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Background: The BBSome is an octameric protein complex crucial for ciliary transport, though it also participates in multiple other cellular processes. These diverse functions may result from the co-option of its ancestral roles. Studying the BBSome in flagellated protists can provide insights into these ancestral functions and their subsequent adaptations. Methods: We examined the functions of the BBSome (LpBBS1 and LpBBS2) in Lotmaria passim, a monoxenous trypanosomatid parasite infecting honey bees. The phenotypes resulting from depletion of LpBBS1 using the auxin-inducible degron system and disruption of LpBBS2 were characterized. Results: Parasites deficient in LpBBS2 are smaller and less motile compared with wild-type. Although intraflagellar transport of a marker membrane protein is only mildly impaired, its association with lipid rafts is significantly disrupted in the mutants. This suggests that the BBSome is essential for maintaining lipid raft integrity in L. passim. Transcriptomic comparisons between wild-type and LpBBS2-deficient parasites reveal that the BBSome may also influence processes related to metabolism, membrane localization of specific proteins, DNA repair, microtubules, and mitochondria. Conclusions: In contrast to Leishmania mexicana, the BBSome in L. passim is crucial for efficient infection of the honey bee gut, demonstrating that its cellular functions vary between related trypanosomatid species. The BBSome is likely an adaptor that links multiple proteins in a species-specific manner under various cellular contexts. [ABSTRACT FROM AUTHOR]

Conserved histone methyltransferases of the DOT1 family are involved in replication regulation, cell cycle progression, stage differentiation, and gene regulation in trypanosomatids. However, the specific functions of these enzymes depend on the host evasion strategies of the parasites. In this study, we investigated the role of DOT1B in Leishmania mexicana , focusing on life cycle progression and infectivity. In contrast to Trypanosoma brucei , in which DOT1B is essential for the differentiation of mammal-infective bloodstream forms to insect procyclic forms, L. mexicana DOT1B (Lmx DOT1B) is not critical for the differentiation of promastigotes to amastigotes in vitro. Additionally, there are no significant differences in the ability to infect or differentiate in macrophages or sand fly vectors between the Lmx DOT1B-depleted and control strains. These findings highlight the divergence of the function of DOT1B in these related parasites, suggesting genus-specific adaptations in the use of histone modifications for life cycle progression and host adaptation processes. [ABSTRACT FROM AUTHOR]

Leishmania spp. cause a wide range of human diseases, localized skin lesions, mucocutaneous and visceral infections. In the present study, the aim was to investigate the potential role of sanguinarine as a specific inhibitor of Leishmania PP2C that can induce apoptosis in the parasite. The results demonstrated that sanguinarine inhibits, in a dose-dependent mode at 72 h, the growth and phosphatase activity of both Leishmania major and Leishmania mexicana promastigotes. Therefore, all assays were performed from this time period onwards. TUNEL assay was used to identify apoptosis and indicated apoptosis in L. major and L. mexicana promastigotes. Similarly, Western blot assay showed that PARP, a DNA damage indicator molecule, was present in L. major and L. mexicana promastigotes incubated with the inhibitor. In addition, differential expression of the proapoptotic protein Bax and the antiapoptotic protein Bcl-2 was observed in both Leishmania species. Finally, the protein phosphatase PP2C expression was not affected, whereas p38 MAPK phosphorylation was increased in L. major promastigotes than in L. mexicana promastigotes. Therefore, sanguinarine proved to be an inhibitor of the growth and PP2C enzymatic activity of L. major and L. mexicana promastigotes, and with it, this inhibition induced apoptosis. [ABSTRACT FROM AUTHOR]

Macrophages represent a fundamental component of the innate immune system that play a critical role in detecting and responding to pathogens as well as danger signals. Leishmania spp. infections lead to a notable alteration in macrophage metabolism, whereby infected cells display heightened energy metabolism that is linked to the integrity of host mitochondria. However, little is known about how different species of Leishmania manipulate host metabolism. Here, we demonstrate that despite differences in their mechanisms for evading host immune responses, L. amazonensis and L. braziliensis induce comparable disruptions in key metabolic pathways. We found that infected macrophages exhibited an overall elevation in energy metabolism regardless of the parasite strain, evidenced by the elevation in glycolysis and oxygen consumption rates, along with increased proton leak and decreased ATP production. We also analyzed the effects of both Leishmania spp. strain infection on mitochondria function, further revealing that infected cells display heightened mitochondrial mass and membrane potential. To investigate the metabolic pathways required for Leishmania amastigotes to persist in BMDMs, we pre-treated cells with small molecule drugs that target major metabolic pathways, revealing that perturbations in several metabolic processes affected parasite survival in a strain-independent manner. Treatments with inhibitors of the oxidative phosphorylation and glycolysis substantially reduced parasite loads. Collectively, our findings suggest that L.amazonensis and L.braziliensis exploit host cell metabolic pathways similarly to survive in macrophages. Author summary: Our study delves into the intricate interplay between macrophages and Leishmania spp. infections, shedding light on how these parasites manipulate host cell metabolism. Despite variations in the mechanisms employed by different Leishmania species to evade immune responses, our findings reveal a striking similarity in their impact on host metabolic pathways. Infected macrophages consistently exhibit heightened energy metabolism, characterized by increased glycolysis and oxygen consumption rates, alongside alterations in mitochondrial function. Importantly, we demonstrate that targeting key metabolic pathways with drugs significantly affects parasite survival, offering promising avenues for therapeutic interventions against leishmaniasis. By elucidating these complex metabolic processes in a clear and accessible manner, our research not only deepens our understanding of host-parasite interactions but also holds significant implications for the development of novel treatment approaches. We aim to bridge the gap between scientific knowledge and its broader implications for both scientists and non-scientists alike, advancing the fight against neglected tropical diseases. [ABSTRACT FROM AUTHOR]

Background: Cutaneous Leishmaniases (CL), highly endemic in Africa and Mediterranean region, are caused by different Leishmania parasite species. Accurate species identification is crucial for effective diagnosis, treatment, and control of these diseases, but traditionally relies on DNA-based methods. High Resolution Melting analysis PCR (HRM PCR) provides rapid results and precise differentiation based on nucleotide variations. We hypothesized that the Strumpellin gene of Leishmania could serve as an effective target for developing a HRM PCR method for the rapid and efficient detection and identification of Leishmania species in CL diagnosis. Methodology: The Strumpellin gene was investigated in Trypanosomatidae family using bioinformatics and phylogenetic approaches to explore its evolutionary conservation and suitability for HRM PCR. HRM PCR target and primers were selected and validated on 73 different Leishmania DNAs. The analytical limit of detection was assessed, and the performance for detecting and identifying parasites in 38 cutaneous lesions aspirates was compared to Direct Examination (DE) and ITS1-PCR RFLP methods. Findings: The developed HRM PCR assay accurately identified promastigote DNAs of L. donovani/L. infantum, L. major, L. aethiopica, L. turanica, L. arabica, L. tarentolae and 3 genotypes of L. tropica. Differentiation was achievable with as little as a single nucleotide difference occurring within or between species. HRM profile interpretations were consistent with sequencing results of the HRM PCR target and identification by ITS1-PCR RFLP. The assay could detect the equivalent of 24 Leishmania parasites. In a small-scale sample, we brought the HRM could detect and identify Leishmania in human cutaneous samples. In comparison to DE, the sensitivity and specificity of the HRM PCR assay on human cutaneous samples were 88% and 84.62%, respectively, while the ITS1-PCR assay evaluation parameters were 84% and 92.31%. Statistical analysis confirmed good correlation among the three tests, with both molecular methods providing congruent parasite identification. Notably, in three samples, only the HRM PCR assay was able to assign them to L. infantum or L. tropica. Conclusions: The HRM PCR assay is a valuable tool for the detection and identification of Old World Leishmania species. Its integration into molecular diagnostic algorithms for CL or in eco-epidemiological studies holds promise for improving disease management and control. Author summary: Cutaneous Leishmaniases (CL) are a group of diseases considered as the most Neglected Tropical Diseases. CL present a complex epidemiology situation in many countries over the World and particularly in Africa and Middle East regions with the proven implication of different Leishmania species causing pleomorphic clinical presentations. Migrations, climate change and variations, agricultural developments are factors complicating eco-epidemiology and driving emerging trends. Leishmania species identification is pivotal for etiological diagnosis and patients' management, and to establish adequate control measures. Molecular techniques are the most accurate methods for detection and identification of Leishmania parasites due to their higher sensitivity and specificity. HRM PCR assay constitutes a valuable test for rapid and reliable diagnosis of infectious diseases including CL. The present study aimed at the development of a HRM PCR targeting the Strumpellin gene, which would be useful for accurate CL diagnosis by detecting and identifying causal Leishmania parasites. Bioinformatic analyses explored conserved regions flanking interspecific polymorphisms to select HRM PCR target fragment and primer pair and develop the assay. We confirmed through this study the relevance of this target and of our new method for CL diagnosis by proving the principle of using it to simultaneously detect and identify Leishmania parasites in human samples, thereby its potential to improve CL-patient management, surveillance and control in well-equipped health centers. [ABSTRACT FROM AUTHOR]

The study of extracellular vesicles has become an incredibly important field of study, but the inherent heterogeneity of these vesicles continues to make their study challenging. The genetic variability and well-documented protocols for the growth and vesicle isolation from Leishmania parasites provide a unique opportunity to compare the heterogeneity of different populations secreted by Leishmania clones. Leishmania mexicana was cultured on solid SDM agar plates and 8 clonal colonies were selected. The EVs collected from the liquid cultures of these 8 clones were assessed by NTA, TEM, and proteomic analysis. We found that all 8 clonal L. mexicana cultures were visually indistinguishable from each other and had similar growth rate, and these physical similarities extended to their EVs. However, proteomic analysis reveals that the EVs collected have unique protein profiles compared to each other and EVs isolated from a heterogeneous liquid culture of L. mexicana. We selected 3 clonal EVs for further mouse infection experiments and found that EVs from CL7 L. mexicana consistently caused reduced footpad swelling in C57BL6 mice footpads compared to EVs from CL1, CL8, and heterogenous L. mexicana. This trend was not observed when infecting Balb/C mice and C57BL6 with the parasites alone, with only CL1 L. mexicana causing significantly increased infection in Balb/c mice. Our results together show that EVs isolated from different clonal colonies of L. mexicana have distinct differences in protein cargo which can lead to varying outcomes on Leishmania infection. Further evaluation will be needed to determine the underlying mechanisms behind this and verify that differences observed in infectivity are directly caused by variations between our L. mexicana clones, especially genetic sequencing and immunoblotting to validate our results. [ABSTRACT FROM AUTHOR]

Background/Objective: Leishmaniasis is the second deadliest parasitic disease in the world, after malaria, with an estimated 1.6 million new cases each year. While cutaneous leishmaniasis can result in permanent scars from lesions after treatment, the mucocutaneous and visceral diseases can result in life-altering and life-threatening complications. Accurate species diagnosis is critical for treatment and follow-up, and while PCR-based diagnostics can provide sensitive parasite detection and species identification, they are slow, expensive, and not suitable for low-resource settings. In this publication, we describe our efforts to develop a simple, affordable, and instrument-free Leishmania DNA diagnostic that can be used in both high-tech settings and the field. Methods: Computational biology was utilized to design region-targeted RPA oligos and the corresponding CRISPR guides for the detection of all Leishmania species as well as the specific identification of L. (V.) panamensis as a predictor of mucocutaneous disease. Then, we executed systematic approaches for parasite lysis, RPA amplification of DNA, and fluorescent CRISPR crRNA detection. Results: We have demonstrated the ability to detect single-digit parasites without compromising the specificity in identifying single species as the proof of concept for a point-of-care diagnostic. Individual assays were carried out in succession, culminating in an unquenched fluorescent signal quantifiable over negative control. Conclusions: The described work is the foundation which will be implemented into a three-track [all Leishmania, mucocutaneous or visceral only, and a human positive control] assay that we plan to utilize in a Funnel Adapted Sensing Tube (FAST) single use, instrument-free, and affordable diagnostic. [ABSTRACT FROM AUTHOR]

Leishmaniasis is recognized as a serious public health problem in Brazil and around the world. The limited availability of drugs for treatment, added to the diversity of side effects and the emergence of resistant strains, shows the importance of research focused on the development of new molecules, thus contributing to treatments. Therefore, this work aimed to identify leishmanicidal compounds using a peptide dimerization strategy, as well as to understand their mechanisms of action. Herein, it was demonstrated that the dimerization of the peptide TSHa, (TSHa)2K, presented higher potency and selectivity than its monomeric form when evaluated against Leishmania mexicana and Leishmania amazonensis. Furthermore, these compounds are capable of inhibiting the parasite cysteine protease, an important target explored for the development of antileishmanial compounds, as well as to selectively interact with the parasite membranes, as demonstrated by flow cytometry, permeabilization, and fluorescence microscopy experiments. Based on this, the identified molecules are candidates for use in in vivo studies with animal models to combat leishmaniasis. [ABSTRACT FROM AUTHOR]

One of the main components of innate defense against invasive parasites is oxidative stress, which is brought on by reactive oxygen species (ROS). On the other hand, oxidative stressors serve two purposes: free radicals aid in the elimination of pathogens, but they can also set off inflammation, which leads to tissue damage. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that controls the expression of numerous genes involved in the body's defense against oxidative stress brought on by aging, inflammation, tissue damage, and other pathological consequences. From cutaneous to visceral forms, Leishmania parasites invade macrophages and cause a wide range of human pathologies. Leishmania parasites have a wide range of adaptive mechanisms that disrupt several macrophage functions by altering host signaling pathways. An increasing amount of data are corroborating the idea that one of the primary antioxidant routes to counteract this oxidative burst against parasites is NRF2 signaling, which also interferes with immune responses. The nature and potency of the host immune response, as well as interactions between the invading Leishmania spp., will ascertain the course of infection and the parasites' eventual survival or eradication. The molecular processes via which Nrf2 coordinates such intricate networks comprising various pathways remain to be completely understood. In light of NRF2's significant contribution to oxidative stress, we examine the NRF2 antioxidant pathway's activation mechanism in Leishmania infection in this review. Thus, this review will examine the relationship between Nrf2 signaling and leishmaniasis, as well as explore potential therapeutic strategies for modifying this system. [ABSTRACT FROM AUTHOR]

Background: Several new species of Leishmania have recently emerged in Europe, probably as the result of global changes and increased human migration from endemic areas. In this study, we tested whether two sand fly species, the Western Mediterranean Phlebotomus perniciosus and the Eastern Mediterranean P. tobbi, are competent vectors of L. donovani, L. major and L. martiniquensis. Methodology/principal findings: Sand flies were infected through the chick skin membrane using Leishmania species and strains of various geographical origins. Leishmania infections were evaluated by light microscopy and qPCR, and the representation of morphological forms was assessed from Giemsa-stained gut smears. Neither P. perniciosus nor P. tobbi supported the development of L. martiniquensis, but L. major and L. donovani in both species survived defecation of blood meal remnants, colonized the stomodeal valve and produced metacyclic stages. The results with L donovani have shown that infection rates in sand flies can be strain-specific; therefore, to determine vector competence or refractoriness, it is optimal to test at least two strains of Leishmania. Conclusions, significance: Both sand fly species tested are potential vectors of L. donovani and L. major in Mediterranean area. However, further studies will be needed to identify European vectors of L. martiniquensis and to test the ability of other European sand fly species to transmit L. major, L. donovani, L. tropica and L. infantum. Author summary: Leishmaniases are serious human and animal diseases caused by protozoa of the genus Leishmania (Kinetoplastida: Trypanosomatidae), transmitted by blood-feeding insects–phlebotomine sand flies (Diptera: Psychodidae). They are mainly distributed in tropical and subtropical regions, but recently the ranges of the vectors have spread more into temperate regions. In Europe, species of Leishmania that were previously absent have emerged, mainly due to the mass migration of the refugees from endemic areas. It is therefore important to know whether local sand flies can transmit these "new" Leishmania species. In our study, we tested two widely distributed sand fly species, P. perniciosus and P. tobbi. In both of them, L. major and L. donovani completed their development, thus P. perniciosus and P. tobbi are potential vectors of these parasites. The third Leishmania species tested, L. martiniquensis, which has been repeatedly recorded in central Europe, did not survive in P. perniciosus and P. tobbi; its vectors have yet to be confirmed. [ABSTRACT FROM AUTHOR]

Promastigote Leishmania mexicana have a complex cell division cycle characterised by the ordered replication of several single-copy organelles, a prolonged S phase and rapid G2 and cytokinesis phases, accompanied by cell cycle stage-associated morphological changes. Here we exploit these morphological changes to develop a high-throughput and semi-automated imaging flow cytometry (IFC) pipeline to analyse the cell cycle in live L. mexicana. Firstly, we demonstrate that, unlike several other DNA stains, Vybrant™ DyeCycle™ Orange (DCO) is non-toxic and enables quantitative DNA imaging in live promastigotes. Secondly, by tagging the orphan spindle kinesin, KINF, with mNeonGreen, we describe KINF's cell cycle-dependent expression and localisation. Then, by combining manual gating of DCO DNA intensity profiles with automated masking and morphological measurements of parasite images, visual determination of the number of flagella per cell, and automated masking and analysis of mNG:KINF fluorescence, we provide a newly detailed description of L. mexicana promastigote cell cycle events that, for the first time, includes the durations of individual G2, mitosis and post-mitosis phases, and identifies G1 cells within the first 12 minutes of the new cell cycle. Our custom-developed masking and gating scheme allowed us to identify elusive G2 cells and to demonstrate that the CDK-inhibitor, flavopiridol, arrests cells in G2 phase, rather than mitosis, providing proof-of-principle of the utility of IFC for drug mechanism-of-action studies. Further, the high-throughput nature of IFC allowed the close examination of promastigote cytokinesis, revealing considerable flexibility in both the timing of cytokinesis initiation and the direction of furrowing, in contrast to the related kinetoplastid parasite, Trypanosoma brucei and many other cell types. Our new pipeline offers many advantages over traditional methods of cell cycle analysis such as fluorescence microscopy and flow cytometry and paves the way for novel high-throughput analysis of Leishmania cell division. [ABSTRACT FROM AUTHOR]

Effective and less risky tools for monitoring the anthropophilic sandfly Lutzomyia (Tricholateralis) cruciata (Coquillett) (Diptera: Psychodidae) are scarce, even though this sandfly species may act as a vector of Leishmania mexicana Biagi (Kinetoplastida: Trypanosomatidae) in the Americas. We evaluated the effectiveness of 1-octen-3-ol, butyric acid, lactic acid, ammonium acetate, and cyclopentanone as attractants for the collection of L. cruciata in field conditions. Chemical lures were placed in CDC light traps installed within an urban forest. A total of 122 (87.8%) individuals of L. cruciata were captured, and 79.5% were females. Other sandfly species such as Micropygomyia cayennensis cayennensis (7.9%), Micropygomyia chiapanensis (3.6%), and Dampfomyia atulapai (0.7%) were also caught. A higher and significant proportion of L. cruciata females were attracted to all compounds evaluated as compared to the other three species. The compound 1-octen-3-ol was the most attractive compound and the only one that attracts females exclusively, followed by cyclopentanone, butyric acid, and ammonium acetate. The use of 1-octen-3-ol could be recommended as an additive to CDC light traps under field conditions. [ABSTRACT FROM AUTHOR]

Drug repositioning is an efficient strategy to search for new treatment alternatives that is especially valuable for neglected parasitic diseases such as leishmaniasis. Tamoxifen and raloxifene are selective estrogen receptor modulators (SERMs) that have shown antileishmanial activity. Clomiphene is a SERM structurally similar to tamoxifen, whose antileishmanial potential is unknown. That is why the objective of the present work was to evaluate its antileishmanial activity in vitro and in vivo in comparison with tamoxifen. The inhibitory effect against promastigotes of L. amazonensis, L. major, and L. mexicana was evaluated for both compounds, as well as the cytotoxicity against mouse peritoneal macrophages, the growth inhibitory activity in intracellular amastigotes of L. mexicana, and the in vivo activity in mice experimentally infected with L. mexicana. Clomiphene was about twice as active as tamoxifen against both promastigotes and intracellular amastigotes, with IC50 values of 1.7–3.3 µM for clomiphene and 2.9–6.4 µM for tamoxifen against all three species of promastigotes and 2.8 ± 0.2 µM and 3.7 ± 0.3 µM, respectively, against L. mexicana amastigotes. Clomiphene structurally affected several parasite organelles in a concentration-dependent fashion, leading to the death of both promastigotes and intracellular amastigotes. Interestingly, the macrophage host cell did not appear damaged by any of the clomiphene concentrations tested. With oral administration at 20 mg/kg for 14 days, both compounds showed similar effects in terms of reducing the growth of the lesions, as well as the weight of the lesions and the parasite load at the end of the follow-up period. The results showed the potential of SERMs as antileishmanial drugs and support further testing of clomiphene and other compounds of this pharmacological group. [ABSTRACT FROM AUTHOR]

Mexico's Yucatan Peninsula is an endemic area of cutaneous leishmaniasis, locally known as the chiclero's ulcer, and Mayan traditional medicine which refers to the use of Thouinia paucidentata Radlk, known as k'an chuunup. Aqueous and organic leaves extracts were evaluated against promastigotes and amastigotes of Leishmania mexicana. Toxicity tests of extracts were performed using Vero and J774A.1 macrophage cell lines. The composition of the most active extracts was analysed by GC-MS. The n-hexane and ethyl acetate extracts showed potent anti-Leishmania activity against the promastigote form, and remarkably, n-hexane extract exhibited potent activity against the amastigote form. Both extracts showed low toxicity on Vero both not on J774A.1 cells. Analysis of both bioactive extracts identified as more abundant compounds, germacrene D-4-ol and thunbergen in n-hexane, and thunbergol in ethyl acetate extracts. Our study presents T. paucidentata as anti-Leishmania phytomedicine supporting its medicinal use and contributes to the understanding of its phytochemical composition. [ABSTRACT FROM AUTHOR]

Background: Localized cutaneous leishmaniasis (LCL) is a serious public health problem in Southern Mexico. Six species of Phlebotominae (Diptera: Psychodidae) have been found to be infected with Leishmania (Leishmania) mexicana, the causative agent of LCL in the region. However, little is known about the biology and potential participation of Psathyromyia cratifer in the Leishmania transmission cycle in Mexico, and the Americas. The present study provides evidence of temporal infection caused by Leishmania in Psathyromyia cratifer as well as data on its population dynamics in a LCL endemic area during the well-known transmission cycle of Leishmania in Southern Mexico. Methodology/Principal findings: Individual specimens of Psathyromyia cratifer were collected in four sites over the course of five months (from November 2020 through March 2021) using animal-baited, human-baited, and light traps. The temporal activity pattern (month + hour) of Psathyromyia cratifer was assessed along with its relationship with environmental variables. Moreover, Leishmania DNA and blood meals were analyzed and detected in female sand flies. This evidenced an infection rate ranging from 8% to 83%, and the record of Homo sapiens and Ototylomys phyllotis as blood hosts of this sand fly species. High abundances of these sand flies in human-baited traps were recorded which revealed the marked anthropophilic behavior of Psathyromyia cratifer. As regards the transmission dynamics of the parasite within the region, it was observed that the potential highest epidemiological risk for Leishmania transmission by Psathyromyia cratifer occurred during the months of January and March. Conclusion: This is the first contribution ever made to both the population dynamic and the temporal Leishmania prevalence patterns in Psathyromyia cratifer. The resulting findings suggest that this sand fly specimen is the sixth potential vector of L. (L.) mexicana in Southern Mexico. Nonetheless, various biology, behavior, and ecology strands are yet to be addressed. The latter, to determine the role it plays in the transmission dynamics of the parasite within the region, and other areas of the country. Author summary: Psathyromyia cratifer is a species of Neotropical dipteran belonging to one of the most medically important groups worldwide wise, the Phlebotomine sand flies. Some sand fly species transmit Leishmania parasites to humans, which leads to one of the most common but neglected tropical diseases known as leishmaniasis. The data on life history traits and involvement of this species in the transmission of Leishmania, is yet limited. This research furnishes evidence about population dynamics and temporal Leishmania infection patterns in female Pa. cratifer specimens collected from four sites part of a transmission area in Yucatan, Mexico. Our findings suggest that females of this species are attracted to and feed on humans as well as an important Leishmania reservoir in the region. Although not all sites recorded infected individuals, the infection and temporal abundance of this species in most sites, were similar. Our findings suggest that this species could be a suspected vector of the parasite in the region, however, it is important to continue with incrimination studies that incorporate other sources of evidence. [ABSTRACT FROM AUTHOR]

Intracellular pathogens that replicate in host myeloid cells have devised ways to inhibit the cell's killing machinery. Pyroptosis is one of the host strategies used to reduce the pathogen replicating niche and thereby control its expansion. The intracellular Leishmania parasites can survive and use neutrophils as a silent entry niche, favoring subsequent parasite dissemination into the host. Here, we show that Leishmania mexicana induces NLRP1- and caspase-1-dependent Gasdermin D (GSDMD)-mediated pyroptosis in neutrophils, a process critical to control the parasite-induced pathology. In the absence of GSDMD, we observe an increased number of infected dermal neutrophils two days post-infection. Using adoptive neutrophil transfer in neutropenic mice, we show that pyroptosis contributes to the regulation of the neutrophil niche early after infection. The critical role of neutrophil pyroptosis and its positive influence on the regulation of the disease outcome was further demonstrated following infection of mice with neutrophil-specific deletion of GSDMD. Thus, our study establishes neutrophil pyroptosis as a critical regulator of leishmaniasis pathology. Author summary: Leishmaniases are neglected infectious diseases with around 1 million new cases reported per year. Neutrophils are rapidly and massively recruited to the site of infection. Several Leishmania species including Leishmania mexicana, inhibit the host neutrophil antimicrobial response, using these cells as an entry shelter. Here, we discover a novel host defense mechanism against Leishmania mexicana that controls neutrophil presence at the onset of infection. The inflammasomes are multiprotein signaling complexes that detect infection and induce pyroptotic cell death. We show that the NLRP1 inflammasome is important in the control of lesion development and parasite burden. We identify NLRP1-driven GSDMD pyroptosis in neutrophils as critical in the control of the early neutrophil pool, impacting the outcome of infection. Taken together, we show that neutrophil pyroptosis is an essential player in cutaneous leishmaniasis. [ABSTRACT FROM AUTHOR]

Phlebotomine sand flies are vectors of the protozoan parasite Leishmania spp. Although the intestinal microbiota is involved in a wide range of biological and physiological processes and has the potential to alter vector competence, little is known about the impact of host species and environment on the gut microbiome. To address this issue, a comparative analysis of the microbiota of sand fly vector populations of Leishmania major and L. tropica in a mixed focus of cutaneous leishmaniasis in Tunisia was performed. Bacterial 16S rRNA gene amplification and Illumina MiSeq sequencing were used to characterize and compare the overall bacterial and fungal composition of field-collected sand flies: Phlebotomus papatasi, Ph. perniciosus, Ph. riouxi, and Ph. sergenti. Thirty-eight bacterial genera belonging to five phyla were identified in 117 female specimens. The similarities and differences between the microbiome data from different samples collected from three collections were determined using principal coordinate analysis (PCoA). Substantial variations in the bacterial composition were found between geographically distinct populations of the same sand fly species, but not between different species at the same location, suggesting that the microbiota content was structured according to environmental factors rather than host species. These findings suggest that host phylogeny may play a minor role in determining the insect gut microbiota, and its potential to affect the transmission of the Leishmania parasite appear to be very low. These results highlight the need for further studies to decode sand fly Leishmania-microbiota interactions, as even the same bacterial species, such as Enterococcus faecalis, can exert completely opposite effects when confronted with different pathogens within various host insects and vice versa. Author summary: Leishmania major and L. tropica are the causative agents of cutaneous leishmaniasis in the Tataouine governorate of southeast Tunisia. In this study, the microbiota of Phlebotomus papatasi, Ph. perniciosus, Ph. riouxi, and Ph. sergenti, which were caught from three diverse ecotypes, were analyzed using 16S rRNA gene amplification and Illumina MiSeq sequencing. In total, 117 Leishmania-free and blood-non-fed female specimens were used individually in this study to avoid any bias caused by pooling samples. We found that the gut microbiota was mainly controlled by environmental habitats and other factors, as remarkable differences in microbiota composition within the same species collected from the same location was revealed. Moreover, the results suggested that host phylogeny may play a minor role in determining the insect gut microbiota, and the potential of the host factor to affect the transmission of the Leishmania parasite appeared to be very low. A better understanding of microorganism-insect-gut bacterial interactions is critical, as the interactions between pathogens and gut microbiota do not adhere to a consistent pattern of synergy or antagonism. Even the same bacterial species, such as Enterococcus faecalis, can exert opposing effects when confronted with different pathogens in various host insects [ABSTRACT FROM AUTHOR]

Cutaneous leishmaniasis is atypical in Sri Lanka because Leishmania donovani, which typically causes visceral disease, is the causative agent. The origins of recently described hybrids between L. donovani and other Leishmania spp. usually responsible for cutaneous leishmaniasis remain unknown. Other endemic dermotropic Leishmania spp. have not been reported in Sri Lanka. Genome analysis of 27 clinical isolates from Sri Lanka and 32 Old World Leishmania spp. strains found 8 patient isolates clustered with L. tropica and 19 with L. donovani. The L. tropica isolates from Sri Lanka shared markers with strain LtK26 reported decades ago in India, indicating they were not products of recent interspecies hybridization. Because L. tropica was isolated from patients with leishmaniasis in Sri Lanka, our findings indicate L. donovani is not the only cause of cutaneous leishmaniasis in Sri Lanka and potentially explains a haplotype that led to interspecies dermotropic L. donovani hybrids. [ABSTRACT FROM AUTHOR]

Conserved histone methyltransferases of the DOT1 family are involved in replication regulation, cell cycle progression, stage differentiation, and gene regulation in trypanosomatids. However, the specific functions of these enzymes depend on the host evasion strategies of the parasites. In this study, we investigated the role of DOT1B in Leishmania mexicana, focusing on life cycle progression and infectivity. In contrast to Trypanosoma brucei, in which DOT1B is essential for the differentiation of mammal-infective bloodstream forms to insect procyclic forms, L. mexicana DOT1B (LmxDOT1B) is not critical for the differentiation of promastigotes to amastigotes in vitro. Additionally, there are no significant differences in the ability to infect or differentiate in macrophages or sand fly vectors between the LmxDOT1B-depleted and control strains. These findings highlight the divergence of the function of DOT1B in these related parasites, suggesting genus-specific adaptations in the use of histone modifications for life cycle progression and host adaptation processes.

LeishGEM is a genome-wide functional annotation community resource for Leishmania mexicana , where deletion mutant growth in vitro and in vivo is measured and protein localisation is determined by endogenous tagging and LOPIT-DC (localisation of organelle proteins by isotope tagging with differential centrifugation) spatial proteomics. Data are being made available pre-publication via http://leishgem.org which allows data-driven identification of the mechanisms for Leishmania parasitism. [ABSTRACT FROM AUTHOR]

Directed enzyme prodrug therapy (DEPT) strategies show promise in mitigating chemotherapy side effects during cancer treatment. Among these, the use of immobilized enzymes on solid matrices as prodrug activating agents (IDEPT) presents a compelling delivery strategy, offering enhanced tumor targeting and reduced toxicity. Herein, we report a novel IDEPT strategy by employing a His-tagged Leishmania mexicana type I 2′-deoxyribosyltransferase (His-LmPDT) covalently attached to glutaraldehyde-activated magnetic iron oxide nanoparticles (MIONPs). Among the resulting derivatives, PDT-MIONP3 displayed the most favorable catalyst load/retained activity ratio, prompting its selection for further investigation. Substrate specificity studies demonstrated that PDT-MIONP3 effectively hydrolyzed a diverse array of 6-oxo and/or 6-amino purine 2′-deoxynucleosides, including 2-fluoro-2′-deoxyadenosine (dFAdo) and 6-methylpurine-2′-deoxyribose (d6MetPRib), both well-known prodrugs commonly used in DEPT. The biophysical characterization of both MIONPs and PDT-MIONPs was conducted by TEM, DLS, and single particle ICPMS techniques, showing an ideal nanosized range and a zeta potential value of −47.9 mV and −78.2 mV for MIONPs and PDT-MIONPs, respectively. The intracellular uptake of MIONPs and PDT-MIONPs was also determined by TEM and single particle ICPMS on HeLa cancer cell lines and NIH3T3 normal cell lines, showing a higher intracellular uptake in tumor cells. Finally, the selectivity of the PDT-MIONP/dFAdo IDEPT system was tested on HeLa cells (24 h, 10 µM dFAdo), resulting in a significant reduction in tumoral cell survival (11% of viability). Based on the experimental results, PDT-MIONP/dFAdo presents a novel and alternative IDEPT strategy, providing a promising avenue for cancer treatment. [ABSTRACT FROM AUTHOR]

In the Americas, L. infantum (syn. chagasi) is the main cause of human visceral leishmaniasis. The role of neutrophils as part of the innate response to Leishmania spp. infection is dubious and varies according to the species causing the infection. Global expression of coding RNAs, microRNAs and long non-coding RNAs changes as part of the immune response against pathogens. Changes in mRNA and non-coding RNA expression resulting from infection by Leishmania spp. are widely studied in macrophages, but scarce in neutrophils, the first cell to encounter the trypanosomatid, especially following infection by L. infantum. Herein, we aimed to understand the expression patterns of coding and non-coding transcripts during acute in vitro infection of human neutrophils by L. infantum. We isolated neutrophils from whole blood of healthy male donors (n = 5) and split into groups: 1) infected with L. infantum (MOI = 5:1), and 2) uninfected controls. After 3 hours of exposure of infected group to promastigotes of L. infantum, followed by 17 hours of incubation, total RNA was extracted and total RNA-Seq and miRNA microarray were performed. A total of 212 genes were differentially expressed in neutrophils following RNA-Seq analysis (log2(FC)±0.58, FDR≤0.05). In vitro infection with L. infantum upregulated the expression of 197 and reduced the expression of 92 miRNAs in human neutrophils (FC±2, FDR≤0.01). Lastly, 5 downregulated genes were classified as lncRNA, and of the 10 upregulated genes, there was only 1 lncRNA. Further bioinformatic analysis indicated that changes in the transcriptome and microtranscriptome of neutrophils, following in vitro infection with L. infantum, may impair phagocytosis, apoptosis and decrease nitric oxide production. Our work sheds light on several mechanisms used by L. infantum to control neutrophil-mediated immune response and identifies several targets for future functional studies, aiming at the development of preventive or curative treatments for this prevalent zoonosis. Author summary: Visceral leishmaniasis is a neglected tropical disease that causes fever, weight loss, anemia and swelling of liver and spleen. About 2500 cases are reported annually in the Americas, with a high mortality rate. Understanding how the immune system of people with visceral leishmaniasis responds to this parasite is essential for the development of preventive and curative methods. In order to understand how gene expression is modulated during visceral leishmaniasis, we infected in vitro cultured human neutrophils, the first immune cells to be recruited in this infection, with Leishmania infantum, the protozoan that causes visceral leishmaniasis in the Americas. Next, we measured the expression of coding RNAs, responsible for the production of proteins required for an effective immune response, and of non-coding RNAs, able to control these coding RNAs, thus helping or hindering host response to infection. Analysis of coding and non-coding RNAs points to an attempt by the parasite to modulate the transcriptome of host cells, influencing the host's response to infection. Our work identifies several targets for future functional studies, aiming at the development of preventive or curative treatments for this prevalent zoonosis. [ABSTRACT FROM AUTHOR]

Equids may be infected by zoonotic Leishmania spp., including Leishmania infantum, in regions where canine leishmaniasis (CanL) is endemic, and Leishmania martiniquensis, which has been reported in horses from Central Europe. This study was designed to evaluate the occurrence of both Leishmania spp. among equids living in CanL endemic areas of Italy, as well as to identify dipteran vectors from the same habitats. From March to October 2023, blood, serum and tissue samples from skin lesions were collected from equids (n = 98; n = 56 donkeys and n = 42 horses) living in Italy, as well as sand flies and biting midges. Blood samples (n = 98) and skin lesions (n = 56) were tested for Leishmania spp. by conventional and real time PCRs and sera were tested by immunofluorescence antibody tests (IFAT) for both L. infantum and L. martiniquensis. Insects were morphologically identified, and female specimens (n = 268 sand flies, n = 7 biting midges) analyzed for Leishmania DNA, as well as engorged sand flies (n = 16) for blood-meal detection. Two animals with skin lesions (i.e., one donkey and one horse) scored positive for Leishmania spp. DNA, and 19 animals (i.e., 19.4%; n = 13 donkeys and n = 6 horses) were seropositive for L. infantum, with five of them also for L. martiniquensis. Most seropositive animals had no dermatological lesions (i.e., 68.4%) while both animals molecularly positive for Leishmania spp. scored seronegative. Of the 356 sand flies collected, 12 females (i.e., n = 8 Sergentomyia minuta; n = 3 Phlebotomus perniciosus, n = 1 Phlebotomus perfiliewi) were positive for Leishmania spp. DNA, and one out of seven biting midges collected was DNA-positive for L. infantum. Moreover, engorged sand flies scored positive for human and equine DNA. Data suggest that equids living in CanL endemic areas are exposed to Leishmania spp., but their role in the circulation of the parasite needs further investigations. Author summary: Horses and donkeys seem to be infected by Leishmania infantum, causative agent of canine leishmaniasis (CanL), which is one of the most important zoonotic vector-borne disease. Moreover, Leishmania martiniquensis, causing visceral and mucocutaneus leishmaniasis in humans, was reported in horses from central Europe. Therefore, we designed this study to investigate the circulation of both Leishmania zoonotic species among equids living in CanL endemic areas and to access the presence of dipteran vectors from the same habitats. Horses and donkeys (n = 98) were sampled and analyzed for Leishmania spp., as well as sand flies and biting midges. Nineteen equids (19.4%) scored seropositive by immunofluorescence antibody test for L. infantum and one donkey and one horse, presenting skin lesions, scored molecularly positive for Leishmania spp. Twelve sand flies and one biting midge were positive for Leishmania spp. DNA. Overall, data herein presented suggest that equids living in CanL endemic areas are exposed to Leishmania spp., potentially contributing to the circulation of the parasite. [ABSTRACT FROM AUTHOR]

Introduction: Casiopeina III-ia (CasIII-ia) is a mixed chelate copper (II) compound capable of interacting with free radicals generated in the respiratory chain through redox reactions, producing toxic reactive oxygen species (ROS) that compromise the viability of cancer cells, bacteria and protozoa. Due to its remarkable effect on protozoa, this study evaluated the effect of CasIII-ia on Leishmania mexicana amastigotes and its potential use as a treatment for cutaneous leishmaniasis in the murine model. Methods: We analyzed the leishmanicidal effect of CasIII-ia on L. mexicana amastigotes and on their survival in bone marrow-derived macrophages. Furthermore, we evaluated the production of ROS in treated parasites and the efficacy of CasIII-ia in the treatment of mice infected with L. mexicana. Results: Our results show that CasIII-ia reduces parasite viability in a dose-dependent manner that correlates with increased ROS production. A decrease in the size of footpad lesions and in parasite loads was observed in infected mice treated with the intraperitoneal administration of CasIII-ia. Conclusions: We propose CasIII-ia as a potential drug for the treatment of cutaneous leishmaniasis. [ABSTRACT FROM AUTHOR]

The risk of vector-borne disease transmission has been increasing due to climate changes observed in recent decades. While most studies on vector-borne diseases focus on identifying their etiological agents, fewer address the vectors responsible for transmission. These vectors are typically arthropods, such as mosquitoes, ticks, or sandflies. In the case of sandflies, information about these often-overlooked vectors is limited and difficult to find. Sandflies are the primary vectors of leishmaniasis, a widespread zoonotic disease caused by protozoa of the genus Leishmania. However, they are also responsible for transmitting other diseases, such as viral infections (e.g., Toscana virus infection, Pappataci fever) and bacterial infections (e.g., bartonellosis). In Romania, eight species of sandflies have been reported in various regions, contributing to an increased risk of disease transmission within the country. These species include Ph. perfiliewi, Ph. neglectus, Ph. balcanicus, Ph. papatasi, Ph. alexandri, Ph. sergenti, Ph. longiductus, and Sergentomyia minuta. Regarding the pathogenicity of sandfly species in Romania, indigenous cases of Toscana virus infection, sandfly fever, and leishmaniasis have been identified in the country. Among the listed species, those responsible for transmitting leishmaniasis are Ph. neglectus, Ph. balcanicus, Ph. papatasi, Ph. perfiliewi, Ph. alexandri, and Ph. sergenti. Ph. perfiliewi is responsible for transmitting Toscana virus, while Ph. papatasi is responsible for transmitting the phleboviruses that causes sandfly fever. This review highlights the pathogenic potential of sandfly species identified in Romania, their ecology, and their significance for both public and animal health. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis is a vector-born neglected parasitic disease belonging to the genus Leishmania. Out of the 30 Leishmania species, 21 species cause human infection that affect the skin and the internal organs. Around, 700,000 to 1,000,000 of the newly infected cases and 26,000 to 65,000 deaths are reported worldwide annually. The disease exhibits three clinical presentations, namely, the cutaneous, muco-cutaneous and visceral Leishmaniasis which affects the skin, mucosal membrane and the internal organs, respectively. The relapsing behavior of the disease limits its diagnosis and treatment efficiency. The common diagnostic approaches follow subjective, error-prone, repetitive processes. Despite, an ever pressing need for an accurate detection of Leishmaniasis, the research conducted so far is scarce. In this regard, the main aim of the current research is to develop an artificial intelligence based detection tool for the Leishmaniasis from the Geimsa-stained microscopic images using deep learning method. Methods: Stained microscopic images were acquired locally and labeled by experts. The images were augmented using different methods to prevent overfitting and improve the generalizability of the system. Fine-tuned Faster RCNN, SSD, and YOLOV5 models were used for object detection. Mean average precision (MAP), precision, and Recall were calculated to evaluate and compare the performance of the models. Results: The fine-tuned YOLOV5 outperformed the other models such as Faster RCNN and SSD, with the MAP scores, of 73%, 54% and 57%, respectively. Conclusion: The currently developed YOLOV5 model can be tested in the clinics to assist the laboratorists in diagnosing Leishmaniasis from the microscopic images. Particularly, in low-resourced healthcare facilities, with fewer qualified medical professionals or hematologists, our AI support system can assist in reducing the diagnosing time, workload, and misdiagnosis. Furthermore, the dataset collected by us will be shared with other researchers who seek to improve upon the detection system of the parasite. The current model detects the parasites even in the presence of the monocyte cells, but sometimes, the accuracy decreases due to the differences in the sizes of the parasite cells alongside the blood cells. The incorporation of cascaded networks in future and the quantification of the parasite load, shall overcome the limitations of the currently developed system. [ABSTRACT FROM AUTHOR]

Herein we report the design and the synthesis of a library of new and more hydrophilic bisindole analogues based on our previously identified antileishmanial compound URB1483 that failed the preliminary in vivo test. The novel bisindoles were phenotypically screened for efficacy against Leishmania infantum promastigotes and simultaneously for toxicity on human macrophage-like THP-1 cells. Among the less toxic compounds, eight bisindoles showed IC50 below 10 μM. The most selective compound 1h (selectivity index = 10.1, comparable to miltefosine) and the most potent compound 2c (IC50 = 2.7 μM) were tested for their efficacy on L. infantum intracellular amastigotes. The compounds also demonstrated their efficacy in the in vitro infection model, showing IC50 of 11.1 and 6.8 μM for 1h and 2c, respectively. Moreover, 1h showed a better toxicity profile than the commercial drug miltefosine. For all these reasons, 1h could be a possible new starting point for hydrophilic antileishmanial agents with low cytotoxicity on human macrophage-like cells. [ABSTRACT FROM AUTHOR]

Objectives: The objective of this study was to analyze the fatty acid content, antioxidant, and antimicrobial activities of Argemone mexicana seed oil growing wild in north East Ethiopia. Materials and Methods: Oils of A mexicana L. were obtained by means of Screw press from seeds. Methyl esters were derived from the oily mixtures by trans-esterification process and were analyzed by GC/ FID and GC/MS systems. This oil was investigated for antioxidant activity using a DPPH radical-scavenging assay and was also tested against a panel of microorganisms. Results: Linolenic acid (49.00%) and oleic acid (28.91%) were the most abundant fatty acids in leaves and stems, respectively. The oil showed moderate to highest antimicrobial activity against Bacillus subtilis, Candida albicans, Shigella dysentery, Staphylococcus aureus, Pseudomonas aeroginosa, and Escherichia coli. The oil also demonstrated the highest antioxidant activity with the percentage of inhibition of 92.5% at a concentration of 1.5 mg/mL, and its IC50 and AAI were 22.4 µg/mL and 4.93 µg/mL, respectively. Conclusion: The results obtained from the present study indicated that the oil of A mexicana seed oil contained a high source of polyunsaturated fatty acids. These results also showed that the extracted oil from this plant has significant antimicrobial and antioxidant activities. [ABSTRACT FROM AUTHOR]

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Background It is well-documented that using immunosuppressive drugs such as corticosteroids or cytokine blockers in treating coronavirus disease 2019 (COVID-19) increases the risk of co-infections. Here we systematically summarized the cases of COVID-19-associated parasitic infections (CAPIs) in Iran. Methods From 19 February 2020 to 10 May 2023, all studies on Iranian patients suffering from CAPIs were collected from several databases using a systematic search strategy. Results Of 540 records, 11 studies remained for data extraction. In this research, most of the studies were related to Lophomonas and Toxoplasma. Of 411 cases of CAPIs, toxoplasmosis (385 [93.7%]) had the highest rate of infection among Iranian patients, followed by blastocystosis (15 [3.6%]), fascioliasis (4 [0.97%]), leishmaniasis (3 [0.7%]), lophomoniasis (3 [0.7%]) and strongyloidiasis (1 [0.2%]). In general, Blastocystis enhanced diarrhoea in patients with COVID-19. Lophomonas, Toxoplasma and Strongyloides increased the severity of COVID-19, but Fasciola decreased its intensity. Patients with a history of cutaneous leishmaniasis showed mild symptoms of COVID-19. Also, patients with a prior history of hydatid cysts were not affected by COVID-19. Conclusions Due to the similar symptoms of some parasitic diseases and COVID-19 and immunosuppressive treatment regimens in these patients that may cause the reactivation or recurrence of parasitic infections, early diagnosis and treatment are required. [ABSTRACT FROM AUTHOR]

Background: The transient receptor potential vanilloid 1 (TRPV1) is well-established in neuronal function, yet its role in immune reactions remains enigmatic. The conflicting data on its inflammatory role, suggesting both pro-inflammatory and anti-inflammatory effects upon TRPV1 stimulation in immune cells, adds complexity. To unravel TRPV1 immunomodulatory mechanisms, we investigated how the TRPV1 agonist capsaicin influences lipopolysaccharide (LPS)-induced pro-inflammatory macrophage phenotypes. Results: Changes in the surface molecules, cytokine production, and signaling cascades linked to the phenotype of M1 or M2 macrophages of the J774 macrophage cell line and bone marrow-derived macrophages, treated with capsaicin before or after the LPS-induced inflammatory reaction were determined. The functional capacity of macrophages was also assessed by infecting the stimulated macrophages with the intracellular parasite Leishmania mexicana. Conclusion: Our findings reveal that TRPV1 activation yields distinct macrophage responses influenced by the inflammatory context. LPS pre-treatment followed by capsaicin activation prompted increased calcium influx, accompanied by a shift toward an anti-inflammatory M2b-like polarization state. [ABSTRACT FROM AUTHOR]

The infectious inoculum of a sand fly, apart from its metacyclic promastigotes, is composed of factors derived from both the parasite and the vector. Vector-derived factors, including salivary proteins and the gut microbiota, are essential for the establishment and enhancement of infection. However, the type and the number of bacteria egested during salivation is unclear. In the present study, sand flies of Phlebotomus papatasi were gathered from three locations in hyperendemic focus of zoonotic cutaneous leishmaniasis (ZCL) in Isfahan Province, Iran. By using the forced salivation assay and targeting the 16S rRNA barcode gene, egested bacteria were characterized in 99 (44%) out of 224 sand flies. Culture-dependent and culture-independent methods identified the members of Enterobacter cloacae and Spiroplasma species as dominant taxa, respectively. Ten top genera of Spiroplasma, Ralstonia, Acinetobacter, Reyranella, Undibacterium, Bryobacter, Corynebacterium, Cutibacterium, Psychrobacter, and Wolbachia constituted >80% of the saliva microbiome. Phylogenetic analysis displayed the presence of only one bacterial species for the Spiroplasma, Ralstonia, Reyranella, Bryobacter and Wolbachia, two distinct species for Cutibacterium, three for Undibacterium and Psychrobacter, 16 for Acinetobacter, and 27 for Corynebacterium, in the saliva. The abundance of microbes in P. papatasi saliva was determined by incorporating the data on the read counts and the copy number of 16S rRNA gene, about 9,000 bacterial cells, per sand fly. Both microbiological and metagenomic data indicate that bacteria are constant companions of Leishmania, from the intestine of the vector to the vertebrate host. This is the first forced salivation experiment in a sand fly, addressing key questions on infectious bite and competent vectors. Author summary: Female sand flies salivate during feeding on vertebrate blood and natural sugars. During salivation, they may release microorganisms associated with the salivary glands and digestive tract, i.e. viruses, bacteria, and other pathogens, into the feeding substrates. However, the type and the number of bacteria egested during salivation is unclear. Here, by sing the forced salivation experiment and targeting the 16S rRNA barcode gene, egested bacteria were characterized in sand flies. Culture-dependent and culture-independent methods identified the members of Enterobacter cloacae and Spiroplasma species as dominant taxa, respectively. The abundance of microbes in the saliva of each P. papatasi sand fly was determined to be around 9,000 bacterial cells. The lack of a vaccine and the failure of treatment in leishmaniasis provide many motivations to better understand the factors cause the inflammatory response. The findings of this study can improve our insight into measuring the effect of vector-derived bacteria on the improvement or deterioration of leishmaniasis. [ABSTRACT FROM AUTHOR]

Leishmania, the dixenous trypanosomatid parasites, are the causative agents of leishmaniasis currently divided into four subgenera: Leishmania, Viannia, Sauroleishmania, and the recently described Mundinia, consisting of six species distributed sporadically all over the world infecting humans and/or animals. These parasites infect various mammalian species and also cause serious human diseases, but their reservoirs are unknown. Thus, adequate laboratory models are needed to enable proper research of Mundinia parasites. In this complex study, we compared experimental infections of five Mundinia species (L. enriettii, L. macropodum, L. chancei, L. orientalis, and four strains of L. martiniquensis) in three rodent species: BALB/c mouse, Chinese hamster (Cricetulus griseus) and steppe lemming (Lagurus lagurus). Culture-derived parasites were inoculated intradermally into the ear pinnae and progress of infection was monitored for 20 weeks, when the tissues and organs of animals were screened for the presence and quantity of Leishmania. Xenodiagnoses with Phlebotomus duboscqi were performed at weeks 5, 10, 15 and 20 post-infection to test the infectiousness of the animals throughout the experiment. BALB/c mice showed no signs of infection and were not infectious to sand flies, while Chinese hamsters and steppe lemmings proved susceptible to all five species of Mundinia tested, showing a wide spectrum of disease signs ranging from asymptomatic to visceral. Mundinia induced significantly higher infection rates in steppe lemmings compared to Chinese hamsters, and consequently steppe lemmings were more infectious to sand flies: In all groups tested, they were infectious from the 5th to the 20th week post infection. In conclusion, we identified two rodent species, Chinese hamster (Cricetulus griseus) and steppe lemming (Lagurus lagurus), as candidates for laboratory models for Mundinia allowing detailed studies of these enigmatic parasites. Furthermore, the long-term survival of all Mundinia species in steppe lemmings and their infectiousness to vectors support the hypothesis that some rodents have the potential to serve as reservoir hosts for Mundinia. Author summary: Leishmania parasites of the subgenus Mundinia are an emerging health and veterinary problem that should not be ignored. Being the most recent of all Leishmania described, many aspects of Mundinia biology are enigmatic. We have very scarce data on their life cycles and biology, thus proper laboratory research must be done to enable their better understanding. One of the most crucial parts of the life cycle of Leishmania is the development in the mammalian host. In the past, we worked on establishment of other laboratory models for the subgenus, but neither Arvicanthis, Mastomys, nor guinea pigs proved to be a good choice. Other authors performed experiments with BALB/c mice using various inoculation techniques, but they also failed. Here we describe the establishment of two new potential laboratory model species, Chinese hamsters and steppe lemmings, which proved to be susceptible to Mundinia and such findings will enable other scientists to continue in research of these parasites. [ABSTRACT FROM AUTHOR]

The multifactorial basis of therapeutic response can obscure the relation between antimicrobial drug susceptibility and clinical outcome. To discern the relationship between parasite susceptibility to meglumine antimoniate (SbV) and therapeutic outcome of cutaneous leishmaniasis, risk factors for treatment failure were considered in evaluating this relationship in ninety-one cutaneous leishmaniasis patients and corresponding clinical strains of Leishmania (Viannia) panamensis. Parasite susceptibility to 32 μg SbV/mL (plasma Cmax) was evaluated in primary human macrophages, PBMCs, and U937 macrophages. Early parasitological response to treatment was determined in lesions of a subgroup of patients, and pathogenicity of Sb-resistant and sensitive clinical strains was compared in BALB/c mice. Parasite survival in cell models and patient lesions was determined by qRT-PCR of Leishmania 7SLRNA transcript. Parasite loads in BALB/c mice were quantified by limiting dilution analysis. The disparate Sb-susceptibility of parasite subpopulations distinguished by isoenzyme profiles (zymodemes) was manifest in all cell models. Notably, Sb-resistance defined by parasite survival, was most effectively discerned in U937 macrophages compared with primary human host cells, significantly higher among strains from patients who failed treatment than cured and, significantly associated with treatment failure. Each unit increase in transformed survival rate corresponded to a 10.6-fold rise in the odds of treatment failure. Furthermore, treatment failure was significantly associated with naturally Sb-resistant zymodeme 2.3 strains, which also produced larger lesions and parasite burdens in BALB/c mice than Sb-sensitive zymodeme 2.2 strains. The confounding effect of host risk factors for treatment failure in discerning this association was evidenced in comparing strains from patients with and without the defined risk factors for treatment failure. These results establish the association of natural resistance to meglumine antimoniate with treatment failure, the importance of host risk factors in evaluating drug susceptibility and treatment outcome, and the clinical and epidemiological relevance of natural Sb-resistance in L. (V.) panamensis subpopulations. Author summary: The relevance of drug susceptibility of clinical strains of Leishmania measured in culture to the outcome of treatment is uncertain. We evaluated the relation between susceptibility of Leishmania isolated from cutaneous leishmaniasis patients to the widely used anti-leishmanial drug, meglumine antimoniate, and the outcome of treatment of the corresponding patients. We considered patient factors associated with treatment failure, such as age, adherence to treatment and concurrent medical conditions, in determining whether drug resistance among parasites isolated from these patients was linked to treatment failure. We found that drug-resistant Leishmania predominantly belong to a widely prevalent subpopulation that is biochemically distinguishable and more pathogenic in mice; and, that treatment failure was significantly more frequent (63%) among patients infected with these Sb-resistant Leishmania than patients infected with drug sensitive parasites (30%). Furthermore, tolerance/resistance to antimony was significantly higher among strains isolated from patients who failed treatment than those cured following treatment. The relationship between drug resistance and treatment failure was not perceived when only patients having the defined risk factors for failure were evaluated separately, evidencing the confounding effect of host factors and the importance of considering these when assessing the relevance of parasite drug resistance in the response to treatment. [ABSTRACT FROM AUTHOR]

Background: Innate immune responses against infectious agents can act as triggers of inflammatory diseases. On the other hand, various pathogens have developed mechanisms for the evasion of the immune response, based on an inhibition of innate immunity and inflammatory responses. Inflammatory diseases could thus be controlled through the administration of pathogens or pathogen-derived molecules, capable of interfering with the mechanisms at the basis of inflammation. In this framework, the NLRP3 inflammasome is an important component in innate antimicrobial responses and a major player in the inflammatory disease. Parasites of the genus Leishmania are master manipulators of innate immune mechanisms, and different species have been shown to inhibit inflammasome formation. However, the exploitation of pathogenic Leishmania species as blockers of NLRP3-based inflammatory diseases poses safety concerns. Methods: To circumvent safety issues associated with pathogenic parasites, we focused on Leishmania tarentolae, a species of Leishmania that is not infectious to humans. Because NLRP3 typically develops in macrophages, in response to the detection and engulfment microorganisms, we performed our experiments on a monocyte-macrophage cell line (THP-1), either wild type or knockout for ASC, a key component of NLRP3 formation, with determination of cytokines and other markers of inflammation. Results: L. tarentolae was shown to possess the capability of dampening the formation of NLRP3 inflammasome and the consequent expression of proinflammatory molecules, with minor differences compared to effects of pathogenic Leishmania species. Conclusion: The non-pathogenic L. tarentolae appears a promising pro-biotic microbe with anti-inflammatory properties or a source of immune modulating cellular fractions or molecules, capable of interfering with the formation of the NLRP3 inflammasome. [ABSTRACT FROM AUTHOR]

Addressing the challenges of quiescence and post-treatment relapse is of utmost importance in the microbiology field. This study shows that Leishmania infantum and L. donovani parasites rapidly enter into quiescence after an estimated 2–3 divisions in both human and mouse bone marrow stem cells. Interestingly, this behavior is not observed in macrophages, which are the primary host cells of the Leishmania parasite. Transcriptional comparison of the quiescent and non-quiescent metabolic states confirmed the overall decrease of gene expression as a hallmark of quiescence. Quiescent amastigotes display a reduced size and signs of a rapid evolutionary adaptation response with genetic alterations. Our study provides further evidence that this quiescent state significantly enhances resistance to treatment. Moreover, transitioning through quiescence is highly compatible with sand fly transmission and increases the potential of parasites to infect cells. Collectively, this work identified stem cells in the bone marrow as a niche where Leishmania quiescence occurs, with important implications for antiparasitic treatment and acquisition of virulence traits. Author summary: Quiescence and post-treatment relapse are crucial aspects of treatment failure across the microbiology field. This study shows that Leishmania infantum and L. donovani parasites rapidly enter into quiescence in both human and mouse bone marrow stem cells, but not in macrophages. Besides a reduced size, quiescent amastigotes show signs of a rapid evolutionary adaptation with notable genetic alterations. Transitioning through a quiescent state allows escape from treatment, efficient transmission by sand flies and the acquisition of an increased cellular infectivity. Transcriptional profiling of quiescent and non-quiescent parasites isolated from the stem cell niche confirmed a generalized transcriptional downregulation as a hallmark of quiescence. [ABSTRACT FROM AUTHOR]

Background: Accuracy of molecular tools for the identification of parasites that cause human cutaneous leishmaniasis (CL) could largely depend on the sampling method. Non-invasive or less-invasive sampling methods such as filter paper imprints and cotton swabs are preferred over punch biopsies and lancet scrapings for detection methods of Leishmania based on polymerase chain reaction (PCR) because they are painless, simple, and inexpensive, and of benefit to military and civilian patients to ensure timely treatment. However, different types of samples can generate false negatives and there is a clear need to demonstrate which sample is more proper for molecular assays. Methodology: Here, we compared the sensitivity of molecular identification of different Leishmania (Viannia) species from Peru, using three types of sampling: punch biopsy, filter paper imprint and lancet scraping. Different composite reference standards and latent class models allowed to evaluate the accuracy of the molecular tools. Additionally, a quantitative PCR assessed variations in the results and parasite load in each type of sample. Principal findings: Different composite reference standards and latent class models determined higher sensitivity when lancet scrapings were used for sampling in the identification and determination of Leishmania (Viannia) species through PCR-based assays. This was consistent for genus identification through kinetoplastid DNA-PCR and for the determination of species using FRET probes-based Nested Real-Time PCR. Lack of species identification in some samples correlated with the low intensity of the PCR electrophoretic band, which reflects the low parasite load in samples. Conclusions: The type of clinical sample can directly influence the detection and identification of Leishmania (Viannia) species. Here, we demonstrated that lancet scraping samples consistently allowed the identification of more leishmaniasis cases compared to filter paper imprints or biopsies. This procedure is inexpensive, painless, and easy to implement at the point of care and avoids the need for anesthesia, surgery, and hospitalization and therefore could be used in resource limited settings for both military and civilian populations. Author summary: Human cutaneous leishmaniasis affects low-income populations living in places far from health services. The early sampling and detection of the parasite are necessary for timely treatment, however there are no uniform sampling criteria, thus the sensitivity of molecular tests may vary due to various factors such as the type of sample, the time of the disease and the parasite load in the lesion. In this study, we compared the performance of three sampling methods for molecular identification of the genus and Leishmania (Viannia) species in Peru. Several analytical methods, including composite reference standards and latent class models, suggested that lancet scraping might be the best approach for parasite genus detection by kDNA-PCR and for parasite species determination by FRET probes-based Nested Real-Time PCR. [ABSTRACT FROM AUTHOR]

Background: With the current treatment options for visceral leishmaniasis (VL), recrudescence of the parasite is seen in a proportion of patients. Understanding parasite dynamics is crucial to improving treatment efficacy and predicting patient relapse in cases of VL. This study aimed to characterize the kinetics of circulating Leishmania parasites in the blood, during and after different antileishmanial therapies, and to find predictors for clinical relapse of disease. Methods: Data from three clinical trials, in which Eastern African VL patients received various antileishmanial regimens, were combined in this study. Leishmania kinetoplast DNA was quantified in whole blood with real-time quantitative PCR (qPCR) before, during, and up to six months after treatment. An integrated population pharmacokinetic-pharmacodynamic model was developed using non-linear mixed effects modelling. Results: Parasite proliferation was best described by an exponential growth model, with an in vivo parasite doubling time of 7.8 days (RSE 12%). Parasite killing by fexinidazole, liposomal amphotericin B, sodium stibogluconate, and miltefosine was best described by linear models directly relating drug concentrations to the parasite elimination rate. After treatment, parasite growth was assumed to be suppressed by the host immune system, described by an Emax model driven by the time after treatment. No predictors for the high variability in onset and magnitude of the immune response could be identified. Model-based individual predictions of blood parasite load on Day 28 and Day 56 after start of treatment were predictive for clinical relapse of disease. Conclusion: This semi-mechanistic pharmacokinetic-pharmacodynamic model adequately captured the blood parasite dynamics during and after treatment, and revealed that high blood parasite loads on Day 28 and Day 56 after start of treatment are an early indication for VL relapse, which could be a useful biomarker to assess treatment efficacy of a treatment regimen in a clinical trial setting. Author summary: After primary treatment of visceral leishmaniasis (VL), relapse of disease occurs in a proportion of patients. Understanding parasite dynamics is crucial to improving treatment efficacy and predicting disease relapse at an early stage. Leishmania kinetoplast DNA loads in blood from East African VL patients from three clinical trials, treated with five different treatment regimens, were used to develop a semi-mechanistic model to integrate in vivo parasite replication in the host, parasite clearance by different VL drug regimens, and suppression of parasite regrowth by the host immune system after treatment. This model deepened our understanding of the parasite-drug-host interaction and described the in vivo parasite growth rate in human for the first time. Moreover, the model revealed that high blood parasite loads on Day 28 and Day 56 after start of treatment are an early indication for VL relapse, which could serve as a biomarker to predict long-term clinical outcome, an important new tool for optimizing future VL treatment regimens. [ABSTRACT FROM AUTHOR]

Leishmania is a trypanosomatid parasite that causes skin lesions in its cutaneous form. Current therapies rely on old and expensive drugs, against which the parasites have acquired considerable resistance. Trypanosomatids are unable to synthesize purines relying on salvaging from the host, and nucleoside analogues have emerged as attractive antiparasitic drug candidates. 4-Methyl-7-β-D-ribofuranosyl-7H-pyrrolo[2,3-d]pyrimidine (CL5564), an analogue of tubercidin in which the amine has been replaced by a methyl group, demonstrates activity against Trypanosoma cruzi and Leishmania infantum. Herein, we investigated its in vitro and in vivo activity against L. amazonensis. CL5564 was 6.5-fold (P = 0.0002) more potent than milteforan™ (ML) against intracellular forms in peritoneal mouse macrophages, and highly selective, while combination with ML gave an additive effect. These results stimulated us to study the activity of CL5564 in mouse model of cutaneous Leishmania infection. BALB/c female and male mice infected by L. amazonensis treated with CL5564 (10 mg kg−1, intralesional route for five days) presented a >93% reduction of paw lesion size likely ML given orally at 40 mg kg−1, while the combination (10 + 40 mg kg−1 of CL5564 and ML, respectively) caused >96% reduction. The qPCR confirmed the suppression of parasite load, but only the combination approach reached 66% of parasitological cure. These results support additional studies with nucleoside derivatives. [ABSTRACT FROM AUTHOR]

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. Author summary: Leishmaniasis is a zoonosis caused by the intracellular parasite of the genus Leishmania. In the Mediterranean region, the human disease can be present mainly in two forms: cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL), which can be fatal if untreated. The clinical presentation of the disease depends on the parasite species and host characteristics. However, the molecular mechanisms underlying the development of the visceral form of leishmaniasis are not yet fully understood. In this study, we have determined the gene expression profile in cells infected by three Leishmania species (L. infantum, L. major, L. tropica) responsible for different forms of the disease. The results have allowed us to identify an expression profile (and the related pathways) shared by cells infected by all Leishmania species. Aside this common response, we also identified a transcriptional response distinguishing cells infected by L. infantum, L. major or L. tropica, as well as the relevant pathways in which these genes participate. Therefore, our results contribute to the knowledge of the host cell mechanisms that are activated along infection with different Leishmania species causing different forms of the disease and, therefore, may help develop new host-directed therapy for Leishmaniasis. [ABSTRACT FROM AUTHOR]

Twenty 2-(4-alkyloxyphenyl)-imidazolines and 2-(4-alkyloxyphenyl)-imidazoles were synthesized, with the former being synthesized in two steps by using MW and ultrasonication energy, resulting in good to excellent yields. Imidazoles were obtained in moderate yields by oxidizing imidazolines with MnO2 and MW energy. In response to the urgent need to treat neglected tropical diseases, a set of 2-(4-alkyloxyphenyl)- imidazolines and imidazoles was tested in vitro on Leishmania mexicana and Trypanosoma cruzi. The leishmanicidal activity of ten compounds was evaluated, showing an IC50 < 10 µg/mL. Among these compounds, 27–31 were the most active, with IC50 values < 1 µg/mL (similar to the reference drugs). In the evaluation on epimastigotes of T. cruzi, only 30 and 36 reached an IC50 < 1 µg/mL, showing better inhibition than both reference drugs. However, compounds 29, 33, and 35 also demonstrated attractive trypanocidal activities, with IC50 values < 10 µg/mL, similar to the values for benznidazole and nifurtimox. [ABSTRACT FROM AUTHOR]

Background Cutaneous leishmaniasis (CL), an emerging vector-borne ailment in Khyber Pakhtunkhwa (KPK), Pakistan, exhibits diverse spread patterns and outbreaks. Methods To comprehend its epidemiology and identify parasite species, we conducted an active survey on suspected CL cases (n=8845) in KPK. Results Microscopy and internal transcribed spacer-1 PCR–restriction fragment length polymorphism (RFLP) molecular techniques detected Leishmania spp. in blood samples. Phylogenetic analysis gauged genetic affinities with other areas. District Bannu displayed the highest CL impact (14.58%), while Swat had the lowest impact (4.33%) among cases. Annual blood examination rate, parasite incidence and slide positivity rate were 4.96 per 1000 people, 0.0233 and 0.047%, respectively. CL infections were prevalent in 1- to 20-y-olds, with males (57.17%) more vulnerable than females (42.82%). Single lesions occurred in 43.73% of patients, while 31.2% people had two lesions, 17.31% had three lesions and 7.74% had more than three lesions. Most had sand-fly exposure but lacked preventive measures like repellents and bed nets. Leishmania tropica was confirmed via RFLP analysis in amplified samples. Phylogenetic analysis unveiled genetic parallels between L. tropica of KPK and isolates from China, Iran, Afghanistan, India, Syria and Morocco. Conclusions Urgent comprehensive control measures are imperative. Early detection, targeted interventions and raising awareness of CL and sand-fly vectors are vital for reducing the disease's impact. International collaboration and monitoring are crucial to tackle Leishmania spp.'s genetic diversity and curtail its cross-border spread. [ABSTRACT FROM AUTHOR]

Parasitic protozoa of the genus Leishmania cycle between the phagolysosome of mammalian macrophages, where they reside as rounded intracellular amastigotes, and the midgut of female sand flies, which they colonize as elongated extracellular promastigotes. Previous studies indicated that protein kinase A (PKA) plays an important role in the initial steps of promastigote differentiation into amastigotes. Here, we describe a novel regulatory subunit of PKA (which we have named PKAR3) that is unique to Leishmania and most (but not all) other Kinetoplastidae. PKAR3 is localized to subpellicular microtubules (SPMT) in the cell cortex, where it recruits a specific catalytic subunit (PKAC3). Promastigotes of pkar3 or pkac3 null mutants lose their elongated shape and become rounded but remain flagellated. Truncation of an N-terminal formin homology (FH)-like domain of PKAR3 results in its detachment from the SPMT, also leading to rounded promastigotes. Thus, the tethering of PKAC3 via PKAR3 at the cell cortex is essential for maintenance of the elongated shape of promastigotes. This role of PKAR3 is reminiscent of PKARIβ and PKARIIβ binding to microtubules of mammalian neurons, which is essential for the elongation of dendrites and axons, respectively. Interestingly, PKAR3 binds nucleoside analogs, but not cAMP, with a high affinity similar to the PKAR1 isoform of Trypanosoma. We propose that these early-diverged protists have re-purposed PKA for a novel signaling pathway that spatiotemporally controls microtubule remodeling and cell shape. Author summary: Leishmania are single cell intracellular parasites of mammals, including humans. During their life, they cycle between macrophage phagolysosomes where they reside as rounded amastigotes, and the midgut of female sand flies, which they colonize as elongated promastigotes. Adaptation of Leishmania parasites to life in their mammalian host includes differentiation-associated changes in morphology from elongated promastigotes to rounded amastigotes. This study shows that a novel trypanosomatid-specific protein kinase A regulatory subunit (PKAR3) is essential for maintenance of the elongated shape of Leishmania promastigotes. PKAR3 is anchored to the subpellicular microtubule in the cell cortex by an N-terminal formin homology (FH)-like domain; where it recruits a developmentally regulated catalytic subunit (PKAC3). Exposure to the differentiation signal results in a rapid downregulation of PKA activity and subsequently cell rounding. Thus, the cAMP-independent PKAR3 appears to have been repurposed to serve an AKAP-like role in spatiotemporal control of morphogenesis, highlighting the evolutionary diversity of this family of signalling proteins. [ABSTRACT FROM AUTHOR]

Cutaneous leishmaniasis is of particular importance in southern Iran. This study aimed to investigate the infection of rodents with Leishmania major in an urban area of Fars Province, located in southern Iran. Rodents were trapped and samples from the liver, spleen, and skin were collected. Impression smears were prepared from these tissues and any skin lesions and were examined microscopically. In addition, a portion of the samples were preserved for subsequent DNA extraction. A total of 41 rodents belonging to three species were caught from 10 trapping stations in gardens or houses within the area. The caught rodent species were Rattus rattus (n = 25, 60.97%), Mus musculus (n = 15, 36.58%), and Meriones persicus (n = 1, 2.5%). Leishmania amastigotes were seen in the spleen tissue smear of 6 (2.43%) of the rodents, including 4 of R. rattus and 2 of M. musculus. Skin lesions were observed on the muzzles of two R. rattus and one M. musculus. Samples taken from these lesions tested positive for Leishmania infection. Leishmania DNA was detected in 18 (43.9%) rodents, including 11 R. rattus, 6 M. musculus, and one M. persicus, based on DNA sequencing of the ITS2 gene and PCR of the kDNA. Phylogenetic reconstruction revealed that the parasite infecting the rodents was L. major. The detection of Leishmania infection in these rodents in urban areas raises concerns about the urbanization of cutaneous leishmaniasis caused by L. major. This urbanization poses unique challenges for control and prevention efforts. [ABSTRACT FROM AUTHOR]

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 actinbinding 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. [ABSTRACT FROM AUTHOR]

Isocitrate dehydrogenase is an enzyme converting isocitrate to α-ketoglutarate in the canonical tricarboxylic acid (TCA) cycle. There are three different types of isocitrate dehydrogenase documented in eukaryotes. Our study points out the complex evolutionary history of isocitrate dehydrogenases across kinetoplastids, where the common ancestor of Trypanosomatidae and Bodonidae was equipped with two isoforms of the isocitrate dehydrogenase enzyme: the NADP+-dependent isocitrate dehydrogenase 1 with possibly dual localization in the cytosol and mitochondrion and NADP+-dependent mitochondrial isocitrate dehydrogenase 2. In the extant trypanosomatids, isocitrate dehydrogenase 1 is present only in a few species suggesting that it was lost upon separation of Trypanosoma spp. and replaced by the mainly NADP+-dependent cytosolic isocitrate dehydrogenase 3 of bacterial origin in all the derived lineages. In this study, we experimentally demonstrate that the omnipresent isocitrate dehydrogenase 2 has a dual localization in both mitochondrion and cytosol in at least four species that possess only this isoform. The apparent lack of the NAD+-dependent isocitrate dehydrogenase activity in trypanosomatid mitochondrion provides further support to the existence of the noncanonical TCA cycle across trypanosomatids and the bidirectional activity of isocitrate dehydrogenase 3 when operating with NADP+ cofactor instead of NAD+. This observation can be extended to all 17 species analyzed in this study, except for Leishmania mexicana , which showed only low isocitrate dehydrogenase activity in the cytosol. The variability in isocitrate oxidation capacity among species may reflect the distinct metabolic strategies and needs for reduced cofactors in particular environments. [ABSTRACT FROM AUTHOR]

Introduction: Leishmaniasis is a neglected disease with high prevalence and incidence in tropical and subtropical areas. Existing drugs are limited due to cost, toxicity, declining efficacy and unavailability in endemic places. Drug repurposing has established as an efficient way for the discovery of drugs for a variety of diseases. Purpose: The objective of the present work was testing the antileishmanial activity of thioridazine, an antipsychotic agent with demonstrated effect against other intracellular pathogens. Methods: The cytotoxicity for mouse peritoneal macrophages as well as the activity against Leishmania amazonensis, Leishmania mexicana and Leishmania major promastigotes and intracellular amastigotes, as well as in a mouse model of cutaneous leishmaniasis, were assessed. Results: Thioridazine inhibited the in vitro proliferation of promastigotes (50% inhibitory concentration—IC50—values in the range of 0.73 µM to 3.8 µM against L. amazonensis, L. mexicana and L. major) and intracellular amastigotes (IC50 values of 1.27 µM to 4.4 µM for the same species). In contrast, in mouse peritoneal macrophages, the 50% cytotoxic concentration was 24.0 ± 1.89 µM. Thioridazine inhibited the growth of cutaneous lesions and reduced the number of parasites in the infected tissue of mice. The dose of thioridazine that inhibited lesion development by 50% compared to controls was 23.3 ± 3.1 mg/kg and in terms of parasite load, it was 11.1 ± 0.97 mg/kg. Conclusions: Thioridazine was effective against the promastigote and intracellular amastigote stages of three Leishmania species and in a mouse model of cutaneous leishmaniasis, supporting the potential repurposing of this drug as an antileishmanial agent. [ABSTRACT FROM AUTHOR]

Immunization with various Leishmania species lacking centrin induces robust immunity against a homologous and heterologous virulent challenge, making centrin mutants a putative candidate for a leishmaniasis vaccine. Centrin is a calcium-binding cytoskeletal protein involved in centrosome duplication in higher eukaryotes and Leishmania spp. lacking centrin are unable to replicate in vivo and are non-pathogenic. We developed a centrin-deficient Leishmania braziliensis (LbCen−/−) cell line and confirmed its impaired survival following phagocytosis by macrophages. Upon experimental inoculation into BALB/c mice, LbCen−/− failed to induce lesions and parasites were rapidly eliminated. The immune response following inoculation with LbCen−/− was characterized by a mixed IFN-γ, IL-4, and IL-10 response and did not confer protection against L. braziliensis infection, distinct from L. major, L. donovani, and L mexicana centrin-deficient mutants. A prime-boost strategy also did not lead to a protective immune response against homologous challenge. On the contrary, immunization with centrin-deficient L. donovani (LdonCen−/−) cross-protected against L. braziliensis challenge, illustrating the ability of LdonCen−/− to induce the Th1-dominant protective immunity needed for leishmaniasis control. In conclusion, while centrin deficiency in L. braziliensis causes attenuation of virulence, and disrupts the ability to cause disease, it fails to stimulate a protective immune response. [ABSTRACT FROM AUTHOR]

This article discusses a surge in imported cases of American cutaneous leishmaniasis (ACL) from Mexico in 2023. ACL is caused by Leishmania (L.) mexicana and is endemic to the Yucatan peninsula in Mexico. The infection is transmitted through the bite of female sandflies, and most cases occur in men engaged in occupational activities in sylvatic areas. The article presents a case series of six patients diagnosed with imported ACL in Germany and France, highlighting the increase in cases compared to previous years. The rise in cases is likely linked to environmental changes, including the construction and deforestation associated with the 'Tren Maya' project in the Yucatan peninsula. [Extracted from the article]