MAPK/ERK Activation in Macrophages Promotes Leishmania Internalization and Pathogenesis.

The obligate intracellular parasite Leishmania binds several receptors to trigger uptake by phagocytic cells, ultimately resulting in visceral or cutaneous leishmaniasis. A series of signaling pathways in host cells, which are critical for establishment and persistence of infection, are activated during Leishmania internalization. Thus, preventing Leishmania uptake by phagocytes could be a novel therapeutic strategy for leishmaniasis. However, the host cellular machinery mediating promastigote and amastigote uptake is not well understood. Here, using small molecule inhibitors of Mitogen-activated protein/Extracellular signal regulated kinases (MAPK/ERK), we demonstrate that ERK1/2 mediates Leishmania amazonensis uptake and (to a lesser extent) phagocytosis of beads by macrophages. We find that inhibiting host MEK1/2 or ERK1/2 leads to inefficient amastigote uptake. Moreover, using inhibitors and primary macrophages lacking spleen tyrosine kinase (SYK) or Abl family kinases, we show that SYK and Abl family kinases mediate Raf, MEK, and ERK1/2 activity and are necessary for uptake. Finally, we demonstrate that trametinib, a MEK1/2 inhibitor used to treat cancer, reduces disease severity and parasite burden in Leishmania-infected mice, even if it is started after lesions develop. Our results show that maximal Leishmania infection requires MAPK/ERK and highlight potential for MAPK/ERK-mediated signaling pathways to be novel therapeutic targets for leishmaniasis.

Leishmania exploits host cAMP/EPAC/calcineurin signaling to induce an IL-33-mediated anti-inflammatory environment for the establishment of infection.

Host anti-inflammatory responses are critical for the progression of visceral leishmaniasis and the pleomorphic cytokine IL-33 was found to be upregulated in infection. The underlying mechanism is not yet known. Here, we documented that IL-33 induction is a consequence of elevated cAMP-mediated EPAC/calcineurin-dependent signaling and is essential for the sustenance of infection. L. donovani-infected RAW and bone marrow-derived macrophages showed significant up-regulation of IL-33 and its neutralization by anti-IL-33 antibody resulted in decreased parasite survival and increased inflammatory responses. Infection-induced elevated cAMP was involved in IL-33 production and of its two downstream effectors PKA and EPAC, only the latter was responsible for elevated IL-33 levels. EPAC initiated Rap-dependent phospholipase C activation, which triggered the release of intracellular calcium followed by calcium/calmodulin complex formation. Screening of calmodulin-dependent enzymes affirmed the involvement of the phosphatase calcineurin in cAMP/EPAC/calcium/calmodulin signaling-induced IL-33 production and parasite survival. Activated calcineurin thereby ensured nuclear localization of the transcription factors NFATc1 and HIF-1α for IL-33 transcription and we further ascertained their role in IL-33 transcription using a ChIP assay. Administering specific inhibitors of NFATc1 and HIF-1α in a BALB/c mouse model of visceral leishmaniasis led to decreased liver and spleen parasite burden with concomitant decrease in IL-33 levels. Splenocyte supernatants of inhibitor-treated infected mice further documented an increase in TNF-α and IL-12 with simultaneous decrease of IL-10, thereby indicating an overall disease-escalating effect of IL-33. Thus, this study demonstrates that cAMP/EPAC/calcineurin signaling is crucial for the activation of IL-33 and in effect creates anti-inflammatory responses, essential for infection.

Identification and Validation of Compounds Targeting Leishmania major Leucyl-Aminopeptidase M17.

Leishmaniasis is a neglected tropical disease; there is currently no vaccine and treatment is reliant upon a handful of drugs suffering from multiple issues including toxicity and resistance. There is a critical need for development of new fit-for-purpose therapeutics, with reduced toxicity and targeting new mechanisms to overcome resistance. One enzyme meriting investigation as a potential drug target in Leishmania is M17 leucyl-aminopeptidase (LAP). Here, we aimed to chemically validate LAP as a drug target in L. major through identification of potent and selective inhibitors. Using RapidFire mass spectrometry, the compounds DDD00057570 and DDD00097924 were identified as selective inhibitors of recombinant Leishmania major LAP activity. Both compounds inhibited in vitro growth of L. major and L. donovani intracellular amastigotes, and overexpression of LmLAP in L. major led to reduced susceptibility to DDD00057570 and DDD00097924, suggesting that these compounds specifically target LmLAP. Thermal proteome profiling revealed that these inhibitors thermally stabilized two M17 LAPs, indicating that these compounds selectively bind to enzymes of this class. Additionally, the selectivity of the inhibitors to act on LmLAP and not against the human ortholog was demonstrated, despite the high sequence similarities LAPs of this family share. Collectively, these data confirm LmLAP as a promising therapeutic target for Leishmania spp. that can be selectively inhibited by drug-like small molecules.

Treatment and Healing of Leishmaniasis in a Wolf in Semi-Captivity Regime from an Educational Center of Zamora Province (Spain).

Leishmaniasis in wild canids is a vector-borne disease caused in Europe by the protozoan parasite Leishmania infantum. To date, there is limited information on clinical signs and laboratory abnormalities in wolves due to leishmaniasis. The current clinical case report described a female Iberian wolf (Canis lupus signatus) housed in semi-captivity conditions at the Centro del Lobo Ibérico "Félix Rodríguez de la Fuente", in Robledo de Sanabria, Zamora (Spain), with an interdigital ulcerous wound at the right forepaw, hyper-gammaglobulinemia, and abnormal liver blood parameters. Definitive serodiagnosis of leishmaniasis was established using antileishmanial serum antibodies and PCR analysis of different biological samples. A gold-standard anti-L. infantum treatment protocol consisting in subcutaneous meglumine antimoniate and oral allopurinol combination was installed. However, the presence of pain at the site of injection due to meglumine antimoniate administration forced its substitution by oral miltefosine. A progressive reduction of the levels of anti-L. infantum serum antibodies and the concentrations of gamma-globulin fraction was detected after antileishmanial treatment as well as a decline of liver GPT. To our knowledge, this is the first case of leishmaniasis diagnosed in a wolf housed in semi-captivity conditions, with the condition subsequently treated and successfully cured.

Nanopore-Based Surveillance of Leishmania Parasites in Culicoides Latrielle (Diptera: Ceratopogonidae) Caught from the Affected Community and Tham Phra Cave in Chiang Rai Province, the Endemic Area of Leishmaniasis in Northern Thailand.

In this research, we elucidated the species composition of Culicoides biting midges, infection prevalence, and genetic diversity of Leishmania parasites circulating in the affected community in Chiang Rai Province, being the most endemic area in Northern Thailand. A total of 146 parous and gravid females, belonging to at least twelve Culicoides species in five subgenera and one species group, were trapped from three collection sites with an overall Leishmania prevalence of 26.7% (39/146). Leishmania was detected, using ITS1-PCR, in C. mahasarakamense (15), C. guttifer (11), C. (Trithecoides) spp. (8), C. jacobsoni (2), C. oxystoma (2), and C. orientalis (1). The evidence of Leishmania infection in these last five species represents new records in Northern Thailand. Given a high infection rate in cavernicolous specimens, this indicates an increased risk of parasite exposure when visiting the cave. Using the nanopore amplicon sequencing, L. martiniquensis was ubiquitously identified in all positives, and more than half of these were also co-infected with L. orientalis. The genetic diversity analysis revealed 13 and 17 unique haplotypes for L. martiniquensis and L. orientalis, respectively. Higher haplotype diversity and relatively low nucleotide diversity were observed in both parasite populations, suggesting recent population divergence. Neutrality tests (Tajima's D and Fu and Li's D) showed to be significantly negative, indicating rapid population growth or a selective sweep. Moreover, dominant haplotypes of both Leishmania species were 100% identical to those in all leishmaniasis patients previously reported from Northern Thailand, strongly supporting the imperative role of Culicoides spp. in disease transmission. Essentially, this research provides the first entomological surveillance data representing the sympatric existence, transmission dynamics, and genetic complexity of two autochthonous Leishmania (Mundinia) parasites in several Culicoides species in the endemic area of Northern Thailand. This would contribute to a more complete understanding of the epidemiology of vector infection and facilitate the development of vector control programs to effectively reduce the transmission of this neglected tropical disease in endemic areas of Northern Thailand.

Human Dendritic Cell Maturation Is Modulated by Leishmania mexicana through Akt Signaling Pathway.

Dendritic cells (DC) along with macrophages are the main host cells of the intracellular parasite Leishmania. DC traverse a process of maturation, passing through an immature state with phagocytic ability to a mature one where they can modulate the immune response through the secretion of cytokines. Several studies have demonstrated that Leishmania inhibits DC maturation. Nevertheless, when cells are subjected to a second stimulus such as LPS/IFN-γ, they manage to mature. In the maturation process of DC, several signaling pathways have been implicated, importantly MAPK. On the other hand, Akt is a signaling pathway deeply involved in cell survival. Some Leishmania species have shown to activate MAPK and Akt in different cells. The aim of this work was to investigate the role of ERK and Akt in the maturation of monocyte-derived DC (moDC) infected with L. mexicana. moDC were infected with L. mexicana metacyclic promastigotes, and the phosphorylation of ERK and Akt, the expression of MHCII and CD86 and IL-12 transcript, and secretion were determined in the presence or absence of an Akt inhibitor. We showed that L. mexicana induces a sustained Akt and ERK phosphorylation, while the Akt inhibitor inhibits it. Moreover, the infection of moDC downregulates CD86 expression but not MHCII, and the Akt inhibitor reestablishes CD86 expression and 12p40 production. Thus, L. mexicana can modulate DC maturation though Akt signaling.

Finding Priority Areas in the Evaluation of Strategies for the Prevention of Leishmaniasis in an Endemic Municipality of Brazil.

Visceral leishmaniasis is a zoonotic disease that affects humans and dogs. The infection is endemic in the municipality of Araçatuba, São Paulo, Brazil. Given the role of dogs in the epidemiology of leishmaniasis, strategies to enhance surveillance and reduce transmission are focused on dogs. In this study, we retrospectively analyzed records of canine visceral leishmaniasis from 2013 to 2022. According to this database, the prevalence of dogs testing positive for leishmaniasis fluctuated, with an average of 65.04% (6590/10,133). Cases were clustered in 10 statistically significant areas. Environmental analyses identified a significant geographical association between animals testing positive and higher vegetation density rates compared with animals testing negative. The period from sample collection to diagnosis and euthanasia, as recommended by the Brazilian Ministry, correlated with disease prevalence and decreased over time. These findings serve to implement different action plans against leishmaniasis for each geographic region and to understand the impact and efforts of strategies in an endemic area.

Anatomical Vascular Differences and Leishmania-Induced Vascular Morphological Changes Are Associated with a High Parasite Load in the Skin of Dogs Infected with Leishmania infantum.

Canine visceral leishmaniasis (CVL), caused by the protozoan Leishmania infantum, affects several organs, including the skin. Dogs are considered the major domestic reservoir animals for leishmaniasis, and through their highly parasitized skin, they can serve as a source of infection for sandfly vectors. Therefore, studies of the skin parasite-host relationship can contribute to the understanding of the infectious dissemination processes of parasites in the dermis and help to identify targets for diagnosis and treatment. Thus, the aim of this study was to evaluate the association of anatomical vascular differences and Leishmania-induced vascular morphological changes with clinical signs and parasite load by analyzing the ear and abdominal skin from dogs naturally infected with L. infantum. Paired samples of ear and abdominal skin from L. infantum-positive dogs (n = 26) were submitted for histological and immunohistochemistry analyses. The ear skin samples showed a more intense and more diffusely distributed granulomatous inflammatory reaction, a higher number and larger diameter of blood vessels, increased parasite load, higher expression of VEGF+ (vascular endothelial growth factor) and MAC 387+ (calprotectin) recently infiltrating cells, and more intense collagen disruption compared to the abdominal skin samples. Intracellular amastigotes were observed in blood vessels and inside endothelial cells and were diffusely distributed throughout the dermis in the ear skin samples. The NOS2/MAC387+ cell ratio was lower in the ear skin samples than in those of the abdomen, suggesting that in the ear dermis, the inflammatory infiltrate was less capable of producing NO and thereby control the parasite load. Together, these findings indicate how parasites and immune cells are distributed in the skin and suggest an important role for dermal vascularization in cellular influx and thereby in parasite dissemination through the skin of naturally infected dogs.

Anti-Leishmania major Properties of Nuphar lutea (Yellow Water Lily) Leaf Extracts and Purified 6,6' Dihydroxythiobinupharidine (DTBN).

Cutaneous leishmaniasis (CL) is a zoonotic disease, manifested as chronic ulcers, potentially leaving unattractive scars. There is no preventive vaccination or optimal medication against leishmaniasis. Chemotherapy generally depends upon a small group of compounds, each with its own efficacy, toxicity, and rate of drug resistance. To date, no standardized, simple, safe, and highly effective regimen for treating CL exists. Therefore, there is an urgent need to develop new optimal medication for this disease. Sesquiterpen thio-alkaloids constitute a group of plant secondary metabolites that bear great potential for medicinal uses. The nupharidines found in Nuphar lutea belong to this group of compounds. We have previously published that Nuphar lutea semi-purified extract containing major components of nupharidines has strong anti-leishmanial activity in vitro. Here, we present in vivo data on the therapeutic benefit of the extract against Leishmania major (L. major) in infected mice. We also expanded these observations by establishing the therapeutic effect of the extract-purified nupharidine 6,6'-dihydroxythiobinupharidine (DTBN) in vitro against promastigotes and intracellular amastigotes as well as in vivo in L. major-infected mice. The results suggest that this novel anti-parasitic small molecule has the potential to be further developed against Leishmania.

Improved Treatment Outcome Following the Use of a Wound Dressings in Cutaneous Leishmaniasis Lesions.

Leishmaniasis, caused by Leishmania parasites, is a neglected tropical disease and Cutaneous Leishmaniasis (CL) is the most common form. Despite the associated toxicity and adverse effects, Meglumine antimoniate (MA) remains the first-choice treatment for CL in Brazil, pressing the need for the development of better alternatives. Bacterial NanoCellulose (BNC), a biocompatible nanomaterial, has unique properties regarding wound healing. In a previous study, we showed that use of topical BNC + systemic MA significantly increased the cure rate of CL patients, compared to treatment with MA alone. Herein, we performed a study comparing the combination of a wound dressing (BNC or placebo) plus systemic MA versus systemic MA alone, in CL caused by Leishmania braziliensis. We show that patients treated with the combination treatment (BNC or placebo) + MA showed improved cure rates and decreased need for rescue treatment, although differences compared to controls (systemic MA alone) were not significant. However, the overall time-to-cure was significantly lower in groups treated with the combination treatment (BNC+ systemic MA or placebo + systemic MA) in comparison to controls (MA alone), indicating that the use of a wound dressing improves CL treatment outcome. Assessment of the immune response in peripheral blood showed an overall downmodulation in the inflammatory landscape and a significant decrease in the production of IL-1a (p < 0.05) in patients treated with topical BNC + systemic MA. Our results show that the application of wound dressings to CL lesions can improve chemotherapy outcome in CL caused by L. braziliensis.

Green-synthesized chitosan­carbon dot nanocomposite as turn-on aptasensor for detection and quantification of Leishmania infantum parasite.

Leishmania is one of the most common diseases between human and animals, caused by Leishmania infantum parasite. Here, we have developed an ultra-selective turn-on fluorescent probe based on an aptamer and Chitosan-CD nanocomposite. The CD used in this study were synthesized using Quercus cap extract and a microwave-assisted approach. The Chitosan-CD nanocomposite was optimized using several microscopic and spectroscopic techniques to possess a bright fluorescence emission before adding aptamer and totally quenched fluorescence after addition of aptamer. The designed probe was proficient in the detection and quantification Leishmania infantum parasite by selective targeting of poly(A) binding protein (PABP) on the surface of the parasite. The designed fluorescent biosensor with high sensitivity, excellent selectivity, and a limit of detection (LOD) of 94 cells/mL of the Leishmania infantum parasite as well as a linear response in the ranges of 188-750 cells/mL and 3000-6000 cells/mL (R2 ≥ 0.98 for both linear ranges). Additionally, the selectivity of the designed probe was evaluated in the presence of different pathogenic species such as Trypanosoma brucei parasite and Staphylococcus aureus bacteria, as well as LiIF2α and LiP2a and BSA proteins as interference substances. The results of this study shows that using Chitosan-CD nanocomposite is a great strategy for developing selective turn-on probes with extraordinary accuracy and sensitivity in identifying Leishmania infantum parasite, especially in the early stages of the disease, and it is promising for the future clinical applications.

Designing Antitrypanosomal and Antileishmanial BODIPY Derivatives: A Computational and In Vitro Assessment.

Leishmaniasis and Human African trypanosomiasis pose significant public health threats in resource-limited regions, accentuated by the drawbacks of the current antiprotozoal treatments and the lack of approved vaccines. Considering the demand for novel therapeutic drugs, a series of BODIPY derivatives with several functionalizations at the meso, 2 and/or 6 positions of the core were synthesized and characterized. The in vitro activity against Trypanosoma brucei and Leishmania major parasites was carried out alongside a human healthy cell line (MRC-5) to establish selectivity indices (SIs). Notably, the meso-substituted BODIPY, with 1-dimethylaminonaphthalene (1b) and anthracene moiety (1c), were the most active against L. major, displaying IC50 = 4.84 and 5.41 µM, with a 16 and 18-fold selectivity over MRC-5 cells, respectively. In contrast, the mono-formylated analogues 2b and 2c exhibited the highest toxicity (IC50 = 2.84 and 6.17 µM, respectively) and selectivity (SI = 24 and 11, respectively) against T. brucei. Further insights on the activity of these compounds were gathered from molecular docking studies. The results suggest that these BODIPYs act as competitive inhibitors targeting the NADPH/NADP+ linkage site of the pteridine reductase (PR) enzyme. Additionally, these findings unveil a range of quasi-degenerate binding complexes formed between the PRs and the investigated BODIPY derivatives. These results suggest a potential correlation between the anti-parasitic activity and the presence of multiple configurations that block the same site of the enzyme.

Corrigendum: Clinical validation of circulating immune complexes for use as a diagnostic marker of canine leishmaniosis.

[This corrects the article DOI: 10.3389/fvets.2024.1368929.].

Arg40 is critical for stability and activity of Adenylosuccinate lyase; a purine salvage enzyme from Leishmania donovani.

Purine salvage enzymes have been of significant interest in anti-Leishmanial drug development due to the parasite's critical dependence on this pathway for the supply of nucleotides in the absence of a de novo purine synthesis pathway. Adenylosuccinate lyase (ADSL) one of the key enzymes in this pathway is a homo-tetramer, where the active site is formed by residues from three distinct subunits. Analysis of the subunit interfaces of LdADSL, revealed a conserved Arg40 forming critical inter-subunit interactions and also involved in substrate binding. We hypothesized that mutating this residue can affect both the structural stability and activity of the enzyme. In our study, we used biochemical, biophysical, and computational simulation approaches to understand the structural and functional role of Arg40 in LdADSL. We have replaced Arg40 with an Ala and Glu using site directed mutagenesis. The mutant enzymes were similar to wild-type enzyme in secondary structure and subunit association. Thermal shift assays indicated that the mutations affected the protein stability. Both mutants showed decreased specific activities in both forward and reverse directions with significantly weakened affinities towards succinyl-adenosine monophosphate (SAMP). The mutations resulted in changes in C3 loop conformation and D3 domain rotation. Consequently, the orientation of the active site amino acid residues changed resulting in compromised activity and stability. Studies so far have majorly focused on the ADSL active site for designing drugs against it. Our work indicates that an alternative inhibitory mechanism for the enzyme can be designed by targeting the inter-subunit interface.

Leishmania donovani mitogen-activated protein kinases as a host-parasite interaction interface.

Leishmaniasis, a major globally re-emerging neglected tropical disease, has a restricted repertoire of chemotherapeutic options due to a narrow therapeutic index, drug resistance, or patient non-compliance due to toxicity. The disease is caused by the parasite Leishmania that resides in two different forms in two different environments: as sessile intracellular amastigotes within mammalian macrophages and as motile promastigotes in sandfly gut. As mitogen-activated protein kinases (MAPKs) play important roles in cellular differentiation and survival, we studied the expression of Leishmania donovani MAPKs (LdMAPKs). The homology studies by multiple sequence alignment show that excepting LdMAPK1 and LdMAPK2, all thirteen other LdMAPKs share homology with human ERK and p38 isoforms. Expression of LdMAPK4 and LdMAPK5 is less in avirulent promastigotes and amastigotes. Compared to miltefosine-sensitive L. donovani parasites, miltefosine-resistant parasites have higher LdMAPK1, LdMAPK3-5, LdMAPK7-11, LdMAPK13, and LdMAPK14 expression. IL-4-treatment of macrophages down-regulated LdMAPK11, in virulent amastigotes whereas up-regulated LdMAPK5, but down-regulated LdMAPK6, LdMAPK12-15, expression in avirulent amastigotes. IL-4 up-regulated LdMAPK1 expression in both virulent and avirulent amastigotes. IFN-γ-treatment down-regulated LdMAPK6, LdMAPK13, and LdMAPK15 in avirulent amastigotes but up-regulated in virulent amastigotes. This complex profile of LdMAPKs expression among virulent and avirulent parasites, drug-resistant parasites, and in amastigotes within IL-4 or IFN-γ-treated macrophages suggests that LdMAPKs are differentially controlled at the host-parasite interface regulating parasite survival and differentiation, and in the course of IL-4 or IFN-γ dominated immune response.

Investigating the effect of parasites (toxoplasma gondii RH strain, Leishmania major (MRHO/IR/75/ER), and hydatid cyst) antigens on Alzheimer's disease: An in vivo evaluation.

This study aimed to investigate the effects of parasite antigens on Alzheimer's symptoms in animal model. Alzheimer's model was induced in Wistar rats using Amyloid-beta peptide, and treated with parasite crude antigens from T. gondii RH strain, L. major (MRHO/IR/75/ER), and HC. Spectrophotometry and real-time PCR were used to evaluate the oxidative stress levels, antioxidant enzyme activity, and gene expression of NLRP3, IL-8, IL-1ß, and Caspase-1. Histological assays were performed to investigate structural changes in the hippocampus. Apoptosis was analyzed using an Annexin V Apoptosis by Flow cytometer. The levels of total oxidant, antioxidant, and SOD increased in the Alzheimer's group compared with the control group, but these factors were lower in the L. major group. The apoptosis in the treated groups was lower compared to the Alzheimer's group. IL-8 expression was significantly higher in all Alzheimer's groups, but decreased in the HC and L. major treated group compared to Alzheimer's. IL-1ß and Caspase-1 expression were similarly increased in all groups compared with the control group, but decreased in the antigen-treated groups compared with Alzheimer's. NLRP3 expression was increased in all groups compared with the control group, with lower expression in HC group, but significantly decreased in L. major group compared with Alzheimer's. In histological results, only L. major group could play a therapeutic role in pathological damage of the hippocampus. The results showed that parasite antigens, specifically L. major antigens, may have neuroprotective effects that reduce oxidative stress, apoptosis, and histopathological changes in response to AD in animal model.

Leishmania spp. amastigotes surrounding sensory nerve fibers in human painless skin ulcers: Evidence of pathogen-neuron proximity and absence of neuronal apoptosis.

In this present study, carried out between November 2020 and July 2023 at Londrina's University Hospital, patients with active lesions of cutaneous leishmaniasis (CL) were analyzed regarding pain perception and anatomopathological aspects of the ulcers. Pain was assessed using a numerical rating scale (NRS) to compare five patients diagnosed with CL with four control patients diagnosed with vascular skin ulcers. Histopathological evaluations were used to investigate the nociceptor neuron-Leishmania interface. Patients with CL ulcers reported less pain compared to patients with vascular ulcers (2.60 ± 2.30 and 7.25 ± 0.95, respectively, p = 0.0072). Histopathology evidenced Leishmania spp. amastigote forms nearby sensory nerve fibers in profound dermis. Schwann cells marker (S100 protein) was detected, and caspase-3 activation was not evidenced in the in the nerve fibers of CL patients' samples, suggesting absence of apoptotic activity in nerve endings. Additionally, samples taken from the active edge of the lesion were negative for bacilli acid-alcohol resistant (BAAR), which excludes concomitant leprosy, in which painless lesions are also observed. Thus, the present data unveil for the first time anatomopathological and microbiological details of painless ulcers in CL patients, which has important clinical implications for a better understanding on the intriguing painless clinical characteristic of CL.

A Modular Cloning Toolkit for the production of recombinant proteins in Leishmania tarentolae.

Modular Cloning (MoClo) is based on libraries of standardized genetic parts that can be directionally assembled via Golden Gate cloning in one-pot reactions into transcription units and multigene constructs. Here, a team of bachelor students established a MoClo toolkit for the protist Leishmania tarentolae in the frame of the international Genetically Engineered Machine (iGEM) competition. Our modular toolkit is based on a domesticated version of a commercial LEXSY expression vector and comprises 34 genetic parts encoding various affinity tags, targeting signals as well as fluorescent and luminescent proteins. We demonstrated the utility of our kit by the successful production of 16 different tagged versions of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein in L. tarentolae liquid cultures. While highest yields of secreted recombinant RBD were obtained for GST-tagged fusion proteins 48 h post induction, C-terminal peptide tags were often degraded and resulted in lower yields of secreted RBD. Fusing secreted RBD to a synthetic O-glycosylation SP20 module resulted in an apparent molecular mass shift around 10 kDa. No disadvantage regarding the production of RBD was detected when the three antibiotics of the LEXSY system were omitted during the 48-h induction phase. Furthermore, the successful purification of secreted RBD from the supernatant of L. tarentolae liquid cultures was demonstrated in pilot experiments. In summary, we established a MoClo toolkit and exemplified its application for the production of recombinant proteins in L. tarentolae.

Visceral leishmaniasis as a rare cause of granulomatous hepatitis.

Visceral leishmaniasis (VL) is a potentially fatal infection caused by species of Leishmania. It is characterized by fever, weight loss, anemia, and enlargement of the spleen and liver. Hepatitis due to VL is one of the causes of granulomatous hepatitis rarely described in the literature. It poses a problem of differential diagnosis with other causes, notably infectious and autoimmune. Hence the need for a global clinical, biological, and histological evaluation to orientate this entity, especially in endemic countries like ours. In the present case study, a 2-year 8-month-old boy was diagnosed with VL and treated with meglumine antimoniate; the evolution was marked after 2 months by the persistence of a large liver; laboratory results showed elevated liver functions and anemia. A liver biopsy was performed, and the histological findings confirmed the diagnosis of granulomatous hepatitis.

Analysis of the current risk of Leishmania infantum transmission for domestic dogs in Spain and Portugal and its future projection in climate change scenarios.

Canine leishmaniosis, caused by the protozoan parasite Leishmania infantum, is a cosmopolitan vector-borne zoonosis, transmitted principally by Phlebotomus perniciosus in Spain and Portugal, where it is considered an endemic disease. Ecoinformatics tools such as ecological niche models (ENM) have been successfully tested to model the distribution of the risk of infection of different parasitosis as they take into account environmental variables vital for their survival. The risk map proposed in this study combines the potential distribution of Ph. perniciosus in the Iberian Peninsula and the calculation of the infection rate of the parasite in the vector to model the risk of contracting the disease in a more realistic way. In fact, this weighting strategy improves the predictive power of the resulting model (R2 = 0.42, p = < 0.01) compared to the Ph. perniciosus ENM model alone (R2 = 0.13, p > 0.05). The places with the highest risk of transmission are the southwest and central peninsular area, as well as the Mediterranean coast, the Balearic Islands and the Ebro basin, places where the ideal habitat of Ph. perniciosus and the infection rate is also high. In the case of future projections under climate change scenarios, an increase in the risk of infection by L. infantum can be observed in most of the territory (4.5% in 2040, 71.6% in 2060 and 63% in 2080), mainly in the northern part of the peninsula. The use of ENMs and their weighting with the infection rate in Ph. perniciosus is a useful tool in predicting the risk of infection for L. infantum in dogs for a given area. In this way, a more complete model can be obtained to facilitate prevention and control.