. The findings of this study indicate that L. donovani has the ability to both evade and suppress the macrophage IL 1 response. Because this monokine provides an obligatory signal during macrophage-dependent T cell activation, evasion of signal transduction for IL 1 synthesis may be related to defects in cell-mediated immunity which occur during infections with this organism.     

Leishmania donovani: characterization and expression of ORFF, a gene amplified from the LDI locus

The LD1 locus is a 27.5-kb region of chromosome 35 that is conserved among all species of Leishmania and is amplified in several different isolates. Here, we report the genomic distribution of ORFF, a gene from the LD1 region, and its expression at the RNA and protein levels in two Indian isolates of Leishmania donovani. In both of these isolates, ORFF was present as a single copy on chromosome 35. Densitometric analysis of ORFF mRNA abundance revealed relative abundance of 0.2 and 1.0 in AG83 and S-Lal, respectively. Antiserum against recombinant ORFF protein detected a protein of the predicted size ( approximately 34 kDa) in both strains. The protein is most abundant in mid-log-phase promastigotes and has a nuclear localization. The ORFF protein is preferentially expressed in L. donovani amastigotes but, in contrast, is expressed at higher levels in L. major promastigotes.     

Metabolic variation during development in culture of Leishmania donovani promastigotes

The genome sequencing of several Leishmania species has provided immense amounts of data and allowed the prediction of the metabolic pathways potentially operating. Subsequent genetic and proteomic studies have identified stage-specific proteins and putative virulence factors but many aspects of the metabolic adaptations of Leishmania remain to be elucidated. In this study, we have used an untargeted metabolomics approach to analyze changes in the metabolite profile as promastigotes of L. donovani develop during in vitro cultures from logarithmic to stationary phase. The results show that the metabolomes of promastigotes on days 3-6 of culture differ significantly from each other, consistent with there being distinct developmental changes. Most notable were the structural changes in glycerophospholipids and increase in the abundance of sphingolipids and glycerolipids as cells progress from logarithmic to stationary phase.