GRASP negatively regulates the secretion of the virulence factor gp63 in Leishmania.

Metalloprotease-gp63 is a virulence factor secreted by Leishmania. However, secretory pathway in Leishmania is not well defined. Here, we cloned and expressed the GRASP homolog from Leishmania. We found that Leishmania expresses one GRASP homolog of 58 kDa protein (LdGRASP) which localizes in LdRab1- and LPG2-positive Golgi compartment in Leishmania. LdGRASP was found to bind with COPII complex, LdARF1, LdRab1 and LdRab11 indicating its role in ER and Golgi transport in Leishmania. To determine the function of LdGRASP, we generated LdGRASP knockout parasites using CRISPR-Cas9. We found fragmentation of Golgi in Ld:GRASPKO parasites. Our results showed enhanced transport of non-GPI-anchored gp63 to the cell surface leading to higher secretion of this form of gp63 in Ld:GRASPKO parasites in comparison to Ld:WT cells. In contrast, we found that transport of GPI-anchored gp63 to the cell surface is blocked in Ld:GRASPKO parasites and thereby inhibits its secretion. The overexpression of dominant-negative mutant of LdRab1 or LdSar1 in Ld:GRASPKO parasites significantly blocked the secretion of non-GPI-anchored gp63. Interestingly, we found that survival of transgenic parasites overexpressing Ld:GRASP-GFP is significantly compromised in macrophages in comparison to Ld:WT and Ld:GRASPKO parasites. These results demonstrated that LdGRASP differentially regulates Ldgp63 secretory pathway in Leishmania.

Characterization of the First Secreted Sorting Nexin Identified in the Leishmania Protists.

Proteins of the sorting nexin (SNX) family present a modular structural architecture with a phox homology (PX) phosphoinositide (PI)-binding domain and additional PX structural domains, conferring to them a wide variety of vital eukaryotic cell's functions, from signal transduction to membrane deformation and cargo binding. Although SNXs are well studied in human and yeasts, they are poorly investigated in protists. Herein, is presented the characterization of the first SNX identified in Leishmania protozoan parasites encoded by the LdBPK_352470 gene. In silico secondary and tertiary structure prediction revealed a PX domain on the N-terminal half and a Bin/amphiphysin/Rvs (BAR) domain on the C-terminal half of this protein, with these features classifying it in the SNX-BAR subfamily of SNXs. We named the LdBPK_352470.1 gene product LdSNXi, as it is the first SNX identified in Leishmania (L.) donovani. Its expression was confirmed in L. donovani promastigotes under different cell cycle phases, and it was shown to be secreted in the extracellular medium. Using an in vitro lipid binding assay, it was demonstrated that recombinant (r) LdSNXi (rGST-LdSNXi) tagged with glutathione-S-transferase (GST) binds to the PtdIns3P and PtdIns4P PIs. Using a specific a-LdSNXi antibody and immunofluorescence confocal microscopy, the intracellular localization of endogenous LdSNXi was analyzed in L. donovani promastigotes and axenic amastigotes. Additionally, rLdSNXi tagged with enhanced green fluorescent protein (rLdSNXi-EGFP) was heterologously expressed in transfected HeLa cells and its localization was examined. All observed localizations suggest functions compatible with the postulated SNX identity of LdSNXi. Sequence, structure, and evolutionary analysis revealed high homology between LdSNXi and the human SNX2, while the investigation of protein-protein interactions based on STRING (v.11.5) predicted putative molecular partners of LdSNXi in Leishmania.

Expression and functional characterization of chimeric recombinant bovine follicle-stimulating hormone produced in Leishmania tarentolae.

Assisted reproductive techniques are routinely used in livestock species to increase and enhance productivity. Ovarian hyperstimulation is a process that currently relies on administering pituitary-derived follicle-stimulating hormone (FSH) or equine chorionic gonadotropin in combination with other hormones to promote the maturation of multiple follicles and thereby achieve superovulation. The use of partially purified preparations of FSH extracted from natural sources is associated with suboptimal and variable results. Recombinant FSH (rFSH) has been produced in a variety of heterologous organisms. However, attaining a bioactive rFSH of high quality and at low cost for use in livestock remains challenging. Here we report the production and characterization of a single chain bovine rFSH consisting of the ß- and α-subunit fused by a polypeptide linker (scbFSH) using Leishmania tarentolae as heterologous expression system. This unicellular eukaryote is non-pathogenic to mammals, can be grown in bioreactors using simple and inexpensive semisynthetic media at 26°C and does not require CO2 or bovine serum supplementation. Stable cell lines expressing scbFSH in an inducible fashion were generated and characterized for their productivity. Different culture conditions and purification procedures were evaluated, and the recombinant product was biochemically and biologically characterized, including bioassays in an animal model. The results demonstrate that L. tarentolae is a suitable host for producing a homogeneous, glycosylated and biologically active form of scbFSH with a reasonable yield.

A novel chemically defined medium for the biotechnological and biomedical exploitation of the cell factory Leishmania tarentolae.

The development of media for cell culture is a major issue in the biopharmaceutical industry, for the production of therapeutics, immune-modulating molecules and protein antigens. Chemically defined media offer several advantages, as they are free of animal-derived components and guarantee high purity and a consistency in their composition. Microorganisms of the genus Leishmania represent a promising cellular platform for production of recombinant proteins, but their maintenance requires supplements of animal origin, such as hemin and fetal bovine serum. In the present study, three chemically defined media were assayed for culturing Leishmania tarentolae, using both a wild-type strain and a strain engineered to produce a viral antigen. Among the three media, Schneider's Drosophila Medium supplemented with Horseradish Peroxidase proved to be effective for the maintenance of L. tarentolae promastigotes, also allowing the heterologous protein production by the engineered strain. Finally, the engineered strain was maintained in culture up to the 12th week without antibiotic, revealing its capability to produce the recombinant protein in the absence of selective pressure.

Sampling is decisive to determination of Leishmania (Viannia) species.

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.

Prokaryotic and eukaryotic skin microbiota modifications triggered by Leishmania infection in localized Cutaneous Leishmaniasis.

Cutaneous Leishmaniasis (CL) is a tropical disease characterized by cutaneous ulcers, sometimes with satellite lesions and nodular lymphangitis. Leishmania parasites, transmitted by sandfly vectors, cause this widespread public health challenge affecting millions worldwide. CL's complexity stems from diverse Leishmania species and intricate host interactions. Therefore, this study aims to shed light on the spatial-temporal distribution of Leishmania species and exploring the influence of skin microbiota on disease progression. We analyzed 40 samples from CL patients at three military bases across Colombia. Using Oxford Nanopore's Heat Shock Protein 70 sequencing, we identified Leishmania species and profiled microbiota in CL lesions and corresponding healthy limbs. Illumina sequencing of 16S-rRNA and 18S-rRNA genes helped analyze prokaryotic and eukaryotic communities. Our research uncovered a spatial-temporal overlap between regions of high CL incidence and our sampling locations, indicating the coexistence of various Leishmania species. L. naiffi emerged as a noteworthy discovery. In addition, our study delved into the changes in skin microbiota associated with CL lesions sampled by scraping compared with healthy skin sampled by brushing of upper and lower limbs. We observed alterations in microbial diversity, both in prokaryotic and eukaryotic communities, within the lesioned areas, signifying the potential role of microbiota in CL pathogenesis. The significant increase in specific bacterial families, such as Staphylococcaceae and Streptococcaceae, within CL lesions indicates their contribution to local inflammation. In essence, our study contributes to the ongoing research into CL, highlighting the need for a multifaceted approach to decipher the intricate interactions between Leishmaniasis and the skin microbiota.

Resolving the polycistronic aftermath: Essential role of topoisomerase IA in preventing R-loops in Leishmania.

Kinetoplastid parasites are "living bridges" in the evolution from prokaryotes to higher eukaryotes. The near-intronless genome of the kinetoplastid Leishmania exhibits polycistronic transcription which can facilitate R-loop formation. Therefore, to prevent such DNA-RNA hybrids, Leishmania has retained prokaryotic-like DNA Topoisomerase IA (LdTOPIA) in the course of evolution. LdTOPIA is an essential enzyme that is expressed ubiquitously and is adapted for the compartmentalized eukaryotic form in harboring functional bipartite nuclear localization signals. Although exhibiting greater homology to mycobacterial TOPIA, LdTOPIA could functionally complement the growth lethality of Escherichia coli TOPIA null GyrB ts strain at non-permissive temperatures. Purified LdTOPIA exhibits Mg2+-dependent relaxation of only negatively supercoiled DNA and preference towards single-stranded DNA substrates. LdTOPIA prevents nuclear R-loops as conditional LdTOPIA downregulated parasites exhibit R-loop formation and thereby parasite killing. The clinically used tricyclic antidepressant, norclomipramine could specifically inhibit LdTOPIA and lead to R-loop formation and parasite elimination. This comprehensive study therefore paves an avenue for drug repurposing against Leishmania.

Clinical and laboratory characterization of cutaneous leishmaniasis in Chinese migrant workers returned from Iraq.

BACKGROUND: Imported cutaneous leishmaniasis (CL) is a growing problem with increasing global travel to endemic areas. Returned travelers with CL are easy to be misdiagnosed and mistreated due to the lack of awareness for the disease to the physicians in non-endemic region that may lead to unfavorable outcome. Our study intends to summarize the characteristics of Leishmania infection imported from Iraq, so as to help Chinese physicians diagnose and treat the disease. All CL patients were treated with intralesional injection of antimony. METHODS: The definitive diagnosis of CL is based on the parasite identification by microscopic examination directly on lesion smear or parasite culture, PCR amplification of Leishmania-specific internal transcribed spacer 1 (ITS-1). The phylogenetic analysis, the immunopathological examination and the cytokine detection were proceeded after the diagnosis. RESULTS: We have identified 25 CL cases in migrant Chinese workers returned from Iraq for the first time with L. major as the major species of infected Leishmania parasite. Clinical features of the Iraq-imported CL include the history of skin exposure to sandflies bite and the lesions mostly on the exposed limbs. More ulcerative wet lesion was observed than nodular dry lesion. PCR is not only used to detect Leishmania parasite with high sensitivity, but also to identify the species of infected parasite through sequencing the amplified Leishmania-specific ITS-1 gene. The phylogenetic analysis based on the amplified ITS-1 sequences revealed that the infected Leishmania was closed related to the species and strains endemic in Iraq. The immunopathological examination revealed the T-cell filtrated cellular immune response with less B cells and NK cells involved. The cytokine profile measured in the skin lesion also confirmed the Th1 cellular response with higher expression levels of IFN-γ, IL-6 and IL-8. The skin lesions in CL patients were healed after being treated locally with antimony. CONCLUSIONS: The clinical and parasitological features of these Chinese CL cases imported from Iraq provide useful information for the diagnosis and treatment of CL that is not commonly seen in Chinese local population.

The perfect storm: Cutaneous leishmaniasis in troops deployed in the Atlantic forest of Argentina.

American cutaneous leishmaniasis (ACL) is the most prevalent form of leishmaniasis, associated with an ulcerative and stigmatizing mucocutaneous pathology. This study assessed the incidence of Leishmania (Viannia) braziliensis in members of the Argentine Army who were exposed to sandfly bites in Iguazú National Park (INP), northeastern Argentina, during an outbreak of ACL in 2019, and the presence of Leishmania in rodents, opossums and phlebotomine sandflies collected in the area of exposure. Samples from military personnel, wild animals and phlebotomine sandflies were analysed. A total of 20 (40%) patients among the Army personnel and two Akodon montensis rodents (11%) were positive for the presence of Leishmania sp. genes by PCR, while Nyssomyia whitmani and Migonemyia migonei, competent vectors of Leishmania, were also found at the same site. Sequences of hsp70 DNA fragments obtained from human samples confirmed the identity of L. (V.) braziliensis. The risk to which military personnel carrying out activities in the forest are exposed is highlighted, and this risk extends to any worker and visitor who circulates without protection in the INP, coming into contact with transmission "hot spots" due to the concentration of vectors, reservoirs and/or parasites.

Chagas Disease and Leishmaniasis, Sympatric Zoonoses Present in Human from Rural Communities of Venezuela.

PURPOSE: Trypanosoma cruzi and Leishmania spp. coexist in several endemic areas, and there are few studies of Chagas disease and leishmaniasis coinfection worldwide; for this reason, the objective of this work was to determine the Chagas disease and leishmaniasis coinfection in several rural communities co-endemic for these diseases. METHODS: A total of 1107 human samples from six co-endemic rural communities of Cojedes state, Venezuela, were analyzed. Serum samples were evaluated by ELISA, indirect hemagglutination, and indirect immunofluorescence for Chagas disease diagnosis, and individuals were evaluated for leishmaniasis by leishmanin skin test (LST). Approximately, 30% of the individuals were also analyzed by PCR (blood clot samples) for T. cruzi and for Leishmania spp. RESULTS: The 14.7% of the individuals were positive to Trypanosoma cruzi infection by serology, and 25.8% were positive to Leishmania spp. current or past infection by LST. Among the group with PCR results, 7.8% were positive for T. cruzi, and 9.4% for Leishmania spp. The coinfection T. cruzi/Leishmania spp. was 6.5%. The T. cruzi DTUs of the positive blood clot samples were TcI, revealed using the molecular markers: (i) intergenic region of the miniexon, (ii) D7 divergent domain of the 24Sα rDNA, (iii) size-variable domain of the 18S rDNA, and (iv) hsp60-PCR-RFLP (EcoRV). The Leishmania species identified were L. (Leishmania) mexicana and L. (Viannia) braziliensis. CONCLUSION: A high prevalence was found for T. cruzi and Leishmania spp. single and coinfections in almost all communities studied, being these results of relevance for the implementation of control programs in co-endemic areas.

Molecular detection of DNA from Trypanosoma spp. and Leishmania spp. in wild boar (Sus scrofa) tissues.

Due to the proximity of humans to the countryside and the progressive increase in populations of invasive species, such as wild boars (Sus scrofa), the risk of disease spread is also exacerbated, some of which are zoonoses caused by protozoa. In the present study, 75 tissue/organ samples from 25 wild boars obtained from authorized hunting in the northern region of Rio Grande do Sul were evaluated to investigate the presence of Trypanosoma spp. using conventional PCR with specific primers and amplification of the ITS1 region for Leishmania spp. detection and species differentiation, multiplex PCR with kDNA minicircle amplification was performed. Trypanosoma spp. DNA was detected in 11 out of 25 hearts, representing 44% of the culled animals. Regarding the detection of Leishmania DNA, L. infantum was detected in one spleen sample, accounting for 4%, and L. amazonensis in one liver sample from the same animal, also representing 4% (1/25) of the samples. It is important to note that this wild boar, with detection for both L. amazonensis and L. infantum, also had Trypanosoma spp. DNA detected in a heart sample, indicating the potential of this species to have multiple infections with these agents. Furthermore, this is the first reported case of multiple infection in a wild boar with these agents. Therefore, the results obtained reinforce the risk posed by invasive species, especially wild boars, as potential sources of infectious agent dissemination and their role as possible reservoirs for numerous diseases.

Using meta-analysis and machine learning to investigate the transcriptional response of immune cells to Leishmania infection.

BACKGROUND: Leishmaniasis is a parasitic disease caused by the Leishmania protozoan affecting millions of people worldwide, especially in tropical and subtropical regions. The immune response involves the activation of various cells to eliminate the infection. Understanding the complex interplay between Leishmania and the host immune system is crucial for developing effective treatments against this disease. METHODS: This study collected extensive transcriptomic data from macrophages, dendritic, and NK cells exposed to Leishmania spp. Our objective was to determine the Leishmania-responsive genes in immune system cells by applying meta-analysis and feature selection algorithms, followed by co-expression analysis. RESULTS: As a result of meta-analysis, we discovered 703 differentially expressed genes (DEGs), primarily associated with the immune system and cellular metabolic processes. In addition, we have substantiated the significance of transcription factor families, such as bZIP and C2H2 ZF, in response to Leishmania infection. Furthermore, the feature selection techniques revealed the potential of two genes, namely G0S2 and CXCL8, as biomarkers and therapeutic targets for Leishmania infection. Lastly, our co-expression analysis has unveiled seven hub genes, including PFKFB3, DIAPH1, BSG, BIRC3, GOT2, EIF3H, and ATF3, chiefly related to signaling pathways. CONCLUSIONS: These findings provide valuable insights into the molecular mechanisms underlying the response of immune system cells to Leishmania infection and offer novel potential targets for the therapeutic goals.

The changing epidemiology of human leishmaniasis in the non-endemic country of Austria between 2000 to 2021, including a congenital case.

BACKGROUND: Leishmaniasis is caused by infection with intracellular protozoans of the genus Leishmania. Transmission occurs predominantly by the bite of phlebotomine sandflies, other routes, including congenital transmission, are rare. The disease manifests as either cutaneous, visceral or mucosal/mucocutaneous leishmaniasis. In recent years, changes in the epidemiological pattern have been reported from Europe. PRINCIPAL FINDINGS: A total of 311 new and 29 published leishmaniasis cases occurring between 01/01/2000 and 12/31/2021 in Austria were collected and analyzed. These encompassed 146 cutaneous (CL), 14 visceral (VL), 4 mucosal, and 3 cases with concurrent VL and CL. In addition, asymptomatic infections, comprising 11 unspecified cases with Leishmania DNA detectable only in the blood and 162 cases with anti-Leishmania antibodies were reported. Particularly since 2016, the incidence of leishmaniasis has steadily risen, mainly attributable to increasing numbers of CL and cases with positive serology against Leishmania species, whereas the incidence of VL has slowly decreased. Analysis revealed that a shift in the causative species spectrum had occurred and that a substantial number of CL cases were caused by members of the Leishmania donovani/infantum complex. Simultaneous occurrence of VL and CL was identified in immunocompromised individuals, but also in a not yet reported case of an immunocompetent child after vertical transmission. CONCLUSIONS: The incidence of leishmaniasis has risen in the recent years. The numbers are anticipated to keep rising due to increasing human mobility, including travel and forced migration, growing reservoir host populations as well as expansion and dispersal of vector species caused by climate and habitat changes, urbanization and globalization. Hence, elevated awareness for the disease, including possible transmission in previously non-endemic regions and non-vector transmission modes, support of sandfly surveillance efforts and implementation and establishment of public health interventions in a One Health approach are pivotal in the global efforts to control and reduce leishmaniasis.

Novel detection of Leishmania RNA virus-1 (LRV-1) in clinical isolates of Leishmania Viannia panamensis.

American tegumentary leishmaniasis comprises a discrete set of clinical presentations endemic to Latin America. Leishmania RNA virus-1 (LRV-1) is a double-stranded RNA virus identified in 20­25% of the Leishmania Viannia braziliensis and L. V. guyanensis, however not in L. V. panamensis. This is the first report of LRV-1 in L. V. panamensis and its associations with clinical phenotypes of ATL. Unique surplus discard clinical isolates of L. V. panamensis were identified from the Public Health Ontario Laboratory (PHOL) and the Leishmania Clinic of the Instituto de Medicina Tropical 'Alexander von Humboldt' between 2012 and 2019 and screened for LRV-1 by real-time polymerase chain reaction. Patient isolates were stratified according to clinical phenotype. Of 30 patients with L. V. panamensis, 14 (47%) and 16 (53%) patients had severe and non-severe ATL, respectively. Five (36%) of 14 severe cases and 2 (12%) of 16 non-severe cases were positive for LRV-1, respectively. No differences in sex were observed for clinical phenotype and LRV-1 status. Although an association between LRV-1 status and clinical phenotype was not demonstrated, this is the first description of the novel detection of LRV-1 in L. V. panamensis, a species that has been documented predominantly in Central America.

Detection of Internal Transcribed Spacer 1 and hsp70 Genetic Markers Using Restriction Fragment Length Polymorphisms and Sequencing in Identification of Leishmania Species Causing Tegumentary Leishmaniasis in Brazil.

The identification of Leishmania species that cause tegumentary leishmaniasis (TL) is important for taxonomic and prognostic purposes. Molecular analysis using different Leishmania genomic targets is the most useful method for identifying Leishmania species. Therefore, we evaluated the performance of ribosomal RNA internal transcribed spacer 1 (ITS1) and heat shock protein (hsp70) genetic markers by polymerase chain reaction (PCR), followed by restriction fragment length polymorphism analysis (RFLP) and sequencing, for identification of Leishmania species. Samples from 84 Brazilian patients were amplified. Internal transcribed spacer 1 PCR followed by RFLP (HaeIII) [ITS1-RFLP (HaeIII)] identified 46.4% (39/84) of the samples as compatible with the Viannia subgenus. Internal transcribed spacer 1 PCR followed by sequencing (ITS1-sequencing) identified Leishmania (Viannia) braziliensis in 91.7% (77/84) of the TL samples, Leishmania (Leishmania) amazonensis in 3.6% (3/84), L. (V.) guyanensis in 2.4% (2/84), and L. (L.) infantum in 1.2% (1/84). One of the samples showed the same proportion of similarity with L. (V.) guyanensis and L. (V.) panamensis. hsp70 nested PCR followed by RFLP (HaeIII) [nested hsp70-RFLP (HaeIII)] identified 91.7% (77/84) of the samples as compatible with L. (V.) braziliensis/L. (V.) naiffi, 3.6% (3/84) with L. (L.) amazonensis, 1.2% (1/84) with L. (L.) infantum, and 3.6% (3/84) with L. (V.) guyanensis. hsp70 PCR followed by sequencing (hsp70-sequencing) identified L. (V.) braziliensis in 91.7% (77/84) of the TL samples, L. (L.) amazonensis in 3.6% (3/84), L. (V.) guyanensis in 3.6% (3/84), and L. (L.) infantum in 1.2% (1/84). Our findings clearly showed that nested hsp70-RFLP (HaeIII) is better than ITS1-RFLP (HaeIII) and that ITS1 or hsp70 PCR followed by sequencing was adequate for identifying Leishmania species. We also found that Leishmania (Viannia) braziliensis is the most common species causing TL in Brazil. Therefore, sequencing multiple target genes such as ITS1 and hsp 70 is more accurate than RFLP for identifying Leishmania species.

Antimony resistance and gene expression in Leishmania: spotlight on molecular and proteomic aspects.

Leishmaniasis is a vector-borne parasitic disease caused by Leishmania parasites with a spectrum of clinical manifestations, ranging from skin lesions to severe visceral complications. Treatment of this infection has been extremely challenging with the concurrent emergence of drug resistance. The differential gene expression and the discrepancies in protein functions contribute to the appearance of 2 distinct phenotypes: resistant and sensitive, but the current diagnostic tools fail to differentiate between them. The identification of gene expression patterns and molecular mechanisms coupled with antimony (Sb) resistance can be leveraged to prompt diagnosis and select the most effective treatment methods. The present study attempts to use comparative expression of Sb resistance-associated genes in resistant and sensitive Leishmania, to disclose their relative abundance in clinical or in vitro selected isolates to gain an understanding of the molecular mechanisms of Sb response/resistance. Data suggest that the analysis of resistance gene expression would verify the Sb resistance or susceptibility only to a certain extent; however, none of the individual expression patterns of the studied genes was diagnostic as a biomarker of Sb response of Leishmania. The findings highlighted will be useful in bridging the knowledge gap and discovering innovative diagnostic tools and novel therapeutic targets.

Disruption of Leishmania flagellum attachment zone architecture causes flagellum loss.

Leishmania are flagellated eukaryotic parasites that cause leishmaniasis and are closely related to the other kinetoplastid parasites such as Trypanosoma brucei. In all these parasites there is a cell membrane invagination at the base of the flagellum called the flagellar pocket, which is tightly associated with and sculpted by cytoskeletal structures including the flagellum attachment zone (FAZ). The FAZ is a complex interconnected structure linking the flagellum to the cell body and has critical roles in cell morphogenesis, function and pathogenicity. However, this structure varies dramatically in size and organisation between these different parasites, suggesting changes in protein localisation and function. Here, we screened the localisation and function of the Leishmania orthologues of T. brucei FAZ proteins identified in the genome-wide protein tagging project TrypTag. We identified 27 FAZ proteins and our deletion analysis showed that deletion of two FAZ proteins in the flagellum, FAZ27 and FAZ34 resulted in a reduction in cell body size, and flagellum loss in some cells. Furthermore, after null mutant generation, we observed distinct and reproducible changes to cell shape, demonstrating the ability of the parasite to adapt to morphological perturbations resulting from gene deletion. This process of adaptation has important implications for the study of Leishmania mutants.

Identification of asymptomatic Leishmania infection in patients undergoing kidney transplant using multiple tests.

OBJECTIVES: In immunocompromised patients, asymptomatic Leishmania infection can reactivate, and evolve to severe disease. To date, no test is considered the gold standard for the identification of asymptomatic Leishmania infection. A combination of methods was employed to screen for Leishmania infection in patients undergoing kidney transplant (KT). METHODS: We employed polymerase chain reaction for the detection of parasitic DNA in peripheral blood, Western blot to identify serum immunoglobulin G and whole blood assay to detect cytokines/chemokines after stimulation of whole blood with parasitic antigen. RESULTS: One-hundred twenty patients residing in Italy were included in the study at the time of KT. Each patient that tested positive to at least one test was considered as Leishmania positive. Fifty out of 120 patients (42%) tested positive for one or more tests. The detection of specific cell-mediated response (32/111, 29%) was the most common marker of Leishmania infection, followed by a positive serology (24/120, 20%). Four patients (3%) harbored parasitic DNA in the blood. CONCLUSION: Our findings underline the high prevalence of asymptomatic Leishmania infection in patients undergoing KT in Italy, who are potentially at-risk for parasite reactivation and can benefit from an increased vigilance. Understanding the clinical relevance of these findings deserves further studies.

Recent Advances in CRISPR/Cas9-Mediated Genome Editing in Leishmania Strains.

BACKGROUND: Genome manipulation of Leishmania species and the creation of modified strains are widely employed strategies for various purposes, including gene function studies, the development of live attenuated vaccines, and the engineering of host cells for protein production. OBJECTIVE: Despite the introduction of novel manipulation approaches like CRISPR/Cas9 technology with significant advancements in recent years, the development of a reliable protocol for efficiently and precisely altering the genes of Leishmania strains remains a challenging endeavor. Following the successful adaptation of the CRISPR/Cas9 system for higher eukaryotic cells, several research groups have endeavored to apply this system to manipulate the genome of Leishmania. RESULTS: Despite the substantial differences between Leishmania and higher eukaryotes, the CRISPR/Cas9 system has been effectively tested and applied in Leishmania.  CONCLUSION: This comprehensive review summarizes all the CRISPR/Cas9 systems that have been employed in Leishmania, providing details on their methods and the expression systems for Cas9 and gRNA. The review also explores the various applications of the CRISPR system in Leishmania, including the deletion of multicopy gene families, the development of the Leishmania vaccine, complete gene deletions, investigations into chromosomal translocations, protein tagging, gene replacement, large-scale gene knockout, genome editing through cytosine base replacement, and its innovative use in the detection of Leishmania. In addition, the review offers an up-to-date overview of all double-strand break repair mechanisms in Leishmania.

Species diversity, barcode, detection of pathogens and blood meal pattern in Phlebotominae (Diptera: Psychodidae) from northeastern Mexico.

More than 90 species of phlebotomines are vectors of parasites, bacteria, and viruses, which cause disease in animals and humans. Therefore, their study is necessary to establish prevention and control strategies. Mexico is an endemic country for leishmaniasis, mostly in the center and southern regions of the country, yet only few studies have been conducted in the northern part of the country. The present study aims to: (a) assess the alpha diversity of Phlebotominae in an annual cycle, (b) to correlate climatic variables with abundance, (c) to generate barcodes of these insects as part of the integrative taxonomy, and (d) to detect Leishmania, Wolbachia and blood sources in an area close to where a case of autochthonous leishmaniasis has been detected in Nuevo Leon, Mexico. A systematic sampling was conducted during three consecutive nights from 17:00 to 22:00 h., placing Shannon traps, CDC traps with incandescent light, and BG Sentinel 2 + BG Lure traps. A total catch effort of 660 nights/traps/hours was achieved, in which a total number of 707 phlebotomines (58% female and 42% male) of six species were collected and identified. The most abundant species were Psathyromyia cratifer (57%) and Psathyromyia shannoni sensu stricto (26%). The highest abundance (72%; 507/707) was collected during March, April and May 2021. Barcodes were generated for four species of phlebotomines, which represent new records for Mexico. For the molecular detection of microorganisms, 302 specimens were analyzed, although no specimens were positive for Leishmania spp. Wolbachia strains were detected in phlebotomines with an infection rate of 1.32% (4/302) and found in Pa. cratifer and Lu. cruciata. Likewise, human DNA was identified in female Lu. cruciata and Pa. cratifer phlebotomines. These findings indicate the presence of potential vector species of the parasite Leishmania spp. This result shows the need for further entomological surveillance to elucidate the transmission mechanisms in these northern areas of the country.