Causative Agent Of Cutaneous Leishmaniasis Research Articles

Cocrystals of a coumarin derivative: an efficient approach towards anti-leishmanial cocrystals against MIL-resistant Leishmania tropica.

Leishmaniasis is a neglected parasitic tropical disease with numerous clinical manifestations. One of the causative agents of cutaneous leishmaniasis (CL) is Leishmania tropica (L. tropica) known for causing ulcerative lesions on the skin. The adverse effects of the recommended available drugs, such as amphotericin B and pentavalent antimonial, and the emergence of drug resistance in parasites, mean the search for new safe and effective anti-leishmanial agents is crucial. Miltefosine (MIL) was the first recommended oral medication, but its use is now limited because of the rapid emergence of resistance. Pharmaceutical cocrystallization is an effective method to improve the physicochemical and biological properties of active pharmaceutical ingredients (APIs). Herein, we describe the cocrystallization of coumarin-3-carboxylic acid (CU, 1a; 2-oxobenzopyrane-3-carboxylic acid, C10H6O4) with five coformers [2-amino-3-bromopyridine (1b), 2-amino-5-(trifluoromethyl)-pyridine (1c), 2-amino-6-methylpyridine (1d), p-aminobenzoic acid (1e) and amitrole (1f)] in a 1:1 stoichiometric ratio via the neat grinding method. The cocrystals 2-6 obtained were characterized via single-crystal X-ray diffraction, powder X-ray diffraction, differential scanning calorimetry and thermogravimetric analysis, as well as Fourier transform infrared spectroscopy. Non-covalent interactions, such as van der Waals, hydrogen bonding, C-H...π and π...π interactions contribute significantly towards the packing of a crystal structure and alter the physicochemical and biological activity of CU. In this research, newly synthesized cocrystals were evaluated for their anti-leishmanial activity against the MIL-resistant L. tropica and cytotoxicity against the 3T3 (normal fibroblast) cell line. Among the non-cytotoxic cocrystals synthesized (2-6), CU:1b (2, IC50 = 61.83 ± 0.59 µM), CU:1c (3, 125.7 ± 1.15 µM) and CU:1d (4, 48.71 ± 0.75 µM) appeared to be potent anti-leishmanial agents and showed several-fold more anti-leishmanial potential than the tested standard drug (MIL, IC50 = 169.55 ± 0.078 µM). The results indicate that cocrystals 2-4 are promising anti-leishmanial agents which require further exploration.

Peptide selection via phage display to inhibit Leishmania-macrophage interactions.

Leishmaniasis comprises a complex group of diseases caused by protozoan parasites from the Leishmania genus, presenting a significant threat to human health. Infection starts by the release into the skin of metacyclic promastigote (MP) form of the parasite by an infected sand fly. Soon after their release, the MPs enter a phagocytic host cell. This study focuses on finding peptides that can inhibit MP-phagocytic host cell interaction. We used a phage display library to screen for peptides that bind to the surface of L. amazonensis (causative agent for cutaneous leishmaniasis) and L. infantum (causative agent for cutaneous and visceral leishmaniasis) MPs. Candidate peptide binding to the MP surface and inhibition of parasite-host cell interaction were tested in vitro. Peptide Inhibition of visceral leishmaniasis development was assessed in BALB/c mice. The selected L. amazonensis binding peptide (La1) and the L. infantum binding peptide (Li1) inhibited 44% of parasite internalization into THP-1 macrophage-like cells in vitro. While inhibition of internalization by La1 was specific to L. amazonensis, Li1 was effective in inhibiting internalization of both parasite species. Importantly, Li1 inhibited L. infantum spleen and liver infection of BALB/c mice by 84%. We identified one peptide that specifically inhibits L. amazonensis MP infection of host cells and another that inhibits both, L. amazonensis and L. infantum, MP infection. Our findings suggest a promising path for the development of new treatments and prevention of leishmaniasis.

Entomological, parasitological and molecular investigations in a new focus of cutaneous leishmaniasis in Youssoufia region, Morocco.

Leishmaniasis is a neglected tropical infection caused by Leishmania parasite that affect human and animal. In Morocco, the cutaneous leishmaniasis has spread substantially to the new areas. The surveillance limited to active foci may underestimate the occurrence of cutaneous leishmaniasis (CL). This study aims to investigate the local transmission of CL in rural districts of Youssoufia province, central Morocco, as a potential focus of CL. For this purpose, parasitological, molecular and entomological investigations were carried out in this area. Data collection concerns potential vectors and human cases. Thus, 402 patients were examined for suspected leishmaniasis lesions in three localities of the province of Youssoufia. In these same localities, 983 sand flies were collected by CDC light traps and sticky paper during one-night per month during 6 months. These sand flies were all identified morphologically using the Moroccan identification key. The results showed that among the 25 skin lesions detected in a population of 402 individuals, 18 were confirmed by kDNA nested PCR as CL positive patients, of which only 25% were positive by direct examination. Leishmania tropica and Leishmania major were identified as causative agents of CL in the study area. Direct parasitological examination showed a low sensitivity (27.78%), especially for L. major, although its specificity was evaluated at 100%. Regarding entomological results, both genera of the Moroccan sand fly were collected in the study area: Genus/Phlebotomus (75.28%) and Sergentomyia (24.72%). Phlebotomus (P) papatasi, the proven vector of L. major, was the most abundant species (33.98%), followed by Paralongicollum sergenti (22.58%), the confirmed vector of L. tropica; while Sergentomyia (S) minuta, P. longicuspis, S. fallax and P. kazeruni were collected with, respectively, 17.60%, 16.99%, 7.12% and 1.73%. This study constitutes the first report of CL in the study areas, as well as the coexistence of L. tropica and L. major in these rural localities. Local transmission of CL is highly probable, as indicated by the prevalence of the two proven vectors of L. major and L. tropica. To control the spread of this disease, our results suggest the use of highly sensitive molecular methods to detect CL cases in potential leishmaniasis foci, which will improve surveillance.

Ethanolaminephosphate cytidylyltransferase is essential for survival, lipid homeostasis and stress tolerance in Leishmania major.

Glycerophospholipids including phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are vital components of biological membranes. Trypanosomatid parasites of the genus Leishmania can acquire PE and PC via de novo synthesis and the uptake/remodeling of host lipids. In this study, we investigated the ethanolaminephosphate cytidylyltransferase (EPCT) in Leishmania major, which is the causative agent for cutaneous leishmaniasis. EPCT is a key enzyme in the ethanolamine branch of the Kennedy pathway which is responsible for the de novo synthesis of PE. Our results demonstrate that L. major EPCT is a cytosolic protein capable of catalyzing the formation of CDP-ethanolamine from ethanolamine-phosphate and cytidine triphosphate. Genetic manipulation experiments indicate that EPCT is essential in both the promastigote and amastigote stages of L. major as the chromosomal null mutants cannot survive without the episomal expression of EPCT. This differs from our previous findings on the choline branch of the Kennedy pathway (responsible for PC synthesis) which is required only in promastigotes but not amastigotes. While episomal EPCT expression does not affect promastigote proliferation under normal conditions, it leads to reduced production of ethanolamine plasmalogen or plasmenylethanolamine, the dominant PE subtype in Leishmania. In addition, parasites with episomal EPCT exhibit heightened sensitivity to acidic pH and starvation stress, and significant reduction in virulence. In summary, our investigation demonstrates that proper regulation of EPCT expression is crucial for PE synthesis, stress response, and survival of Leishmania parasites throughout their life cycle.

Immunopathological investigation of a gerbil model of cutaneous leishmaniasis

Leishmaniasis, caused by Leishmania species (intracellular protozoans), is a chronic, systemic disease that causes skin (cutaneous) and internal organ infections (visceral). Its prevalence has increased in recent years. Leishmania species are considered important pathogens that affect public health. After infecting an individual, the pathogen disrupts the immune system, but, there are not enough studies on which immune mechanisms are affected. The aim of this study was to establish a Leishmania major infection model (the causative agent of cutaneous leishmaniasis) in gerbils (Meriones unguiculatus) and to investigate the immune response in this model by examining the expression of important inflammatory genes (IL-1β, IL-2, IL-6, IFN-ɣ and TNF-α). The presence of parasites was confirmed by microscopic examination of samples taken from the lesions and culture studies. The expression of inflammatory cytokine genes was significantly increased in infected gerbils. The changes indicated that both the Th1 and Th2 pathways are activated in cutaneous leishmaniasis infection. Hence, different immunopathological mechanisms should be evaluated in the pathogenesis of the disease.

Visceral Leishmaniasis Caused by Leishmania Tropica.

In Turkey, the main causative agent of visceral leishmaniasis (VL) is Leishmania. infantum and the main causative agent of cutaneous leishmaniasis (CL) is Leishmania tropica. In this study, we aimed to discuss the possible mechanisms, clinical aspects, and threat of visceralizing L. tropica. This study includes seven cases of VL caused by L. tropica.Five patients were male (71%) and four were adults (57%). All the VL patients complained of fever and splenomegaly. Fatigue, pancytopenia, and hepatomegaly were present in six patients each (86%), while weight loss and gastrointestinal system (GIS) symptoms were present in 5 patients (71%). In this study, we have evaluated seven cases of visceralized L. tropica (VLT) in the context of the changing leishmaniasis epidemiology in Turkey. We have evaluated the possible mechanisms of visceralization; inter- and intraspecies genetic exchange with all the old world leishmaniasis agents present in the region, stress induced by inappropriate use of drugs, and possible ongoing adaptation mechanisms of Leishmania spp. The threat posed by VLT is significant as L. tropica is the most widespread and most common cause of leishmaniasis in Turkey. We do not know the vectorial capacity of the sand flies for the transmission of VLT strains or if these strains are in circulation in Turkey. Future studies should be carried out to investigate these issues as the transition of L. tropica from a mild disease-causing agent to a mortal one poses a significant public health concern for Turkey and Europe.

The in vitro and in vivo effects of Lucilia sericata larval secretions on Leishmania major.

The emerging of drug resistant against Leishmania parasites prompts scientists to seek for novel therapeutic strategies against theses infectious protozoan parasites. Among different strategies, the use of larvae secretions could be suggested as a possible therapy with low side effects. Accordingly, the current study evaluated the in vitro and in vivo effects of Lucilia sericata larval secretions on Leishmania major, the causative agent of cutaneous leishmaniasis (CL). After preparation of L. sericata larval stages (L2 and L3) secretions, the potential effects of secretions were evaluated against L. major promastigotes and amastigotes (in vitro) using MTT assay. The cytotoxicity effects of secretions were also checked on uninfected macrophages. In addition, in vivo experiments were also conducted to investigate the effects of larvae's secretions on the CL lesions induced in the BALB/c mice. Although the increased concentration of larvae secretions exhibited a direct effect on the promastigotes proliferation (viability), contrarily, L2 secretions at a concentration of 96μg/ml represented the highest inhibitory effect on parasite (amastigotes) burden in infected macrophages. Interestingly, L3 secretions > 60μg/ml induced inhibitory effects on amastigotes. The results relevant to the cytotoxicity effects of L2 and L3 secretions on uninfected-macrophages showed a dose dependent correlation. In vivo results were also significant, compared to the positive control group. This study suggested the plausible inhibitory effects of L. sericata larvae's secretions on the L. major amastigotes and CL lesions progression. It seems that the characterization of all effective components/proteins in the larvae secretions and their specific targets in parasite structure or in cell (macrophage) responses could further reveal more details regarding the anti-leishmanial properties of these compounds.

In Vitro Determination of Antileshmanial Activities of Benzimidazolium Derivatives on L. major Promastigotes and Amastigotes.

Leishmaniasis is a serious public health problem infecting millions of people worldwide. An effective and reliable treatment method to be used in the treatment of the disease has not been developed yet. In this article, the anti-leishmanial activities of two benzimidazolium derivatives (B.A and B.B) against Leishmania major promastigotes and amastigotes, which are known to cause cutaneous leishmaniasis, were investigated for the first time. The immunostimulatory activity of the developed formulations was determined using the J774 murine macrophage cell line. B.A and B.B compounds were found to have a much higher cytotoxic effect than Amphotericin B (IC50 value 0.75μM ± 0.03), which is used as the reference drug. The IC50 value was determined as 2.02µM ± 0.52 for B.A and 1.83µM ± 0.71 for B.B in Leishmania promastigotes. In addition, IC50 values of B. A and B.B Leishmania amastigotes were found to be 1.01µM and 0.67µM, respectively. It was found that B.B was 81.12 times more selective than Amphotericin B and showed the highest selectivity against L. major promastigotes (359.09) and amastigotes (980.80). Considering the selectivity indices (SI) of B.A and B.B, both compounds tested are more promising than Amphotericin B. The results showed that benzimidazolium derivatives have anti-leishmanial potential against L. major, which is the causative agent of cutaneous leishmaniasis. Thus, we can say that the obtained results will help the development of effective and safe antileishmanial drug formulations against cutaneous leishmaniasis.

Patterns of cutaneous leishmaniasis during the COVID-19 pandemic in four endemic regions of Iran.

Both zoonotic cutaneous leishmaniasis (ZCL) and anthroponotic cutaneous leishmaniasis are endemic in different regions of Iran. It is important to know the type of cutaneous leishmaniasis (CL) for prevention strategies. On the other hand, Iran is one of the countries that was severely affected by the coronavirus 2019 (COVID-19) pandemic. However, little is known about the causative agents of CL during the COVID-19 pandemic in Iran. In this study, a total of 181 samples were isolated from patients with CL lesions in four different endemic cities of Iran (Jahrom, Juyom, Kashan and Shoushtar) during the COVID-19 pandemic (the summer and autumn of 2021). The nested-PCR targeting kinetoplast DNA (kDNA) minicircles was applied for species identification of the parasite and the internal transcribed spacer-1 (ITS-1) gene was applied for sequencing and phylogenetic analysis. Molecular detection of kDNA minicircles revealed that all isolates from the four cities were Leishmania major, indicating the ZCL pattern. Ten isolates were sequenced by the ITS-1 gene and deposited in GenBank [accession numbers: OL627363-72]. Sequencing and phylogenetic analysis displayed a high similarity rate of the isolates with sequences from other parts of Iran and Iraq. Over half of the patients (53.59%) had a single lesion, while 17.12, 14.92, 2.21 and 12.16% of cases had two, three, four and more than four lesions, respectively. About half of the patients (50.27%) had lesions on their hands, while the rest had lesions on multiple locations (19.34%), legs (16.58%), face (9.94%), ears (1.66%) and waist (2.21%). This study revealed the occurrence of ZCL during the COVID-19 pandemic in four endemic regions of Iran. Strategies for prevention and control of the disease should be considered to mitigate the occurrence of ZCL.

Treatment of pediatric cutaneous leishmaniasis with liposomal amphotericin B.

The use of liposomal amphotericin B (L-AmB) in the treatment of cutaneous leishmaniasis (CL) is increasing. However, few data are available regarding the efficacy and safety of L-AmB in pediatric CL patients. Our aim in this study is to evaluate the efficacy and safety of L-AmB in pediatric CL patients. Pediatric patients admitted to a tertiary training and research hospital in a hyperendemic region for CL between January 2019 and May 2021 and receiving L-AmB therapy for CL were included in this retrospective study. L-AmB treatment was administered as 3mg/kg for 5 consecutive days and on the 10th day, in a total of 6 doses (18 mg/kg total dose). A total of 52 pediatric patients who received L-AmB therapy for CL were included in the study. In the follow-up 3months after L-AmB treatment, 16 (31%) patients showed complete clinical recovery, while treatment failure was detected in 36 (69%) patients. In conclusion, considering the low treatment success rate in our study, we think that the L-AmB dose used in our study is not an appropriate treatment option for the treatment of pediatric CL patients. However, we think that prospective studies with a large number of patients treated with higher doses of L-AmB and in whom the causative agents of CL were determined are needed.

Effect of hybridization on lipophosphoglycan expression in Leishmania major.

Leishmania major is the causative agent of cutaneous leishmaniasis. It is one of the most studied Leishmania species not only during vector interaction but also in the vertebrate host. Lipophosphoglycan (LPG) is the Leishmania multifunctional virulence factor during host-parasite interaction, whose polymorphisms are involved in the immunopathology of leishmaniasis. Although natural hybrids occur in nature, hybridization of L. major strains in the laboratory was successfully demonstrated. However, LPG expression in the hybrids remains unknown. LPGs from parental (Friedlin, Fn and Seidman,Sd) and hybrids (FnSd3, FnSd4A, FnSd4B, and FnSd6F) were extracted, purified, and their repeat units analyzed by immunoblotting and fluorophore-assisted carbohydrate electrophoresis. Parental strains have distinct profiles in LPG expression, and a mixed profile was observed for all hybrids. Variable levels of NO production by macrophages were detected after LPG exposure (parental and hybrids) and were strain specific.

Predominance of Leishmania (Leishmania) amazonensis DNA in Lutzomyia longipalpis sand flies (Diptera: Psychodidae) from an endemic area for leishmaniasis in Northeastern Brazil.

ABSTRACTLeishmaniasis is a serious public health concern in the Northeastern region of Brazil, where the sand fly fauna is well studied, although few species have been identified as competent vectors. The detection of Leishmania spp. parasites in wild-caught sand flies could help sanitary authorities draw strategies to avoid the transmission of the parasites and, therefore, the incidence of leishmaniases. We detected Leishmania DNA in wild-caught sand flies and correlated that data with aspects of sand fly ecology in the Caxias municipality, Maranhao State, Brazil. The sand flies were sampled in the peridomicile (open areas in the vicinity of human residences) and intradomicile (inside the residences) from July/2019 to March/2020. Leishmania DNA was detected in females, targeting a fragment of the Internal Transcribed Spacer (ITS1) from ribosomal DNA. Among the fourteen species of sand flies identified, five (Lutzomyia longipalpis, Nyssomyia whitmani, Evandromyia evandroi, Micropygomyia trinidadensis, and Micropygomyia quinquefer) harbored DNA of Leishmania (Leishmania) amazonensis. The most abundant species in rural (Ny. whitmani: 35.2% and Ev. evandroi: 32.4%) and urban areas (Lu. longipalpis: 89.8%) are the permissive vectors of L. (L.) amazonensis, especially Ny. whitmani, a known vector of causative agents of cutaneous leishmaniasis. Although Lu. longipalpis is the vector of L. (L.) infantum, which was not detected in this study, its permissiveness for the transmission of L. (L.) amazonensis has been reported. We suspect that visceral leishmaniasis and cutaneous leishmaniasis are caused by L. (L.) amazonensis, and the transmission may be occurring through Lu. longipalpis, at least in the urban area.

Differential activity on trypanosomatid parasites of a novel recombinant defensin type 1 from the insect Triatoma (Meccus) pallidipennis

Defensins are one of the major families of antimicrobial peptides (AMPs) that are widely distributed in insects. In Triatomines (Hemiptera: Reduviidae) vectors of Trypanosoma cruzi the causative agent of Chagas disease, two large groups of defensin isoforms have been described: type 1 and type 4. The aim of this study was to analyze the trypanocidal activity of a type 1 recombinant defensin (rDef1.3) identified in Triatoma (Meccus) pallidipennis, an endemic specie from México. The trypanocidal activity of this defensin was evaluated in vitro, against the parasites T. cruzi, T. rangeli, and two species of Leishmania (L. mexicana and L. major) both causative agents of cutaneous leishmaniasis. Our data demonstrated that the defensin was active against all the parasites although in different degrees. The defensin altered the morphology, reduced the viability and inhibited the growth of T.cruzi. When tested against T. rangeli (a parasite that infects a variety of mammalian species), stronger morphological effects where observed. Surprisingly the greatest effects were observed against the two Leishmania species, of which L. major was the parasite most affected with 50% of dead cells or with damaged membranes, in addition of a reduction in its proliferative capacity in culture. These results suggest that rDef1.3 has an important antimicrobial effect against trypanosomatids which cause some of the more important neglected tropical diseases transmitted by insect vectors.

Gene Annotation and Transcriptome Delineation on a De Novo Genome Assembly for the Reference Leishmania major Friedlin Strain.

Leishmania major is the main causative agent of cutaneous leishmaniasis in humans. The Friedlin strain of this species (LmjF) was chosen when a multi-laboratory consortium undertook the objective of deciphering the first genome sequence for a parasite of the genus Leishmania. The objective was successfully attained in 2005, and this represented a milestone for Leishmania molecular biology studies around the world. Although the LmjF genome sequence was done following a shotgun strategy and using classical Sanger sequencing, the results were excellent, and this genome assembly served as the reference for subsequent genome assemblies in other Leishmania species. Here, we present a new assembly for the genome of this strain (named LMJFC for clarity), generated by the combination of two high throughput sequencing platforms, Illumina short-read sequencing and PacBio Single Molecular Real-Time (SMRT) sequencing, which provides long-read sequences. Apart from resolving uncertain nucleotide positions, several genomic regions were reorganized and a more precise composition of tandemly repeated gene loci was attained. Additionally, the genome annotation was improved by adding 542 genes and more accurate coding-sequences defined for around two hundred genes, based on the transcriptome delimitation also carried out in this work. As a result, we are providing gene models (including untranslated regions and introns) for 11,238 genes. Genomic information ultimately determines the biology of every organism; therefore, our understanding of molecular mechanisms will depend on the availability of precise genome sequences and accurate gene annotations. In this regard, this work is providing an improved genome sequence and updated transcriptome annotations for the reference L. major Friedlin strain.

Comparison of Three Commonly Used Genetic Markers for Detection of Leishmania Major: An Experimental Study.

Leishmaniasis is a vector-borne disease caused by an intracellular protozoan parasite called Leishmania spp. Different species produce different clinical outcomes; the majority of cases are cutaneous forms. Leishmania major is one of the main causative agents of cutaneous leishmaniasis (CL). Various methods are being using to diagnose CL, including microscopic examination, culture, and molecular detection of the parasite genome. In the current study, we tried to compare three common molecular markers, including Kinetoplast DNA (kDNA), Cytochrome b (Cyt b), and Internal transcribed space 1 (ITS1), for the detection of Leishmania major. After cultivation of standard strain of L. major MHOM/IR/75/ER in RPMI 1640, certain number of promastigotes was subjected to DNA extraction and different PCR reactions. The lowest number of the parasite (5 promastigotes) can be detected by kDNA-PCR, followed by Cyt b-PCR (10 promastigotes), and ITS1-PCR (50 promastigotes). In conclusion, kDNA-PCR was the most sensitive marker and may provide more reliable data in the initial screening, especially in false-negative results provided by parasitological methods due to the low number of parasites.

Protein methyltransferase 7 deficiency in Leishmania major increases neutrophil associated pathology in murine model.

Leishmania major is the main causative agent of cutaneous leishmaniasis in the Old World. In Leishmania parasites, the lack of transcriptional control is mostly compensated by post-transcriptional mechanisms. Methylation of arginine is a conserved post-translational modification executed by Protein Arginine Methyltransferase (PRMTs). The genome from L. major encodes five PRMT homologs, including the cytosolic protein associated with several RNA-binding proteins, LmjPRMT7. It has been previously reported that LmjPRMT7 could impact parasite infectivity. In addition, a more recent work has clearly shown the importance of LmjPRMT7 in RNA-binding capacity and protein stability of methylation targets, demonstrating the role of this enzyme as an important epigenetic regulator of mRNA metabolism. In this study, we unveil the impact of PRMT7-mediated methylation on parasite development and virulence. Our data reveals that higher levels of LmjPRMT7 can impair parasite pathogenicity, and that deletion of this enzyme rescues the pathogenic phenotype of an attenuated strain of L. major. Interestingly, lesion formation caused by LmjPRMT7 knockout parasites is associated with an exacerbated inflammatory reaction in the tissue correlated with an excessive neutrophil recruitment. Moreover, the absence of LmjPRMT7 also impairs parasite development within the sand fly vector Phlebotomus duboscqi. Finally, a transcriptome analysis shed light onto possible genes affected by depletion of this enzyme. Taken together, this study highlights how post-transcriptional regulation can affect different aspects of the parasite biology.

Effect of DODAB Nano-Sized Cationic Bilayer Fragments against Leishmania amazonensis.

The dioctadecyldimethylammonium bromide (DODAB) is a double-chained cationic lipid with potent bactericide and fungistatic activities; however, its toxicity on protozoan parasites is still unknown. Here, we show the antileishmanial activity of DODAB nano-sized cationic bilayer fragments on stationary-phase promastigotes and amastigotes of Leishmania amazonensis, the causative agent of cutaneous leishmaniasis. Upon treatment with DODAB, we analyzed the parasite surface zeta-potential, parasite viability, cellular structural modifications, and intracellular proliferation. The DODAB cytotoxic effect was dose-dependent, with a median effective concentration (EC50) of 25 µM for both life-cycle stages, comparable to the reported data for bacteria and fungi. The treatment with DODAB changed the membrane zeta-potential from negative to positive, compromised the parasite’s morphology, affected the cell size regulation, caused a loss of intracellular organelles, and probably dysregulated the plasma membrane permeability without membrane disruption. Moreover, the parasites that survived after treatment induced small parasitophorous vacuoles and failed to proliferate inside macrophages. In conclusion, DODAB displayed antileishmanial activity, and it remains to be elucidated how DODAB acts on the protozoan membrane. Understanding this mechanism can provide insights into the development of new parasite-control strategies.

Antileishmanial activity of 4-phenyl-1-[2-(phthalimido-2-yl)ethyl]-1H-1,2,3-triazole (PT4) derivative on Leishmania amazonensis and Leishmania braziliensis: In silico ADMET, in vitro activity, docking and molecular dynamic simulations

Organic compounds obtained by click chemistry reactions have demonstrated a broad spectrum of biological activities being widely applied for the development of molecules against pathogens of medical and veterinary importance. Cutaneous leishmaniasis (CL), caused by intracellular protozoa parasite of genus Leishmania, comprises a complex of clinical manifestations that affect the skin and mucous membranes. The available drugs for the treatment are toxic and costly, with long periods of treatment, and the emergence of resistant strains has been reported. In this study we investigated the in vitro effects of a phthalimide-1,2,3-triazole derivative, the 4-Phenyl-1-[2-(phthalimido-2-yl)ethyl]-1H-1,2,3-triazole (PT4) obtained by click chemistry, on mammalian cells and on L. amazonensis and L. braziliensis, the causative agents of CL in Brazil. In silico ADMET evaluation of PT4 showed that this molecule has good pharmacokinetic properties with no violation of Lipinski’s rules. The in vitro assays showed that PT4 was more selective for both Leishmania species than to mammalian cells. This compound also presented low cytotoxicity to mammalian cells with CC50 > 500 μM. Treatment of promastigote forms with different concentrations of PT4 resulted in ultrastructural alterations, such as plasma membrane wrinkling, shortening of cell body, increased cell volume and cell rupture. The molecular dynamic simulations showed that PT4 interacts with Lanosterol 14 α-demethylase from Leishmania, an essential enzyme of lipid synthesis pathway in this parasite. Our results demonstrated PT4 was effective against both species of Leishmania. PT4 caused a decrease of mitochondrial membrane potential and increased production of reactive oxygen species, which may lead to parasite death. Taken together, our results pointed PT4 as promissing therapeutic agent against CL.

Enhanced production of pro-inflammatory cytokines and chemokines in Ethiopian cutaneous leishmaniasis upon exposure to Leishmania aethiopica

The clinical course and outcome of cutaneous leishmaniasis (CL) vary due to the infecting Leishmania species and host genetic makeup that result in different immune responses against the parasites. The host immune response to Leishmania aethiopica (L.aethiopica), the causative agent of CL in Ethiopia, is poorly understood. To contribute to the understanding of the protective immune response in CL due to L.aethiopica, we characterized the cytokine response to L. aethiopica in patients with the localized form of CL (LCL) and age-and sex-matched apparently healthy controls. By applying a whole blood based in vitro culture we found enhanced release of TNF, IL–6, MCP-1 or CCL2, IP-10 or CXCL10, MIP-1β or CCL4 and IL-8 or CXCL8- but not of IL–10CL patients in response to L. aethiopica compared to the controls. No difference was observed between LCL cases and controls in the secretion of these cytokines and chemokines in whole blood cultures treated with the TLR-ligands LPS, MALP-2 or polyI: C. The observed increased secretion of the pro-inflammatory cytokines/chemokines reflects an enhanced response against the parasites by LCL patients as compared to healthy controls rather than a generally enhanced ability of blood leukocytes from LCL patients to respond to microbial constituents. Our findings suggest that the enhanced production of pro-inflammatory cytokines/chemokines is associated with localized cutaneous leishmaniasis caused by L.aethiopica.

Expanded genome-wide comparisons give novel insights into population structure and genetic heterogeneity of Leishmania tropica complex.

Leishmania tropica is one of the main causative agents of cutaneous leishmaniasis (CL). Population structures of L. tropica appear to be genetically highly diverse. However, the relationship between L. tropica strains genomic diversity, protein coding gene evolution and biogeography are still poorly understood. In this study, we sequenced the genomes of three new clinical L. tropica isolates, two derived from a recent outbreak of CL in camps hosting Syrian refugees in Lebanon and one historical isolate from Azerbaijan to further refine comparative genome analyses. In silico multilocus microsatellite typing (MLMT) was performed to integrate the current diversity of genome sequence data in the wider available MLMT genetic population framework. Single nucleotide polymorphism (SNPs), gene copy number variations (CNVs) and chromosome ploidy were investigated across the available 18 L. tropica genomes with a main focus on protein coding genes. MLMT divided the strains in three populations that broadly correlated with their geographical distribution but not populations defined by SNPs. Unique SNPs profiles divided the 18 strains into five populations based on principal component analysis. Gene ontology enrichment analysis of the protein coding genes with population specific SNPs profiles revealed various biological processes, including iron acquisition, sterols synthesis and drug resistance. This study further highlights the complex links between L. tropica important genomic heterogeneity and the parasite broad geographic distribution. Unique sequence features in protein coding genes identified in distinct populations reveal potential novel markers that could be exploited for the development of more accurate typing schemes to further improve our knowledge of the evolution and epidemiology of the parasite as well as highlighting protein variants of potential functional importance underlying L. tropica specific biology.