Role of Macrophage PIST Protein in Regulating Leishmania major Infection.

PDZ protein interacting specifically with Tc10 or PIST is a mammalian trans-Golgi resident protein that regulates subcellular sorting of plasma membrane receptors. PIST has recently emerged as a key player in regulating viral pathogenesis. Nevertheless, the involvement of PIST in parasitic infections remains unexplored. Leishmania parasites infiltrate their host macrophage cells through phagocytosis, where they subsequently multiply within the parasitophorous vacuole (PV). Host cell autophagy has been found to be important in regulating this parasite infection. Since PIST plays a pivotal role in triggering autophagy through the Beclin 1-PI3KC3 pathway, it becomes interesting to identify the status of PIST during Leishmania infection. We found that while macrophage cells are infected with Leishmania major (L. major), the expression of PIST protein remains unaltered; however, it traffics from the Golgi compartment to PV. Further, we identified that in L. major-infected macrophage cells, PIST associates with the autophagy regulatory protein Beclin 1 within the PVs; however, PIST does not interact with LC3. Reduction in PIST protein through siRNA silencing significantly increased parasite burden, whereas overexpression of PIST in macrophages restricted L. major infectivity. Together, our study reports that the macrophage PIST protein is essential in regulating L. major infectivity.

Neutrophil extracellular traps formation: effect of Leishmania major promastigotes and salivary gland homogenates of Phlebotomus papatasi in human neutrophil culture.

BACKGROUND: Leishmaniasis as a neglected tropical disease (NTD) is caused by the inoculation of Leishmania parasites via the bite of phlebotomine sand flies. After an infected bite, a series of innate and adaptive immune responses occurs, among which neutrophils can be mentioned as the initiators. Among the multiple functions of these fighting cells, neutrophil extracellular traps (NETs) were studied in the presence of Leishmania major promastigotes and salivary gland homogenates (SGH) of Phlebotomus papatasi alone, and in combination to mimic natural conditions of transmission. MATERIAL & METHODS: The effect of L. major and SGH on NETs formation was studied in three different groups: neutrophils + SGH (NS), neutrophils + L. major (NL), neutrophils + L. major + SGH (NLS) along with negative and positive controls in 2, 4 and 6 h post-incubation. Different microscopic methods were used to visualize NETs comprising: fluorescence microscopy by Acridine Orange/ Ethidium Bromide staining, optical microscopy by Giemsa staining and scanning electron microscopy. In addition, the expression level of three different genes NE, MPO and MMP9 was evaluated by Real-Time PCR. RESULTS: All three microscopical methods revealed similar results, as in NS group, chromatin extrusion as a sign of NETosis, was not very evident in each three time points; but, in NL and especially NLS group, more NETosis was observed and the interaction between neutrophils and promastigotes in NL and also with saliva in NLS group, gradually increased over times. Real-time reveals that, the expression of MPO, NE and MMP9 genes increased during 2 and 4 h after exposure, and then decreased at 6 h in most groups. CONCLUSION: Hence, it was determined that the simultaneous presence of parasite and saliva in NLS group has a greater impact on the formation of NETs compared to NL and NS groups.

Transcriptional signatures in human macrophage-like cells infected by Leishmania infantum, Leishmania major and Leishmania tropica.

BACKGROUND: In the Mediterranean basin, three Leishmania species have been identified: L. infantum, L. major and L. tropica, causing zoonotic visceral leishmaniasis (VL), zoonotic cutaneous leishmaniasis (CL) and anthroponotic CL, respectively. Despite animal models and genomic/transcriptomic studies provided important insights, the pathogenic determinants modulating the development of VL and CL are still poorly understood. This work aimed to identify host transcriptional signatures shared by cells infected with L. infantum, L. major, and L. tropica, as well as specific transcriptional signatures elicited by parasites causing VL (i.e., L. infantum) and parasites involved in CL (i.e., L. major, L. tropica). METHODOLOGY/PRINCIPAL FINDINGS: U937 cells differentiated into macrophage-like cells were infected with L. infantum, L. major and L. tropica for 24h and 48h, and total RNA was extracted. RNA sequencing, performed on an Illumina NovaSeq 6000 platform, was used to evaluate the transcriptional signatures of infected cells with respect to non-infected cells at both time points. The EdgeR package was used to identify differentially expressed genes (fold change > 2 and FDR-adjusted p-values < 0.05). Then, functional enrichment analysis was employed to identify the enriched ontology terms in which these genes are involved. At 24h post-infection, a common signature of 463 dysregulated genes shared among all infection conditions was recognized, while at 48h post-infection the common signature was reduced to 120 genes. Aside from a common transcriptional response, we evidenced different upregulated functional pathways characterizing L. infantum-infected cells, such as VEGFA-VEGFR2 and NFE2L2-related pathways, indicating vascular remodeling and reduction of oxidative stress as potentially important factors for visceralization. CONCLUSIONS: The identification of pathways elicited by parasites causing VL or CL could lead to new therapeutic strategies for leishmaniasis, combining the canonical anti-leishmania compounds with host-directed therapy.

Phylogenetic analysis and antimony resistance of Leishmania major isolated from humans and rodents.

Cutaneous Leishmaniasis (CL) is one of the world's neglected diseases which is caused by Leishmania spp. The aim of this study was to assess molecular profile and antimony resistance of Leishmania isolated from human and rodent hosts. Samples were collected from suspected CL patients referred to health centres and wild rodent's traps in Gonbad-e-Qabus region, north-eastern Iran. Smears were subjected to PCR-RFLP to identify Leishmania species. In addition, ITS1-PCR products were sequenced for phylogenetic analysis. Clinical isolates and rodent samples were subjected to MTT assay to determine IC50 values and in vitro susceptibilities. Expression levels of antimony resistance-related genes were determined in CL isolates. Out of 1,949 suspected patients with CL and 148 rodents, 1,704 (87.4%) and 6 (4.05%) were positive with direct smear, respectively. Digestion patterns of BusRI (HaeIII) endonuclease enzyme were similar to what expected for Leishmania major. Phylogenetic analysis revealed that the highest interspecies similarity was found between current L. major sequences with L. major obtained from Russia and Uzbekistan. Out of 20 L. major samples tested, 13 (65%) were resistant to meglumine antimoniate (MA) treatment, with an activity index (AI) exceeding 4. The remaining 7 samples (35%) responded to MA treatment and were classified as sensitive isolates, with a confirmed sensitive phenotype based on their AI values. The comparison expression analysis of three major antimony resistance-associated genes in unresponsive clinical isolates demonstrated significant fold changes for TDR1 (4.78-fold), AQP1 (1.3-fold), and γ-GCS (1.17-fold) genes (P < 0.05). Herein, we demonstrate genetic diversity and antimony resistance of L. major isolated from human and reservoir hosts in north-eastern Iran, which could be the basis for planning future control strategies.

In silico prediction of CD8+ and CD4+ T cell epitopes in Leishmania major proteome: Using immunoinformatics.

The leishmaniases are NDTs (neglected tropical diseases) that affect people all over the world. They are brought on by protozoans from the genus Leishmania and disseminated by phlebotomine flies that are afflicted with the disease. The best option to manage and lower the incidence of these diseases has been thought by the creation of a safe and effective vaccination. This research used an in silico based mining approach to look for high potential epitopes that might bind to MHC Class I and MHC Class II molecules (mainly; HLA-A*02:01 & HLA-DRB1*03:01) from human population in order to promote vaccine development. Based on the presence of signal peptides, GPI anchors, antigenicity predictions, and a subtractive proteomic technique, we have screened 17 putative antigenic proteins from the 8083 total proteins of L. major. After that thorough immunogenic epitope prediction were done using IEDB-AR tools. We isolated five immunogenic epitopes (three 9-mer & two 15-mer) from five antigenic proteins through docking and MD simulation analysis. Finally, these five anticipated epitopes, viz., TLPEIPVNV, ELMAPVFGL, TLAAAVALL, NSINIRLDGVTSAGF and NVPLVVDASSLFRVA have considerably stronger binding potential with their respective alleles and may trigger immunological responses. The goal of this work was to identify MHC restricted epitopes for CD8+ and CD4+ T cells activation using immunoinformatics in order to identify potential vaccine candidates against L. major parasites.

Leishmania profilin interacts with actin through an unusual structural mechanism to control cytoskeletal dynamics in parasites.

Diseases caused by Leishmania and Trypanosoma parasites are a major health problem in tropical countries. Because of their complex life cycle involving both vertebrate and insect hosts, and >1 billion years of evolutionarily distance, the cell biology of trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is divergent when compared with other eukaryotes. To understand how actin dynamics are regulated in trypanosomatid parasites, we focused on a central actin-binding protein profilin. Co-crystal structure of Leishmania major actin in complex with L. major profilin revealed that, although the overall folds of actin and profilin are conserved in eukaryotes, Leishmania profilin contains a unique α-helical insertion, which interacts with the target binding cleft of actin monomer. This insertion is conserved across the Trypanosomatidae family and is similar to the structure of WASP homology-2 (WH2) domain, a small actin-binding motif found in many other cytoskeletal regulators. The WH2-like motif contributes to actin monomer binding and enhances the actin nucleotide exchange activity of Leishmania profilin. Moreover, Leishmania profilin inhibited formin-catalyzed actin filament assembly in a mechanism that is dependent on the presence of the WH2-like motif. By generating profilin knockout and knockin Leishmania mexicana strains, we show that profilin is important for efficient endocytic sorting in parasites, and that the ability to bind actin monomers and proline-rich proteins, and the presence of a functional WH2-like motif, are important for the in vivo function of Leishmania profilin. Collectively, this study uncovers molecular principles by which profilin regulates actin dynamics in trypanosomatids.

Adipose-Derived Stem Cells' Secretome Attenuates Lesion Size and Parasite Loading in Leishmaniasis Caused by Leishmania Major in Mice.

Background: Stem cell-derived secretome (SE) released into the extracellular space contributes to tissue repair. The present study aimed to investigate the impact of isolated secretome (SE) from adipose-derived mesenchymal stem cells (ASCs) on Leishmania major (L. major) lesions in BALB/c mice. Methods: This experimental study was conducted at Ahvaz University of Medical Sciences (Ahvaz, Iran) in 2021. Forty female BALB/c mice were infected with stationary phase promastigotes through intradermal injection in the bottom of their tail and randomly divided into four groups (n=10 per group). The mice were given SE (20 mg/mL), either alone or in combination with Glucantime (GC, 20 mg/mL/Kg), meglumine antimoniate (20 mg/mL/Kg) for the GC group, and phosphate-buffered saline (PBS) for the control group. After eight weeks, the lesion size, histopathology, the levels of Interleukin 10 (IL-10), and Interleukin 12 (IL-12) were assessed. For the comparison of values between groups, the parametric one-way ANOVA was used to assess statistical significance. Results: At the end of the experiment, the mice that received SE had smaller lesions (4.56±0.83 mm versus 3.62±0.59 mm, P=0.092), lower levels of IL-10 (66.5±9.7 pg/mL versus 285.4±25.2 pg/mL, P<0.001), and higher levels of IL-12 (152.2±14.2 pg/mL versus 24.2±4.4 pg/mL, P<0.001) than the control. Histopathology findings revealed that mice treated with SE had a lower parasite burden in lesions and spleen than the control group. Conclusion: The current study demonstrated that ADSC-derived SE could protect mice infected with L. major against leishmaniasis.

Molecular effects of photodynamic therapy with curcumin on Leishmania major promastigotes.

Leishmaniasis is a neglected disease mainly affecting low-income populations. Conventional treatment involves several side effects, is expensive, and, in addition, protozoa can develop resistance. Photodynamic therapy (PDT) is a promising alternative in treating the disease. PDT involves applying light at a specific wavelength to activate a photosensitive compound (photosensitizer, PS), to produce reactive oxygen species (ROS). Curcumin and its photochemical characteristics make it a good candidate for photodynamic therapy. Studies evaluating gene expression can help to understand the molecular events involved in the cell death caused by PDT. In the present study, RNA was extracted from promastigotes from the control and treated groups after applying PDT. RT-qPCR was performed to verify the expression of the putative ATPase beta subunit (ATPS), ATP synthase subunit A (F0F1), argininosuccinate synthase 1 (ASS), ATP-binding cassette subfamily G member 2 (ABCG2), glycoprotein 63 (GP63), superoxide dismutase (FeSODA), and glucose-6-phosphate dehydrogenase (G6PDH) genes (QR). The results suggest that PDT altered the expression of genes that participate in oxidative stress and cell death pathways, such as ATPS, FeSODA, and G6PD. The ATP-F0F1, ASS, and GP63 genes did not have their expression altered. However, it is essential to highlight that other genes may be involved in the molecular mechanisms of oxidative stress and, consequently, in the death of parasites.

Single-cell ATAC sequencing identifies sleepy macrophages during reciprocity of cytokines in L. major infection.

The hallmark characteristic of macrophages lies in their inherent plasticity, allowing them to adapt to dynamic microenvironments. Leishmania strategically modulates the phenotypic plasticity of macrophages, creating a favorable environment for intracellular survival and persistent infection through regulatory cytokine such as interleukin (IL)-10. Nevertheless, these effector cells can counteract infection by modulating crucial cytokines like IL-12 and key components involved in its production. Using sophisticated tool of single-cell assay for transposase accessible chromatin (ATAC) sequencing, we systematically examined the regulatory axis of IL-10 and IL-12 in a time-dependent manner during Leishmania major infection in macrophages Our analysis revealed the cellular heterogeneity post-infection with the regulators of IL-10 and IL-12, unveiling a reciprocal relationship between these cytokines. Notably, our significant findings highlighted the presence of sleepy macrophages and their pivotal role in mediating reciprocity between IL-10 and IL-12. To summarize, the roles of cytokine expression, transcription factors, cell cycle, and epigenetics of host cell machinery were vital in identification of sleepy macrophages, which is a transient state where transcription factors controlled the epigenetic remodeling and expression of genes involved in pro-inflammatory cytokine expression and recruitment of immune cells.IMPORTANCELeishmaniasis is an endemic affecting 99 countries and territories globally, as outlined in the 2022 World Health Organization report. The disease's severity is compounded by compromised host immune systems, emphasizing the pivotal role of the interplay between parasite and host immune factors in disease regulation. In instances of cutaneous leishmaniasis induced by L. major, macrophages function as sentinel cells. Our findings indicate that the plasticity and phenotype of macrophages can be modulated to express a cytokine profile involving IL-10 and IL-12, mediated by the regulation of transcription factors and their target genes post-L. major infection in macrophages. Employing sophisticated methodologies such as single-cell ATAC sequencing and computational genomics, we have identified a distinctive subset of macrophages termed "sleepy macrophages." These macrophages exhibit downregulated housekeeping genes while expressing a unique set of variable features. This data set constitutes a valuable resource for comprehending the intricate host-parasite interplay during L. major infection.

Effects of Leishmania major infection on the gut microbiome of resistant and susceptible mice.

Cutaneous leishmaniasis, a parasitic disease caused by Leishmania major, is a widely frequent form in humans. To explore the importance of the host gut microbiota and to investigate its changes during L. major infection, two different groups of mouse models were assessed. The microbiome of two parts of the host gut-ileum and colon-from infected and non-infected mice were characterised by sequencing of 16S rDNA using an Ion Torrent PGM platform. Microbiome analysis was performed to reveal changes related to the susceptibility and the genetics of mice strains in two different gut compartments and to compare the results between infected and non-infected mice. The results showed that Leishmania infection affects mainly the ileum microbiota, whereas the colon bacterial community was more stable. Different biomarkers were determined in the gut microbiota of infected resistant mice and infected susceptible mice using LEfSe analysis. Lactobacillaceae was associated with resistance in the colon microbiota of all resistant mice strains infected with L. major. Genes related to xenobiotic biodegradation and metabolism and amino acid metabolism were primarily enriched in the small intestine microbiome of resistant strains, while genes associated with carbohydrate metabolism and glycan biosynthesis and metabolism were most abundant in the gut microbiome of the infected susceptible mice. These results should improve our understanding of host-parasite interaction and provide important insights into the effect of leishmaniasis on the gut microbiota. Also, this study highlights the role of host genetic variation in shaping the diversity and composition of the gut microbiome. KEY POINTS: • Leishmaniasis may affect mainly the ileum microbiota while colon microbiota was more stable. • Biomarkers related with resistance or susceptibility were determined in the gut microbiota of mice. • Several pathways were predicted to be upregulated in the gut microbiota of resistant or susceptible mice.

Designing of a new multi-epitope vaccine against Leishmania major using Leish-F1 epitopes: An In-silico study.

Cutaneous leishmaniasis (CL) is the most common form of the disease which can cause malignant lesions on the skin. Vaccination for the prevention and treatment of leishmaniasis can be the most effective way to combat this disease. In this study, we designed a novel multi-epitope vaccine against Leishmania major (L. major) using immunoinformatics tools to assess its efficacy in silico. Sequences of Leish-F1 protein (TSA, Leif, and LMSTI1) of L. major were taken from GenBank. The helper T (Th) and cytotoxic T (Tc) epitopes of the protein were predicted. The final multi-epitope consisted of 18 CTL epitopes joined by AAY linker. There were also nine HTL epitopes in the structure of the vaccine construct, joined by GPGPG linker. The profilin adjuvant (the toll-like receptor 11 agonist) was also added into the construct by AAY Linker. There were 613 residues in the structure of the vaccine construct. The multi-epitope vaccine candidate was stable and non-allergic. The data obtained from the binding of final multi-epitope vaccine-TLR11 residues (band lengths and weighted scores) unveiled the ligand and the receptor high score of binding affinity. Moreover, in silico assessment of the vaccine construct cloning achieved its suitable expression in E. coli host. Based on these results, the current multi-epitope vaccine prevents L. major infection in silico, while further confirmatory assessments are required.

Comparison of the long-term and short-term protection in mouse model of Leishmania major infection following vaccination with Live Iranian Lizard Leishmania mixed with chitin microparticles.

Inducing long-term immunity is the primary goal of vaccination. Leishmanisation using non-pathogenic to human Leishmania spp. could be considered a reliable approach to immunising subjects against Leishmania infection. Here, we evaluated the long-term immune responses (14 weeks) after immunisation with either live- or killed-Iranian Lizard Leishmania (ILL) mixed with chitin microparticles (CMPs) against L. major infection in BALB/c mice. In total, nine groups of mice were included in the study. To evaluate short-term immunity, mice were immunised with live-ILL and CMPs and 3 weeks later were challenged with L. majorEGFP . To evaluate the long-term immunity, mice were immunised with either live- or killed-ILL both mixed with CMPs, and 14 weeks after immunisation, mice were challenged with L. majorEGFP . A group of healthy mice who received no injection was also included in the study. Eight weeks after the challenge with L. majorEGFP , all subjects were sacrificed and the parasite burden (quantitative real-time PCR and in vivo imaging), cytokines levels (IFN-γ, IL-4 and IL-10), Leishmania-specific antibody concentration, and total levels of IgG1 and IgG2a were measured. In addition, nitric oxide concentration and arginase activity were evaluated. Results showed that in mice that were immunised using live-ILL+CMP, the induced protective immune response lasted at least 14 weeks; since they were challenged with L. majorEGFP at the 14th -week post-immunisation, no open lesion was formed during the 8-week follow-up, and the footpad swelling was significantly lower than controls. They also showed a significant reduction in the parasite burden in splenocytes, compared to the control groups including the group that received killed-ILL+CMP. The observed protection was associated with a higher IFN-γ and a lower IL-10 production by splenocytes. Additionally, the results demonstrated that arginase activity was decreased in the ILL+CMP group compared to other groups. Immunisation with ILL alone reduced the parasite burden compared to non-immunised control; however, it was still significantly higher than the parasite burden in the ILL+CMP groups. In conclusion, the long-term immune response against L. major infection induced by Live-ILL+CMP was more competent than the response elicited by killed-ILL+CMP to protect mice against infection with L. majorEGFP .

Interleukin-7 potentiates MAPK10-elicited host-protective vaccine against Leishmania donovani.

Leishmania donovani causes the potentially fatal disease visceral leishmaniasis for which neither a vaccine nor an adjuvant for human use exists. Although interleukin-7 (IL-7) is implicated in CD4+ T-cell response stabilization, its anti-leishmanial function is uncertain. Therefore, we examined whether IL-7 would potentiate the efficacy of Leishmania major-expressed MAPK10 (LmjMAPK10; M10)-elicited anti-leishmanial host-protective response. We observed that aligning with IL-7R expression, IL-7 increased IFN-γ-secreting TH1 cell but reduced IL-4-producing TH2 cells and production of IL-10 and TGF-ß effectuating anti-leishmanial functions in susceptible BALB/c mouse-derived macrophages. Co-culturing IL-7-pre-treated L. donovani-infected macrophages with L. donovani-infected BALB/c-derived T cells induced IFN-γ-dominated TH1 type anti-leishmanial function. IL-7 treatment of L. donovani-infected BALB/c mice significantly reduced splenic and hepatic parasite loads. Co-culturing CD4+ T cells from IL to 7-treated mice with L. donovani-infected macrophages reduced amastigote numbers suggesting IL-7-elicited host-protective effector T cells. Priming BALB/c with M10 + IL-7 reduced the splenic parasite burden more effectively than that was observed in M10-primed mice. An enhanced protection against L. donovani infection was accompanied by enhanced IL-12 and IFN-γ, but suppressed IL-10 and IL-4, response and host-protective TH1 and memory T cells. These results indicate IL-7-induced leishmanial antigen-specific memory T cell response that protects a susceptible host against L. donovani infection.

Importance of aspartate 4 in the Mg2+ dependent regulation of Leishmania major PAS domain-containing phosphoglycerate kinase.

Magnesium is an important divalent cation for the regulation of catalytic activity. Recently, we have described that the Mg2+ binding through the PAS domain inhibits the phosphoglycerate kinase (PGK) activity in PAS domain-containing PGK from Leishmania major (LmPAS-PGK) at neutral pH 7.5, but PGK activity is derepressed at acidic pH 5.5. The acidic residue within the PAS domain of LmPAS-PGK is expected to bind the cofactor Mg2+ ion at neutral pH, but which specific acidic residue(s) is/are responsible for the Mg2+ binding is still unknown. To identify the residues, we exploited mutational studies of all acidic (twelve Asp/Glu) residues in the PAS domain for plausible Mg2+ binding. Mg2+ ion-dependent repression at pH 7.5 is withdrawn by substitution of Asp-4 with Ala, whereas other acidic residue mutants (D16A, D22A, D24A, D29A, D43A, D44A, D60A, D63A, D77A, D87A, and E107A) showed similar features compared to the wild-type protein. Fluorescence spectroscopic studies and isothermal titration calorimetry analysis showed that the Asp-4 is crucial for Mg2+ binding in the absence of both PGK's substrates. These results suggest that Asp-4 residue in the regulatory (PAS) domain of wild type enzymes is required for Mg2+ dependent repressed state of the catalytic PGK domain at neutral pH.

New ψ-Santonin Derivatives from Crossostephium chinense and Their Anti-Proliferative Activities against Leishmania major and Human Cancer Cells A549.

Previously, we reported two cytotoxic ψ-santonin-amino acid conjugates isolated from the EtOAc layer of Crossostephium chinense. However, a further phytochemical investigation seems to be required because of the few reports of similar derivatives. In this study, we targeted the 1-BuOH layer, which resulted in the isolation of seven new ψ-santonin derivatives (1-7) together with ten known compounds (8-17). The structures of 1-7 were elucidated based on spectroscopic methods, including 1D and 2D NMR experiments (1H, 13C, DEPT, COSY, HSQC, and HMBC), IR spectrum, and high-resolution electrospray ionization-mass spectrometry (HR-ESI-MS). The stereochemistry of new compounds was confirmed by NOESY and ECD calculations. All isolated compounds were evaluated by in vitro experiments for their anti-proliferative activities against Leishmania major, human lung cancer cell line A549, and Vero cells. As a result, most of the ψ-santonin derivatives, especially 1-5, showed significant cytotoxicity against L. major with a lower IC50 than the positive control we used (miltefosine).

Antigen recognition reinforces regulatory T cell mediated Leishmania major persistence.

Cutaneous Leishmania major infection elicits a rapid T cell response that is insufficient to clear residually infected cells, possibly due to the accumulation of regulatory T cells in healed skin. Here, we used Leishmania-specific TCR transgenic mice as a sensitive tool to characterize parasite-specific effector and immunosuppressive responses in vivo using two-photon microscopy. We show that Leishmania-specific Tregs displayed higher suppressive activity compared to polyclonal Tregs, that was mediated through IL-10 and not through disrupting cell-cell contacts or antigen presentation. In vivo expansion of endogenous Leishmania-specific Tregs resulted in disease reactivation that was also IL-10 dependent. Interestingly, lack of Treg expansion that recognized the immunodominant Leishmania peptide PEPCK was sufficient to restore robust effector Th1 responses and resulted in parasite control exclusively in male hosts. Our data suggest a stochastic model of Leishmania major persistence in skin, where cellular factors that control parasite numbers are counterbalanced by Leishmania-specific Tregs that facilitate parasite persistence.

In Silico and In Vitro Search for Dual Inhibitors of the Trypanosoma brucei and Leishmania major Pteridine Reductase 1 and Dihydrofolate Reductase.

The parasites Trypanosoma brucei (Tb) and Leishmania major (Lm) cause the tropical diseases sleeping sickness, nagana, and cutaneous leishmaniasis. Every year, millions of humans, as well as animals, living in tropical to subtropical climates fall victim to these illnesses' health threats. The parasites' frequent drug resistance and widely spread natural reservoirs heavily impede disease prevention and treatment. Due to pteridine auxotrophy, trypanosomatid parasites have developed a peculiar enzyme system consisting of dihydrofolate reductase-thymidylate synthase (DHFR-TS) and pteridine reductase 1 (PTR1) to support cell survival. Extending our previous studies, we conducted a comparative study of the T. brucei (TbDHFR, TbPTR1) and L. major (LmDHFR, LmPTR1) enzymes to identify lead structures with a dual inhibitory effect. A pharmacophore-based in silico screening of three natural product databases (approximately 4880 compounds) was performed to preselect possible inhibitors. Building on the in silico results, the inhibitory potential of promising compounds was verified in vitro against the recombinant DHFR and PTR1 of both parasites using spectrophotometric enzyme assays. Twelve compounds were identified as dual inhibitors against the Tb enzymes (0.2 µM < IC50 < 85.1 µM) and ten against the respective Lm enzymes (0.6 µM < IC50 < 84.5 µM). These highly promising results may represent the starting point for the future development of new leads and drugs utilizing the trypanosomatid pteridine metabolism as a target.

Antiparasitic effect of Farnesol against Leishmania major: A rationale from in vitro and in silico investigations.

Leishmaniasis is a vector-borne parasitic infection caused by the infective bite of female Phlebotomine sandflies. Treatment of leishmaniasis by conventional synthetic compounds is met by challenges pertaining to adverse effects which call for the discovery of newer anti-leishmanial molecules. This study was performed to evaluate the effect and modes of action of a sesquiterpene alcoholic molecule Farnesol on Leishmania major, the causative agent of Zoonotic CL. The cytotoxic effect of Farnesol against L.major promastigotes, amastigotes and macrophages was assessed by MTT test and counting. The IC50 on promastigotes by Farnesol on L.major was also evaluated by flow cytometry. In the findings, promastigotes were reduced at 167µM. The mean numbers of L.major amastigotes in macrophages were significantly decreased on exposure to Farnesol at 172µM. In addition, Farnesol induced significant apoptosis dose-dependent on L.major promastigotes. In silico protein-ligand_binding analyses indicated the effect of Farnesol in perturbation of the ergosterol synthesis pathway of Leishmania with attributes suggesting inhibition of Lanosterol-α-demethylase, the terminal enzyme of ergosterol synthesis machinery. Findings from flow cytometry reveal the role of Farnesol in apoptosis-induced killing in promastigotes. Farnesol was effective at very lower concentrations when compared to Paromomycin. Further studies are crucial to evaluate the therapeutic potential of Farnesol alone or in combination with other conventional drugs in animal models.

The dynamic subcellular localisation of Rad1 is cell cycle dependent in Leishmania major.

The subcellular localisation of Rad1, a subunit of the Leishmania major 9-1-1 complex, remains unexplored. Herein, we reveal that Rad1 localises predominantly to the nucleus. Upon hydroxyurea treatment, the diffuse nuclear localisation of Rad1 becomes more punctate, suggesting that Rad1 is responsive to replication stress. Moreover, Rad1 localisation correlates with cell cycle progression. In the majority of G1 to early S-phase cells, Rad1 localises predominantly to the nucleus. As cells progress from late-S phase to mitosis, Rad1 relocalizes to both the nucleus and the cytoplasm in ∼90 % of cells. This pattern of distribution is different from Rad9 and Hus1, which remain nuclear throughout the cell cycle, suggesting Leishmania Rad1 may regulate 9-1-1 activities and/or perform relevant functions outside the 9-1-1 complex.

Mitochondrial Acyl Carrier Protein of Leishmania major Displays Features Distinct from the Canonical Type II ACP.

Prokaryotes synthesize fatty acids using a type II synthesis pathway (FAS). In this process, the central player, i.e., the acyl carrier protein (ACP), sequesters the growing acyl chain in its internal hydrophobic cavity. As the acyl chain length increases, the cavity expands in size, which is reflected in the NMR chemical shift perturbations and crystal structures of the acyl-ACP intermediates. A few eukaryotic organelles, such as plastids and mitochondria, also harbor type II fatty acid synthesis machinery. Plastid FAS from spinach and Plasmodium falciparum has been characterized at the molecular level, but the mitochondrial pathway remains unexplored. Here, we report NMR studies of the mitochondrial acyl-acyl carrier protein intermediates of Leishmania major (acyl-LmACP). Our studies show that LmACP experiences remarkably small conformational changes upon acylation, with perturbations confined to helices II and III only. CastP determined that the cavity size of apo-LmACP (PDB entry 5ZWT) is less than that of Escherichia coli ACP (PDB 1T8K). Thus, the small chemical shift perturbations observed in the LmACP intermediates, coupled with CastP results, suggest an unusually small cavity when fully expanded. The faster rate of C8-LmACP chain hydrolysis compared to E. coli ACP (EcACP) also supports these convictions. Structure comparison of LmACP with other type II ACP disclosed unique differences in the helix I and loop I conformations, as well as several residues present there. Numerous hydrophobic residues in helix I and loop I (conserved in all mitochondrial ACPs) are substituted with hydrophilic residues in the bacterial/plastid type II ACP. For instance, Phe and leucine at positions 14 and 34 in LmACP are substituted with a hydrophilic residue and Ala in bacterial/plastid type II ACP. Mutation of Leu 34 to Ala (corresponding residue in EcACP) resulted in a complete loss of structure, underscoring its importance in maintaining the ACP fold. Thus, our NMR studies, combined with insights from the crystal structure, highlight several unique features of LmACP, distinct from the prokaryote and plastid type II ACP. Given the high sequence identity, the features might be conserved in all mitochondrial ACPs.