The sterol-binding antibiotic nystatin inhibits entry of non-opsonized Leishmania donovani into macrophages

Leishmania donovani is an obligate intracellular parasite that infects macrophages of the vertebrate host resulting in visceral leishmaniasis in humans, a major public health problem worldwide. The molecular mechanisms involved in internalization of Leishmania are still poorly characterized. We report here that cholesterol sequestration by the sterol-binding antifungal polyene antibiotic nystatin markedly inhibits binding and entry of non-opsonized L. donovani promastigotes into macrophages. Interestingly, these effects are not observed when serum-opsonized L. donovani are used for infectivity studies thus pointing the essential role of cholesterol in mediating entry of the parasite via the non-opsonic pathway. Based on our earlier results where leishmanial infectivity was shown to be sensitive to physical depletion of cholesterol from macrophages, these results indicate that the mere sequestration of cholesterol in the host plasma membrane is sufficient to inhibit the binding and entry of non-opsonized L. donovani. These results represent the first report on the effect of a cholesterol-sequestering agent on the entry of Leishmania parasites to host macrophages. More importantly, these findings offer the possibility of reevaluating the mechanism behind the effectiveness of current therapeutic strategies to treat leishmaniasis.     

Amphotericin B inhibits entry of Leishmania donovani into primary macrophages

Visceral leishmaniasis is a vector-borne disease caused by an obligate intra-macrophage protozoan parasite Leishmania donovani. The molecular mechanisms involved in internalization of Leishmania are still poorly understood. Amphotericin B and its formulations are considered as the best existing drugs against visceral leishmaniasis and are being increasingly used. The reason for its antileishmanial activity is believed to be its ability to bind ergosterol found in parasite membranes. In case of in vivo amphotericin B treatment, both host macrophages and parasites are exposed to amphotericin B. The effect of amphotericin B treatment could therefore be due to a combination of its interaction with both sterols i.e., ergosterol of Leishmania and cholesterol of host macrophages. We report here that cholesterol complexation by amphotericin B markedly inhibits binding of L. donovani promastigotes to macrophages. These results represent one of the first reports on the effect of amphotericin B on the binding of Leishmania parasites to host macrophages. Importantly, these results offer the possibility of reevaluating the mechanism behind the effectiveness of current therapeutic strategies that employ sterol-complexing agents such as amphotericin B to treat leishmaniasis.     

IL-10- and TGF-beta-mediated susceptibility in kala-azar and post-kala-azar dermal leishmaniasis: the significance of amphotericin B in the control of Leishmania donovani infection in India

Visceral leishmaniasis (VL) or kala-azar is known to be associated with a mixed Th1-Th2 response, and effective host defense requires the induction of IFN-gamma and IL-12. We address the role of the differential decline of IL-10 and TGF-beta in response to sodium antimony gluconate (SAG) and amphotericin B (AmB), the therapeutic success of SAG and AmB in Indian VL, and the significance of IL-10 and TGF-beta in the development and progression of post-kazla-azar dermal leishmaniasis (PKDL). In the active disease, PBMC from VL patients showed suppressed Ag-specific lymphoproliferation, IFN-gamma and IL-12 production, and elevation of IL-10 and TGF-beta. Cure corresponded with an elevation in IFN-gamma and IL-12 production and down-regulation of IL-10 and TGF-beta. Both CD4(+) and CD8(+) T cells were involved in IFN-gamma and IL-10 production. Interestingly, the retention and maintenance of residual IL-10 and TGF-beta in some SAG-treated individuals and the elevation of IL-10 and TGF-beta in PKDL, a sequel to kala-azar, probably reflects the role of these cytokines in reactivation of the disease in the form of PKDL. Contrastingly, AmB treatment of VL resulted in negligible TGF-beta levels and absolute elimination of IL-10, reflecting the better therapeutic activity of AmB and its probable role in the recent decline in PKDL occurrences in India. Moreover, elucidation of immune responses in Indian PKDL patients revealed a spectral pattern of disease progression where disease severity could be correlated inversely with lymphoproliferation and directly with TGF-beta, IL-10, and Ab production. In addition, the enhancement of CD4(+)CD25(+) T cells in active VL, their decline at cure, and reactivation in PKDL suggest their probable immunosuppressive role in these disease forms.     

Persistent dermal lesions in a patient with previous history of visceral leishmaniasis

Post-kala-azar dermal leishmaniasis (PKDL) is a complication of visceral leishmaniasis (VL) that most frequently occurs after an episode of VL caused by Leishmania donovani. In this case report, we present a 21-year-old male patient with persistent skin lesions and recurrent visceral leishmaniasis (VL) due to Leishmania infantum. The patient did not respond to multiple lines of anti-leishmanial treatment (including Liposomal amphotericin B and miltefosine) and later died from cerebral lesions presumed to be secondary to persistent VL.     

Hyperlipidemia offers protection against Leishmania donovani infection: role of membrane cholesterol

Leishmania donovani (LD), the causative agent of visceral leishmaniasis (VL), extracts membrane cholesterol from macrophages and disrupts lipid rafts, leading to their inability to stimulate T cells. Restoration of membrane cholesterol by liposomal delivery corrects the above defects and offers protection in infected hamsters. To reinforce further the protective role of cholesterol in VL, mice were either provided a high-cholesterol (atherogenic) diet or underwent statin treatment. Subsequent LD infection showed that an atherogenic diet is associated with protection, whereas hypocholesterolemia due to statin treatment confers susceptibility to the infection. This observation was validated in apolipoprotein E knockout mice (AE) mice that displayed intrinsic hypercholesterolemia with hepatic granuloma, production of host-protective cytokines, and expansion of antileishmanial CD8⁺IFN- γ ⁺ and CD8⁺IFN- γ ⁺TNF- α ⁺ T cells in contrast to the wild-type C57BL/6 (BL/6) mice when infected with LD. Normal macrophages from AE mice (N-AE-M ϕ) showed 3-fold higher membrane cholesterol coupled with increased fluorescence anisotropy (FA) compared with wild-type macrophage (N-BL/6-M ϕ). Characterization of in vitro LD-infected AE macrophage (LD-AE-M ϕ) revealed intact raft architecture and ability to stimulate T cells, which were compromised in LD-BL/6-Mϕ. This study clearly indicates that hypercholesterolemia, induced intrinsically or extrinsically, can control the pathogenesis of VL by modulating immune repertoire in favor of the host.     

Cholesterol: a potential therapeutic target in Leishmania infection?

Leishmania are obligate intracellular parasites that invade and survive within host macrophages and can result in visceral leishmaniasis, a major public health problem worldwide. The entry of intracellular parasites, in general, involves interaction with the plasma membrane of host cells. Cholesterol in host cell membranes was recently shown to be necessary for binding and internalization of Leishmania and for the efficient presentation of leishmanial antigens in infected macrophages. This article describes the need to explore cyclodextrin-based compounds, which modulate host membrane cholesterol levels, as a possible therapeutic strategy against leishmaniasis in addition to other intracellular parasites.     

Hexadecylphosphocholine interaction with lipid monolayers

The phospholipid analogue miltefosine or hexadecylphosphocholine (HePC) is a drug of high interest in the treatment for fatal visceral leishmaniasis (VL) due to Leishmania donovani particularly because of its activity by oral route. In this study, the interaction of HePC with a monolayer of beta-palmitoyl-gamma-oleyl-phosphatidylcholine (POPC) as membrane model or sterol (ergosterol or cholesterol) was investigated. At a constant pressure of 25 mN/m, the adsorption kinetics of HePC into the monolayers showed that HePC molecules are inserted into the monolayer of lipids as monomers until the critical micellar concentration (CMC). At HePC concentrations superior to the CMC, the micelles of HePC are deployed at the interface as groups of monomers into the POPC or sterol monolayer. The study of mixture of HePC/(POPC or sterol), spread at the air-water interface, shows that a simple miscibility between HePC and POPC is observed, whereas a high condensation appears between HePC and sterols showing a high affinity between HePC and sterols. In addition, HePC does not act as detergent disturbing membrane integrity.     

Integrity of the Actin Cytoskeleton of Host Macrophages is Essential for Leishmania donovani Infection

Visceral leishmaniasis is a vector-borne disease caused by an obligate intracellular protozoan parasite Leishmania donovani. The molecular mechanism involved in internalization of Leishmania is poorly understood. The entry of Leishmania involves interaction with the plasma membrane of host cells. We have previously demonstrated the requirement of host membrane cholesterol in the binding and internalization of L. donovani into macrophages. In the present work, we explored the role of the host actin cytoskeleton in leishmanial infection. We observed a dose-dependent reduction in the attachment of Leishmania promastigotes to host macrophages upon destabilization of the actin cytoskeleton by cytochalasin D. This is accompanied by a concomitant reduction in the intracellular amastigote load. We utilized a recently developed high resolution microscopy-based method to quantitate cellular F-actin content upon treatment with cytochalasin D. A striking feature of our results is that binding of Leishmania promastigotes and intracellular amastigote load show close correlation with cellular F-actin level. Importantly, the binding of Escherichia coli remained invariant upon actin destabilization of host cells, thereby implying specific involvement of the actin cytoskeleton in Leishmania infection. To the best of our knowledge, these novel results constitute the first comprehensive demonstration on the specific role of the host actin cytoskeleton in Leishmania infection. Our results could be significant in developing future therapeutic strategies to tackle leishmaniasis.     

Structure-guided approach to identify a novel class of anti-leishmaniasis diaryl sulfide compounds targeting the trypanothione metabolism

Leishmania protozoans are the causative agent of leishmaniasis, a neglected tropical disease consisting of three major clinical forms: visceral leishmaniasis (VL), cutaneous leishmaniasis, and mucocutaneous leishmaniasis. VL is caused by Leishmania donovani in East Africa and the Indian subcontinent and by Leishmania infantum in Europe, North Africa, and Latin America, and causes an estimated 60,000 deaths per year. Trypanothione reductase (TR) is considered to be one of the best targets to find new drugs against leishmaniasis. This enzyme is fundamental for parasite survival in the human host since it reduces trypanothione, a molecule used by the tryparedoxin/tryparedoxin peroxidase system of Leishmania to neutralize the hydrogen peroxide produced by host macrophages during infection. Recently, we solved the X-ray structure of TR in complex with the diaryl sulfide compound RDS 777 (6-(sec-butoxy)-2-((3-chlorophenyl)thio)pyrimidin-4-amine), which impairs the parasite defense against the reactive oxygen species by inhibiting TR with high efficiency. The compound binds to the catalytic site and engages in hydrogen bonds the residues more involved in the catalysis, namely Glu466′, Cys57 and Cys52, thereby inhibiting the trypanothione binding. On the basis of the RDS 777–TR complex, we synthesized structurally related diaryl sulfide analogs as TR inhibitors able to compete for trypanothione binding to the enzyme and to kill the promastigote in the micromolar range. One of the most active among these compounds (RDS 562) was able to reduce the trypanothione concentration in cell of about 33% via TR inhibition. RDS 562 inhibits selectively Leishmania TR, while it does not inhibit the human homolog glutathione reductase.     

In vitro activity of amphotericin B cochleates against Leishmania chagasi

Cochleate delivery vehicles are a novel lipid-based system with potential for delivery of amphotericin B (AmB). In this study, the efficacy of cochleates was evaluated by examining the in vitro activity of AmB cochleates (CAMB) against Leishmania chagasi in a macrophage model of infection. We demonstrate that CAMB is nontoxic to macrophages at concentrations as high as 2.5 μg/mL, whereas the conventional formulation, AmB deoxycholate, showed high toxicity at this concentration. The in vitro activity of CAMB against L. chagasi was found to be similar to that of the reference drug AmB deoxycholate, with ED50s of 0.017 μg/mL and 0.021 μg/mL, respectively. Considering that L. chagasi affects organs amenable to cochleate-mediated delivery of AmB, we hypothesize that CAMB will be an effective lipid system for the treatment of visceral leishmaniasis.     

Increased calcium permeability is not responsible for the rapid lethal effects of amphotericin B on Leishmania sp

The mode of action of the polyene antibiotic amphotericin B (AmB), the drug of choice for the treatment of systemic fungal infections and visceral leishmaniasis, is still unclear. An increase in intracellular Ca2+ concentration [( Ca2+]i), toxic in many cases, has been postulated as a possible lethal mechanism for AmB. Cell permeabilization to ethidium bromide (EB) was used as a criterion of viability. Kinetics of the DNA-EB fluorescent complex formation was studied in ergosterol-containing Leishmania promastigotes. Intracellular Ca2+ concentration was measured using quin-2 fluorescence in parallel aliquots. It is shown in this work that AmB can act as an efficient Ca2+ ionophore. However, the rapid permeabilization effect induced by AmB on these cells was not dependent on an increase in [Ca2+]i. On the contrary, it was found that leishmanicidal effect of AmB was enhanced in the absence of external calcium. Furthermore, A23187 a Ca2+ ionophore did not provoke cell permeabilization to EB.     

The interplay between environmental and host factors during an outbreak of visceral leishmaniasis in eastern Sudan

Parasitic diseases, including human visceral leishmaniasis, are multifactorial. Factors that are expected to play an important role in the parasite-human interaction are exposure, parasite "virulence" and host resistance factors. In populations exposed to Leishmania donovani most subjects do not allow the parasites to establish themselves or remain asymptomatic. Some individuals, however, fail to control parasite expansion and dissemination and develop a visceral disease. We report here the results of a longitudinal survey whose aims were to identify risk factors underlying visceral leishmaniasis (VL) susceptibility during an outbreak that occurred in a Sudanese village between 1995 and 1999. Most of the 660 subjects (90%) living in the central district were exposed to Leishmania and 20.9% (n = 138), mostly teenagers, developed VL. VL cases increased markedly in adults late in the outbreak, suggesting some changes in adult resistance status or in Leishmania "virulence" during the epidemic. Age and ethnic origin of the patients were the most important critical risk factors to account for the distribution of the VL cases that were recorded during the whole epidemic. This and the high frequency of VL in certain families suggest that host genetic factors played an important role in shaping the outbreak in this village. However, environmental factors (the presence of cows and neems in the households) that increase/decrease exposure to the parasite had significant effects on the distribution of VL cases in the village in the first phase of the outbreak.     

Hexadecylphosphocholine interaction with lipid monolayers

The phospholipid analogue miltefosine or hexadecylphosphocholine (HePC) is a drug of high interest in the treatment for fatal visceral leishmaniasis (VL) due to Leishmania donovani particularly because of its activity by oral route. In this study, the interaction of HePC with a monolayer of β-palmitoyl-γ-oleyl-phosphatidylcholine (POPC) as membrane model or sterol (ergosterol or cholesterol) was investigated. At a constant pressure of 25 mN/m, the adsorption kinetics of HePC into the monolayers showed that HePC molecules are inserted into the monolayer of lipids as monomers until the critical micellar concentration (CMC). At HePC concentrations superior to the CMC, the micelles of HePC are deployed at the interface as groups of monomers into the POPC or sterol monolayer. The study of mixture of HePC/(POPC or sterol), spread at the air-water interface, shows that a simple miscibility between HePC and POPC is observed, whereas a high condensation appears between HePC and sterols showing a high affinity between HePC and sterols. In addition, HePC does not act as detergent disturbing membrane integrity.     

Case Report: No Response to Liposomal Daunorubicin in a Patient with Drug-Resistant HIV-Associated Visceral Leishmaniasis

Visceral leishmaniasis (VL) in patients with HIV co-infection presents a significant therapeutic challenge due to the lessened chance of achieving long-term cure. We report a case of VL in a 60-year-old man with HIV infection who became refractory to anti-leishmania treatment due to multi-drug resistance. In the face of a worsening clinical situation, and with no other options available, he was treated with an experimental regimen of liposomal daunorubicin, which has previously been shown to have in vitro activity against Leishmania donovani and to be effective treatment of VL in animal studies. To our knowledge, he was the first patient with VL and HIV co-infection to have this treatment evaluated. We report on the lack of response to this treatment and possible causes for its failure.     

Mechanism of Action of Amphotericin B at the Cellular Level. Its Modulation by Delivery Systems

Abstract

Amphotericin B (AmB) is the drug of choice for the treatment of systemic fungal infections, but its use is hampered by its severe side-effects. A better understanding of its mechanisms of action is needed to develop new AmB formulations with an optimal selectivity between fungal and mammalian cells. Interactions between AmB and cells depend on the concentration of the drug. Stimulatory effects, modulation of the activity of immunocompetent cells and inhibition of yeast adherence are early events that precede the actual cellular toxicity. If membrane permeability alterations are considered to be the first toxic step, cell death results not only from osmotic imbalances, but also from additional mechanisms, such as lipid peroxidation, inhibition of membrane enzymes and blockade of endocytosis. The selectivity between fungal and mammalian cells takes its origin from the difference in the nature of the membrane sterol: ergosterol in fungi, cholesterol in mammalian cells. Transmembrane pores result from different mechanisms according to the sterol: ergosterol-AmB complexes are formed from monomelic AmB in solution, which is the only form present in aqueous medium at low AmB concentrations, whereas pores in the cholesterol containing membrane result from the adsorption onto the membrane surface of aqueous self-associated AmB, that appears in medium when AmB concentration increases. The liposomes seem to sequester AmB in a manner which makes it unavailable for mammalian cells, but maintains its access to fungal cells. The transfer of AmB by progressive diffusion of free AmB through the aqueous phase could explain the enhancement of the therapeutic index of the drug by liposomes, since the induction of pore formation needs a higher threshold of drug for host cell than for fungal cell membranes. The closed structure of the vehicle is not required to enhance the selectivity of the drug: esters of sucrose or high concentration of sodium deoxycholate afford a protective effect as well. Macrophages, after phagocytosis of liposomal AmB, may be considered as a reservoir of AmB, from which the drug is progressively released. Finally, the strong binding of AmB to the delivery system reduces the amount of drug bound to serum components and thus the endocytosis of AmB through the LDL receptor, resulting in lower toxicity.     

Anfotericina B liposomal en el tratamiento de la leishmaniasis visceral

A review on the current evidence of the efficacy and security of liposomal amphotericin B (L-AmB) for the treatment of visceral leishmaniasis (VL) has been performed. In the Indian subcontinent, a single dose of 10 mg/kg has shown effectiveness in the treatment of VL due to Leishmania donovani. In contrast, higher doses of L-AmB (up to 30 mg/kg) are required in Africa to treat a VL of the same etiology. When treating VL by Leishmania infantum acquired in the Americas and Europe the usual dose of L-AmB is 20-21 mg/kg. In HIV co-infected patients the required doses are usually higher, up to 60 mg/kg, and if it is administered in a prophylactic schedule after the treatment of VL relapses are reduced. L-AmB has shown synergism with other antiparasitic drugs, especially with paromomycin in the Indian subcontinent and with miltefosin in patients coinfected with HIV in East Africa. Due to its efficacy and safety profile, L-AmB is the first therapeutic option for VL.     

Upregulation of surface proteins in Leishmania donovani isolated from patients of post kala-azar dermal leishmaniasis

Five to fifteen percent of visceral leishmaniasis (VL) patients in India develop post kala-azar dermal leishmaniasis (PKDL), usually 1-2 years after apparent clinical cure. There is evidence pointing to a role played by the host immune responses in the disease pathogenesis, however, the contribution of changes in parasite gene expression has not been explored. Highly sensitive gene expression microarray technology was employed to identify genes that are differentially expressed in Leishmania parasites isolated from PKDL patients in comparison with those from VL. Hybridization on Leishmania donovani genomic microarray comprised of unique clones allowed us to identify 46/2268 (2%) clones that showed statistically significant (P<0.05) changes in expression (1.5-3.5-fold) in parasites of PKDL origin compared to those of VL origin. Sequence analysis of six genomic clones, consistently showing approximately 2-fold higher expression in PKDL parasites, revealed significant homology with gp63, gp46, putative amastin, a putative reductase and a possible calpain-like protein. The gene products showing upregulated expression in PKDL isolates may be candidates playing a role in the altered clinical manifestation in PKDL. Such differentially expressed genes hold the key to understanding the parasite genetic factors that contribute to the persistence after clinical cure of VL.     

Comparative molecular dynamics simulations of amphotericin B-cholesterol/ergosterol membrane channels

Amphotericin B (AmB) is a very effective anti-fungal polyene macrolide antibiotic whose usage is limited by its toxicity. Lack of a complete understanding of AmB's molecular mechanism has impeded attempts to design less toxic AmB derivatives. The antibiotic is known to interact with sterols present in the cell membrane to form ion channels that disrupt membrane function. The slightly higher affinity of AmB toward ergosterol (dominant sterol in fungal cells) than cholesterol (mammalian sterol) is regarded as the most essential factor on which antifungal chemotherapy is based. To study these differences at the molecular level, two realistic model membrane channels containing molecules of AmB, sterol (cholesterol or ergosterol), phospholipid, and water were studied by molecular dynamics (MD) simulations. Comparative analysis of the simulation data revealed that the sterol type has noticeable effect on the properties of AmB membrane channels. In addition to having a larger size, the AmB channel in the ergosterol-containing membrane has a more pronounced pattern of intermolecular hydrogen bonds. The interaction between the antibiotic and ergosterol is more specific than between the antibiotic and cholesterol. These observed differences suggest that the channel in the ergosterol-containing membrane is more stable and, due to its larger size, would have a higher ion conductance. These observations are in agreement with experiments.     

Leishmania donovani: immunostimulatory cellular responses of membrane and soluble protein fractions of splenic amastigotes in cured patient and hamsters

Visceral leishmaniasis (VL), caused by the intracellular parasite Leishmania donovani, L. chagasi and L. infantum is characterized by defective cell-mediated immunity (CMI) and is usually fatal if not treated properly. An estimated 350 million people worldwide are at risk of acquiring infection with Leishmania parasites with approximately 500,000 cases of VL being reported each year. In the absence of an efficient and cost-effective antileishmanial drug, development of an appropriate long-lasting vaccine against VL is the need of the day. In VL, the development of a CMI, capable of mounting Th1-type of immune responses, play an important role as it correlate with recovery from and resistance to disease. Resolution of infection results in lifelong immunity against the disease which indicates towards the feasibility of a vaccine against the disease. Most of the vaccination studies in Leishmaniasis have been focused on promastigote--an infective stage of parasite with less exploration of pathogenic amastigote form, due to the cumbersome process of its purified isolation. In the present study, we have isolated and purified splenic amastigotes of L. donovani, following the traditional protocol with slight modification. These were fractionated into five membranous and soluble subfractions each i.e MAF1-5 and SAF1-5 and were subjected for evaluation of their ability to induce cellular responses. Out of five sub-fractions from each of membrane and soluble, only four viz. MAF2, MAF3, SAF2 and SAF3 were observed to stimulate remarkable lymphoproliferative, IFN-γ, IL-12 responses and Nitric Oxide production, in Leishmania-infected cured/exposed patients and hamsters. Results suggest the presence of Th-1 type immunostimulatory molecules in these sub-fractions which may further be exploited for developing a successful subunit vaccine from the less explored pathogenic stage against VL.