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1.
PLoS Negl Trop Dis ; 18(3): e0012028, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38452055

RESUMO

BACKGROUND: India is going through the maintenance phase of VL elimination programme which may be threatened by the persistence of hidden parasite pools among asymptomatic leishmanial infection (ALI) and PKDL. The present work was designed to determine the burden of VL, PKDL, and ALI and to assess the role of treatment of ALI in maintaining post-elimination phase. METHODS AND FINDING: The study was undertaken in Malda district, West Bengal, India during October 2016 to September 2021. Study areas were divided into 'Study' and 'Control' arms. VL and PKDL cases of both the arms were diagnosed by three active mass surveys with an interval of one year and treated as per National guideline. ALI of 'Study' arm was treated like VL. ALI of 'Control' arm was followed up to determine their fate. Fed sand-fly pools were analysed for parasitic DNA. No significant difference was noted between the incidence of VL and PKDL in both the arms. Incidence of ALI declined sharply in 'Study' arm but an increasing trend was observed in 'Control' arm. Significantly higher rate of sero-conversion was noted in 'Control' arm and was found to be associated with untreated ALI burden. Parasitic DNA was detected in 22.8% ALI cases and 2.2% sand-fly pools. CONCLUSION: Persistence of a significant number of PKDL and ALI and ongoing transmission, as evidenced by new infection and detection of leishmanial DNA in vector sand-flies, may threaten the maintenance of post-elimination phase. Emphasis should be given for elimination of pathogen to prevent resurgence of VL epidemics.


Assuntos
Leishmania donovani , Leishmaniose Cutânea , Leishmaniose Visceral , Phlebotomus , Psychodidae , Animais , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/prevenção & controle , Leishmaniose Visceral/complicações , Areia , Psychodidae/parasitologia , Infecções Assintomáticas/epidemiologia , Índia/epidemiologia , DNA , Leishmaniose Cutânea/epidemiologia
2.
Sci Rep ; 14(1): 3246, 2024 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-38332162

RESUMO

Leishmania donovani is the causal organism of leishmaniasis with critical health implications affecting about 12 million people around the globe. Due to less efficacy, adverse side effects, and resistance, the available therapeutic molecules fail to control leishmaniasis. The mitochondrial primase of Leishmania donovani (LdmtPRI1) is a vital cog in the DNA replication mechanism, as the enzyme initiates the replication of the mitochondrial genome of Leishmania donovani. Hence, we target this protein as a probable drug target against leishmaniasis. The de-novo approach enabled computational prediction of the three-dimensional structure of LdmtPRI1, and its active sites were identified. Ligands from commercially available drug compounds were selected and docked against LdmtPRI1. The compounds were chosen for pharmacokinetic study and molecular dynamics simulation based on their binding energies and protein interactions. The LdmtPRI1 gene was cloned, overexpressed, and purified, and a primase activity assay was performed. The selected compounds were verified experimentally by the parasite and primase inhibition assay. Capecitabine was observed to be effective against the promastigote form of Leishmania donovani, as well as inhibiting primase activity. This study's findings suggest capecitabine might be a potential anti-leishmanial drug candidate after adequate further studies.


Assuntos
Antiprotozoários , Leishmania donovani , Leishmaniose Visceral , Leishmaniose , Humanos , Leishmania donovani/genética , DNA Primase , DNA Mitocondrial , Capecitabina/uso terapêutico , Reposicionamento de Medicamentos , Leishmaniose/tratamento farmacológico , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Antiprotozoários/química
3.
Acta Trop ; 252: 107139, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38307362

RESUMO

Clotrimazole is an FDA approved drug and is widely used as an antifungal agent. An extensive body of research is available about its mechanism of action on various cell types but its mode of killing of Leishmania donovani parasites is unknown. L. donovani causes Visceral Leishmaniasis which is a public health problem with limited treatment options. Its present chemotherapy is expensive, has adverse effects and is plagued with drug resistance issues. In this study we have explored the possibility of repurposing clotrimazole as an antileishmanial drug. We have assessed its efficacy on the parasites and attempted to understand its mode of action. We found that it has a half-maximal inhibitory concentration (IC50) of 35.75 ± 1.06 µM, 12.75 ± 0.35 µM and 73 ± 1.41 µM in promastigotes, intracellular amastigotes and macrophages, respectively. Clotrimazole is 5.73 times more selective for the intracellular amastigotes as compared to the mammalian cell. Effect of clotrimazole was reduced by ergosterol supplementation. It leads to impaired parasite morphology. It alters plasma membrane permeability and disrupts plasma membrane potential. Mitochondrial function is compromised as is evident from increased ROS generation, depolarized mitochondrial membrane and decreased ATP levels. Cell cycle analysis of clotrimazole treated parasites shows arrest at sub-G0 phase suggesting apoptotic mode of cell death.


Assuntos
Antiprotozoários , Leishmania donovani , Leishmaniose Visceral , Animais , Clotrimazol/farmacologia , Clotrimazol/metabolismo , Clotrimazol/uso terapêutico , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Macrófagos , Pontos de Checagem do Ciclo Celular , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Mamíferos
4.
Parasitol Int ; 100: 102865, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38341021

RESUMO

In visceral and mucocutaneous leishmaniasis, humoral immune response can reflect disease severity and parasite burden. Cutaneous leishmaniasis (CL) in Sri Lanka is caused by a usually visceralizing parasite, Leishmania donovani. We assessed the parasite burden (relative quantity-RQ) in 190 CL patients using quantitative real-time PCR (qPCR-with primers designed for this study) and smear microscopy, then correlated it with clinical parameters and IgG response. RQ of parasite DNA was determined with human-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal control. The qPCR sensitivity was tested with serially diluted DNA from cultured L. donovani parasites. Smears were assigned a score based on number of parasites per high power field. Data from previous studies were used for comparison and correlation; nested Internal Transcribed Spacer 1 (ITS1) PCR as reference standard (RS) and IgG antibody titers to the Leishmania rKRp42 antigen as the immune response. The qPCR amplified and quantified 86.8% of the samples while demonstrating a fair and significant agreement with ITS1-PCR and microscopy. Parasite burden by qPCR and microscopy were highly correlated (r = 0.76; p = 0.01) but showed no correlation with the IgG response (r = 0.056; p = 0.48). Corresponding mean RQs of IgG titers grouped by percentiles, showed no significant difference (p = 0.93). Mean RQ was higher in early lesions (p = 0.04), decreased with lesion size (p = 0.12) and slightly higher among papules, nodules and wet ulcers (p = 0.72). Our study established qPCR's efficacy in quantifying parasite burden in Sri Lankan CL lesions but no significant correlation was observed between the parasite burden and host IgG response to the Leishmania rKRP42 antigen.


Assuntos
Leishmania donovani , Leishmaniose Cutânea , Parasitos , Animais , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Sri Lanka/epidemiologia , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/parasitologia , Leishmania donovani/genética , DNA , Imunoglobulina G
5.
Exp Parasitol ; 259: 108710, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38350521

RESUMO

Sri Lanka reports a large focus of Leishmania donovani caused cutaneous leishmaniasis (CL). Subsequent emergence of visceral leishmaniasis (VL) was also reported recently. Expansion of the on-going disease outbreak and many complexities indicate urgent need to enhance early case detection methods. In vitro cultivation (IVC) of parasites causing visceral leishmaniasis (VL) is important for disease confirmation and to obtain sufficient quantities of parasites required in many scientific studies. IVC is carried out as a useful second line investigation for direct microscopy negative patients with CL in this setting. Along with the emergence of VL, current study was carried out to evaluate in vitro growth of local VL parasites and to identify their differences associated with in vitro growth characteristics. Routine parasitological diagnostic methods, i.e., light microscopy (LM), polymerase chain reaction (PCR) were used for confirmation of suspected cases. Lesion samples from 125 suspected CL cases and bone marrow or splenic aspirations from 125 suspected VL patients were used to inoculate IVCs. Media M199 (about 70 µl) supplemented with 15-20% of heat inactivated fetal bovine serum was used for initial culturing procedures in capillaries. Capillary cultures were monitored daily. Total of 44 different compositions/conditions were used for evaluating in vitro growth of VL causing parasite. Daily records on parasite counts, morphological appearance (size, shape, and wriggly movements) were maintained. In vitro transformation of Leishmania promastigotes to amastigotes and outcome of the attempts on recovery of live Leishmania from culture stabilates was also compared between CL and VL parasites. Proportion of cultures showing a transformation of promastigotes were 40/45 (88.9%) and 4/10 (40.0%) for CL and VL respectively. In the transformed cultures, parasites showing typical shape, size and movement patterns were less in VL (1/4, 25.0%) compared to CL (28/40, 70.0%). CL cultures showed a growth up to mass culturing level with mean duration of two weeks while it was about five weeks for VL cultures. Proportion of cultures that reached a parasite density of 1 × 106 cells/ml (proceeded to mass cultures) was significantly low in VL (4/10, 40%) as compared to CL (28/40, 70.0%). None of media compositions/conditions were successful for mass culturing of VL parasites while all of them were shown to be useful for growing CL strains. Also in vitro transformation to amastigote form and recovering of culture stabilates were not successful compared to CL. There were clear differences between in vitro growth of Leishmania parasites causing local CL and VL. Further studies are recommended for optimization of in vitro culturing of VL parasite which will be invaluable to enhance case detection in future.


Assuntos
Leishmania donovani , Leishmaniose Cutânea , Leishmaniose Visceral , Parasitos , Animais , Humanos , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Sri Lanka/epidemiologia , Leishmaniose Cutânea/parasitologia , Biópsia
6.
Int Immunopharmacol ; 129: 111644, 2024 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-38330797

RESUMO

Residing obligatorily as amastigotes within the mammalian macrophages, the parasite Leishmania donovani inflicts the potentially fatal, globally re-emerging disease visceral leishmaniasis (VL) by altering intracellular signaling through kinases and phosphatases. Because the phosphatases that modulate the VL outcome in humans remained unknown, we screened a human phosphatase siRNA-library for anti-leishmanial functions in THP-1, a human macrophage-like cell line. Of the 251 phosphatases, the screen identified the Ca++-activated K+-channel-associated phosphatase myotubularin-related protein-6 (MTMR6) as the only phosphatase whose silencing reduced parasite load and IL-10 production in human macrophages. Virulent, but not avirulent, L. donovani infection increased MTMR6 expression in macrophages. As virulent L. donovani parasites expressed higher lipophosphoglycan, a TLR2-ligand, we tested the effect of TLR2 stimulation or blockade on MTMR6 expression. TLR1/TLR2-ligand Pam3CSK4 enhanced, but TLR2 blockade reduced, MTMR6 expression. L. donovani infection of macrophages ex vivo increased, but miltefosine treatment reduced, MTMR6 expression. Corroboratively, compared to endemic controls, untreated VL patients had higher, but miltefosine-treated VL patients had reduced, MTMR6 expression. The phosphatase siRNA-library screening thus identified MTMR6 as the first TLR2-modulated ion channel-associated phosphatase with significant implications in VL patients and anti-leishmanial functions.


Assuntos
Leishmania donovani , Leishmaniose Visceral , Fosforilcolina , Animais , Humanos , Canais Iônicos , Leishmaniose Visceral/parasitologia , Ligantes , Mamíferos , Fosforilcolina/análogos & derivados , Proteínas Tirosina Fosfatases não Receptoras , RNA Interferente Pequeno/genética , Receptor 2 Toll-Like
7.
Emerg Infect Dis ; 30(3): 611-613, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38407178

RESUMO

We sequenced Leishmania donovani genomes in blood samples collected in emerging foci of visceral leishmaniasis in western Nepal. We detected lineages very different from the preelimination main parasite population, including a new lineage and a rare one previously reported in eastern Nepal. Our findings underscore the need for genomic surveillance.


Assuntos
Leishmania donovani , Leishmaniose Visceral , Humanos , Leishmania donovani/genética , Leishmaniose Visceral/epidemiologia , Nepal/epidemiologia , Genômica
8.
Front Cell Infect Microbiol ; 14: 1332381, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38357442

RESUMO

Visceral leishmaniasis (VL) is the most severe type of leishmaniasis which is caused by infection of Leishmania donovani complex. In the BALB/c mouse model of VL, multinucleated giant cells (MGCs) with heavy parasite infection consist of the largest population of hemophagocytes in the spleen of L. donovani-infected mice, indicating that MGCs provide the parasites a circumstance beneficial for their survival. Although ATP6V0D2 is a demonstrated factor inducing the formation of hemophagocytic MGCs during L. donovani infection, functions of this protein in shaping the infection outcome in macrophages remain unclear. Here we evaluated the influence of upregulated ATP6V0D2 on intracellular survival of the parasites. L. donovani infection-induced hemophagocytosis of normal erythrocytes by macrophages was suppressed by RNAi-based knockdown of Atp6v0d2. The knockdown of Atp6v0d2 did not improve the survival of amastigotes within macrophages when the cells were cultured in the absence of erythrocytes. On the other hand, reduced intracellular survival of amastigotes in macrophages by the knockdown was observed when macrophages were supplemented with antibody-opsonized erythrocytes before infection. There, increase in cytosolic labile iron pool was observed in the L. donovani-infected knocked-down macrophages. It suggests that ATP6V0D2 plays roles not only in upregulation of hemophagocytosis but also in iron trafficking within L. donovani-infected macrophages. Superior access to iron in macrophages may be how the upregulated expression of the molecule brings benefit to Leishmania for their intracellular survival in the presence of erythrocytes.


Assuntos
Leishmania donovani , Leishmaniose Visceral , Animais , Camundongos , Eritrócitos , Ferro/metabolismo , Leishmaniose Visceral/parasitologia , Macrófagos/metabolismo , Camundongos Endogâmicos BALB C , Regulação para Cima
9.
PLoS Negl Trop Dis ; 18(1): e0011920, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38295092

RESUMO

Sand fly transmitted Leishmania species are responsible for severe, wide ranging, visceral and cutaneous leishmaniases. Genetic exchange can occur among natural Leishmania populations and hybrids can now be produced experimentally, with limitations. Feeding Phlebotomus orientalis or Phlebotomus argentipes on two strains of Leishmania donovani yielded hybrid progeny, selected using double drug resistance and fluorescence markers. Fluorescence activated cell sorting of cultured clones derived from these hybrids indicated diploid progeny. Multilocus sequence typing of the clones showed hybridisation and nuclear heterozygosity, although with inheritance of single haplotypes in a kinetoplastid target. Comparative genomics showed diversity of clonal progeny between single chromosomes, and extraordinary heterozygosity across all 36 chromosomes. Diversity between progeny was seen for the HASPB antigen, which has been noted previously as having implications for design of a therapeutic vaccine. Genomic diversity seen among Leishmania strains and hybrid progeny is of great importance in understanding the epidemiology and control of leishmaniasis. As an outcome of this study we strongly recommend that wider biological archives of different Leishmania species from endemic regions should be established and made available for comparative genomics. However, in parallel, performance of genetic crosses and genomic comparisons should give fundamental insight into the specificity, diversity and limitations of candidate diagnostics, vaccines and drugs, for targeted control of leishmaniasis.


Assuntos
Leishmania donovani , Leishmaniose Cutânea , Leishmaniose Visceral , Phlebotomus , Psychodidae , Animais , Phlebotomus/genética , Leishmania donovani/genética , Psychodidae/genética , Cruzamentos Genéticos , Genômica , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/prevenção & controle , Leishmaniose Visceral/epidemiologia
10.
Acta Trop ; 251: 107114, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38190929

RESUMO

BACKGROUND: Visceral leishmaniasis is a neglected tropical disease affecting millions of people worldwide. Macrophages serve as the primary host cells for L. donovani, the immune response capability of these host cells is crucial for parasites' intracellular survival. L. donovani peptidyl-prolyl cis/trans isomerase Cyclophilin A (LdCypA) is a key protein for L. donovani intracellular proliferation, while the molecular mechanism conducive to intracellular survival of parasites remains elusive. METHODS: In this study, we generated a macrophage cell line overexpressing LdCyPA to investigate its role in controlling host immunity and promoting intracellular immune escape of L. donovani. RESULTS: It was discovered that the overexpression of the LdCyPA cell line regulated the host immune response following infection by downregulating the proportion of M1-type macrophages, promoting the secretion of the anti-inflammatory factor IL-4, and inhibiting the secretion of pro-inflammatory factors like IL-12, IFN-γ, TNF-α, and INOS. Transcriptome sequencing and mechanistic validation, meanwhile, demonstrated that cells overexpressing LdCyPA controlled the immune responses that followed infection by blocking the phosphorylation of P38 and JNK1/2 proteins in the MAPK signaling pathway and simultaneously increasing the phosphorylation of ERK proteins, which helped the L. donovani escape immune recognition. CONCLUSION: Our findings thus pave the way for the development of host-directed antiparasitic drugs by illuminating the pro-Leishmania survival mechanism of L. donovani cyclophilin A and exposing a novel immune escape strategy for L. donovani that targets host cellular immune regulation.


Assuntos
Leishmania donovani , Leishmaniose Visceral , Parasitos , Humanos , Animais , Leishmania donovani/genética , Ciclofilina A , Leishmaniose Visceral/parasitologia , Macrófagos , Interleucina-12
11.
Int Immunopharmacol ; 129: 111589, 2024 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-38295542

RESUMO

The protozoan parasite Leishmania donovani resides within mammalian macrophages and alters its antigen-presenting functions to negatively regulate host-protective T cell responses. This negative regulation of human T cell responses in vitro is attributed to myotubularin-related protein-6 (MTMR6), an ion channel-associated phosphatase. As mouse and human MTMR6 share homology, we studied whether MTMR6 silencing by lentivirally expressed MTMR6shRNA (Lv-MTMR6shRNA) reduced Leishmania growth in macrophages and whether MTMR6 silencing in Leishmania-susceptible BALB/c mice reduced the infection and reinstated host-protective T cell functions. MTMR6 silencing reduced amastigote count and IL-10 production, increased IL-12 expression and, induced IFN-γ-secreting T cells with anti-leishmanial activity in macrophage-T cell co-cultures. Lv-MTMR6shRNA reduced the infection, accompanied by increased IFN-γ expression, in susceptible BALB/c mice. Delays in Lv-MTMR6shRNA treatment by 7 days post-infection significantly reduced the infection suggesting MTMR6 as a plausible therapeutic target. Priming of BALB/c mice with avirulent parasites and Lv-MTMR6shRNA reduced parasite burden in challenge infection. These results indicate that MTMR6 is the first receptor-regulated ion channel-associated phosphatase regulating anti-leishmanial immune responses.


Assuntos
Leishmania donovani , Leishmaniose Visceral , Leishmaniose , Camundongos , Humanos , Animais , Proteínas Tirosina Fosfatases não Receptoras/genética , Camundongos Endogâmicos BALB C , Canais Iônicos , Mamíferos
12.
Infect Immun ; 92(2): e0050423, 2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38193711

RESUMO

The intracellular protozoan parasite Leishmania donovani causes debilitating human diseases that involve visceral and dermal manifestations. Type 3 interferons (IFNs), also referred to as lambda IFNs (IFNL, IFN-L, or IFN-λ), are known to play protective roles against intracellular pathogens at the epithelial surfaces. Herein, we show that L. donovani induces IFN-λ3 in human as well as mouse cell line-derived macrophages. Interestingly, IFN-λ3 treatment significantly decreased parasite load in infected cells, mainly by increasing reactive oxygen species production. Microscopic examination showed that IFN-λ3 inhibited uptake but not replication, while the phagocytic ability of the cells was not affected. This was confirmed by experiments that showed that IFN-λ3 could decrease parasite load only when added to the medium at earlier time points, either during or soon after parasite uptake, but had no effect on parasite load when added at 24 h post-infection, suggesting that an early event during parasite uptake was targeted. Furthermore, the parasites could overcome the inhibitory effect of IFN-λ3, which was added at earlier time points, within 2-3 days post-infection. BALB/c mice treated with IFN-λ3 before infection led to a significant increase in expression of IL-4 and ARG1 post-infection in the spleen and liver, respectively, and to different pathological changes, especially in the liver, but not to changes in parasite load. Treatment with IFN-λ3 during infection did not decrease the parasite load in the spleen either. However, IFN-λ3 was significantly increased in the sera of visceral leishmaniasis patients, and the IFNL genetic variant rs12979860 was significantly associated with susceptibility to leishmaniasis.


Assuntos
Leishmania donovani , Leishmaniose Visceral , Parasitos , Animais , Humanos , Camundongos , Linhagem Celular , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Macrófagos/parasitologia , Camundongos Endogâmicos BALB C
13.
Free Radic Biol Med ; 213: 371-393, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-38272324

RESUMO

Understanding the unique metabolic pathway of L. donovani is crucial for comprehending its biology under oxidative stress conditions. The de novo cysteine biosynthetic pathway of L. donovani is absent in humans and its product, cysteine regulates the downstream components of trypanothione-based thiol metabolism, important for maintaining cellular redox homeostasis. The role of serine o-acetyl transferase (SAT), the first enzyme of this pathway remains unexplored. In order to investigate the role of SAT protein, we cloned SAT gene into pXG-GFP+ vector for episomal expression of SAT in Amphotericin B sensitive L. donovani promastigotes. The SAT overexpression was confirmed by SAT enzymatic assay, GFP fluorescence, immunoblotting and PCR. Our study unveiled an upregulated expression of both LdSAT and LdCS of cysteine biosynthetic pathway and other downstream thiol pathway proteins in LdSAT-OE promastigotes. Additionally, there was an increase in enzymatic activities of LdSAT and LdCS proteins in LdSAT-OE, which was found similar to the Amp B resistant parasites, indicating a potential role of SAT protein in modulating drug resistance. We observed that the overexpression of SAT in Amp B sensitive parasites increases tolerance to drug pressure and oxidative stress via trypanothione-dependent antioxidant mechanism. Moreover, the in vitro J774A.1 macrophage infectivity assessment showed that SAT overexpression augments parasite infectivity. In LdSAT-OE promastigotes, antioxidant enzyme activities like APx and SOD were upregulated, intracellular reactive oxygen species were reduced with a corresponding increase in thiol level, emphasizing SAT's role in stress tolerance and enhanced infectivity. Additionally, the ROS mediated upregulation in the expression of LdSAT, LdCS, LdTryS and LdcTXNPx proteins reveals an essential cross talk between SAT and proteins of thiol metabolism in combating oxidative stress and maintaining redox homeostasis. Taken together, our results provide the first insight into the role of SAT protein in parasite infectivity and survival under drug pressure and oxidative stress.


Assuntos
Leishmania donovani , Humanos , Leishmania donovani/genética , Leishmania donovani/metabolismo , Compostos de Sulfidrila/metabolismo , Serina O-Acetiltransferase/metabolismo , Cisteína/metabolismo , Antioxidantes/metabolismo , Estresse Oxidativo , Oxirredução , Resistência a Medicamentos/genética
14.
PLoS Negl Trop Dis ; 18(1): e0011559, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38166146

RESUMO

The parasite Leishmania donovani is one of the species causing visceral leishmaniasis in humans, a deadly infection claiming up to 40,000 lives each year. The current drugs for leishmaniasis treatment have severe drawbacks and there is an urgent need to find new anti-leishmanial compounds. However, the search for drug candidates is complicated by the intracellular lifestyle of Leishmania. Here, we investigate the use of human induced pluripotent stem cell (iPS)-derived macrophages (iMACs) as host cells for L. donovani. iMACs obtained through embryoid body differentiation were infected with L. donovani promastigotes, and high-content imaging techniques were used to optimize the iMACs seeding density and multiplicity of infection, allowing us to reach infection rates up to 70% five days after infection. IC50 values obtained for miltefosine and amphotericin B using the infected iMACs or mouse peritoneal macrophages as host cells were comparable and in agreement with the literature, showing the potential of iMACs as an infection model for drug screening.


Assuntos
Antiprotozoários , Células-Tronco Pluripotentes Induzidas , Leishmania donovani , Leishmaniose Visceral , Animais , Humanos , Camundongos , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Leishmaniose Visceral/parasitologia , Macrófagos/parasitologia
15.
Parasitol Int ; 100: 102863, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38272301

RESUMO

Leishmania, a protozoan parasite, is responsible for the occurrence of leishmaniasis, a disease that is prevalent in tropical regions. Visceral Leishmaniasis (VL), also known as kala-azar in Asian countries, is one of the most significant forms of VL, along with Cutaneous Leishmaniasis (CL) and Mucocutaneous Leishmaniasis (ML). Management of this condition typically entails the use of chemotherapy as the sole therapeutic option. The current treatments for leishmaniasis present several drawbacks, including a multitude of side effects, prolonged treatment duration, disparate efficacy across different regions, and the emergence of resistance. To address this urgent need, it is imperative to identify alternative treatments that are both safer and more effective. The identification of appropriate pharmacological targets in conjunction with biological pathways constitutes the initial stage of drug discovery. In this review, we have addressed the key metabolic pathways that represent potential pharmacological targets as well as prominent treatment options for leishmaniasis.


Assuntos
Leishmania donovani , Leishmania , Leishmaniose Cutânea , Leishmaniose Mucocutânea , Leishmaniose Visceral , Leishmaniose , Animais , Leishmaniose/tratamento farmacológico , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Ásia
16.
Biol Trace Elem Res ; 202(1): 210-220, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37088826

RESUMO

In leishmaniasis, the protective immunity is largely mediated by proinflammatory cytokine producing abilities of T cells and an efficient parasite killing by phagocytic cells. Notwithstanding a substantial progress that has been made during last decades, the mechanisms or factors involved in establishing protective immunity against Leishmania are not identified. In ancient Indian literature, metallic "bhasma," particularly that of "swarna" or gold (fine gold particles), is indicated as one of the most prominent metal-based therapeutic medicine, which is known to impart protective and curative properties in various health issues. In this work, we elucidated the potential of swarna bhasma (SB) on the effector properties of phagocytes and antigen-activated CD4+ T cells in augmenting the immunogenicity of L. donovani antigens. The characterization of SB revealing its shape, size, composition, and measurement of cytotoxicity established the physiochemical potential for its utilization as an immunomodulator. The activation of macrophages with SB enhanced their capacity to produce nitric oxide and proinflammatory cytokines, which eventually resulted in reduced uptake of parasites and their proliferation in infected cells. Further, in Leishmania-infected animals, SB administration reduced the generation of IL-10, an anti-inflammatory cytokine, and enhanced pro-inflammatory cytokine generation by antigen activated CD4+ T cells with increased frequency of double (IFNγ+/TNFα+) and triple (IFNγ+TNFα+IL-2+) positive cells and abrogated disease pathogeneses at the early days of infection. Our results also suggested that cow-ghee (A2) emulsified preparation of SB, either alone or with yashtimadhu, a known natural immune modulator which enhances the SB's potential in enhancing the immunogenicity of parasitic antigens. These findings suggested a definite potential of SB in enhancing the effector functions of phagocytes and CD4+ T cells against L. donovani antigens. Therefore, more studies are needed to elucidate the mechanistic details of SB and its potential in enhancing vaccine-induced immunity.


Assuntos
Apresentação de Antígeno , Antígenos de Protozoários , Linfócitos T CD4-Positivos , Calotropis , Ouro , Látex , Leishmania donovani , Macrófagos , Ayurveda , Células Th1 , Arsênio , Combinação de Medicamentos , Ouro/administração & dosagem , Ouro/farmacologia , Látex/administração & dosagem , Látex/farmacologia , Chumbo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Linfócitos T CD4-Positivos/imunologia , Fagócitos/efeitos dos fármacos , Fagócitos/imunologia , Leishmaniose/imunologia , Leishmaniose/parasitologia , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/crescimento & desenvolvimento , Leishmania donovani/imunologia , Antígenos de Protozoários/imunologia , Células Th1/imunologia , Animais , Camundongos , Células RAW 264.7 , Feminino , Camundongos Endogâmicos BALB C
17.
J Biomol Struct Dyn ; 42(3): 1293-1306, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-37054523

RESUMO

Visceral leishmaniasis (VL) is a tropical disease that causes severe public health problems in humans when untreated. As no licensed vaccine exists against VL, we aimed to formulate a potential MHC-restricted chimeric vaccine construct against this dreadful parasitic disease. Amastin-like protein derived from L. donovani is considered to be stable, immunogenic and non-allergic. A comprehensive established framework was used to explore the set of immunogenic epitopes with estimated population coverage of 96.08% worldwide. The rigorous assessment revealed 6 promiscuous T-epitopes which can plausibly be presented by more than 66 diverse HLA alleles. Further docking and simulation study of peptide receptor complexes identified a strong and stable binding interaction with better structural compactness. The predicted epitopes were combined with appropriate linkers and adjuvant molecules and their translation efficiency was evaluated in pET28+(a), an bacterial expression vector using in-silico cloning. Molecular docking followed by MD simulation study revealed a stable interaction between chimeric vaccine construct with TLRs. Immune simulation of the chimeric vaccine constructs showed an elevated Th1 immune response against both B and T epitopes. With this, the detailed computational analysis suggested that the chimeric vaccine construct can evoke a robust immune response against Leishmania donovani infection. Future studies are required to validate the role of amastin as a promising vaccine target.Communicated by Ramaswamy H. Sarma.


Assuntos
Leishmania donovani , Leishmaniose Visceral , Vacinas , Humanos , Leishmania donovani/genética , Epitopos , Simulação de Acoplamento Molecular , Vacinologia , Leishmaniose Visceral/parasitologia , Epitopos de Linfócito T , Epitopos de Linfócito B , Vacinas de Subunidades , Biologia Computacional
18.
Int J Biol Macromol ; 254(Pt 2): 127756, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37907177

RESUMO

Aminoacyl-tRNA synthetases (aaRSs), essential components of the protein synthesizing machinery, have been often chosen for devising therapeutics against parasitic diseases. Due to their relevance in drug development, the current study was designed to explore functional and structural aspects of Leishmania donovani glutamyl-tRNA synthetase (LdGluRS). Hence, LdGluRS was cloned into an expression vector and purified to homogeneity using chromatographic techniques. Purified protein showed maximum enzymatic activity at physiological pH, with more binding capacity towards its cofactor (Adenosine triphosphate, 0.06 ± 0.01 mM) than the cognate substrate (L-glutamate, 9.5 ± 0.5 mM). Remarkably, salicylate inhibited LdGluRS competitively with respect to L-glutamate and exhibited druglikeness with negligible effect on human macrophages. The protein possessed more α-helices (43 %) than ß-sheets (12 %), whereas reductions in thermal stability and cofactor-binding affinity, along with variation in mode of inhibition after mutation signified the role of histidine (H60) as a catalytic residue. LdGluRS could also generate a pro-inflammatory milieu in human macrophages by upregulating cytokines. The docking study demonstrated the placement of salicylate into LdGluRS substrate-binding site, and the complex was found to be stable during molecular dynamics (MD) simulation. Altogether, our study highlights the understanding of molecular inhibition and structural features of glutamyl-tRNA synthetase from kinetoplastid parasites.


Assuntos
Aminoacil-tRNA Sintetases , Leishmania donovani , Humanos , Glutamato-tRNA Ligase/química , Glutamato-tRNA Ligase/genética , Glutamato-tRNA Ligase/metabolismo , Ácido Glutâmico , Aminoacil-tRNA Sintetases/química , Trifosfato de Adenosina , Leishmania donovani/metabolismo , Salicilatos
19.
Chemistry ; 30(3): e202303316, 2024 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-37926692

RESUMO

Balgacyclamide A-C are a family of cyanobactin natural products isolated from freshwater cyanobacteria Microcystis aeruginosa. These macrocyclic peptides are characterized by their oxazoline-thiazole core, their 7 or 8 stereocenters, and their antiparasitic activities. Balgacyclamide B is known for its activity towards Plasmodium falciparum chloroquine-resistant strain K1, Trypanosoma brucei rhodesiense, and Leishmania donovani. In this report, the first total synthesis of Balgacyclamide B is described in a 17-steps pathway and a 2 % overall yield. The synthetic pathway toward balgacyclamide B can be adapted for the future syntheses of balgacyclamide A and C. In addition, a brief history background of oxazolines syntheses is shown to emphasize the importance of the cyclization conditions used to interconvert or retain configuration of ß-hydroxy amides via dehydrative cyclization.


Assuntos
Antiparasitários , Leishmania donovani , Peptídeos Cíclicos , Testes de Sensibilidade Parasitária , Trypanosoma brucei rhodesiense , Plasmodium falciparum
20.
Future Microbiol ; 19: 33-49, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37830931

RESUMO

Aim: Leishmaniasis is characterized by a spectrum of diseases with two main clinical forms, cutaneous and visceral, caused by Leishmania tropica and Leishmania donovani, respectively. Studying Leishmania's interaction with the epithelial barrier at the initial site of a bite is crucial to understanding the establishment of the disease. Materials & methods: To discern parasite-host epithelial interaction, we developed in vitro cellular models involving co-cultures of Leishmania and MDCK epithelial cells. Results: Both L. donovani-MDCK and L. tropica-MDCK co-culture models demonstrated a phenomenon known as atypical anoikis apoptosis, typically identified by distinctive 'flipping in' of cell membranes and disordered cytoskeletal frameworks. Conclusion: This study bridges the gap in the fundamental understanding of the intricate latticework involving vector-Leishmania-host and may inform drug development strategies.


Small parasites called Leishmania are passed to humans through the bites of sandflies. These parasites cause three deadly forms of disease: one that affects the organs, one that causes skin lesions and one that affects organ linings. This study looked at how Leishmania parasites behave when they enter through the skin. We found that when the parasites were in contact with cells, the cells changed their shape and lost contact with neighboring cells. This led to a type of cell death known as anoikis, a Greek term meaning 'homelessness'.


Assuntos
Pessoas Mal Alojadas , Leishmania donovani , Leishmania tropica , Leishmaniose Cutânea , Leishmaniose Visceral , Humanos , Anoikis , Células Epiteliais
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