Cutaneous Leishmania mexicana infections in the United States: defining strains through endemic human pediatric cases in northern Texas.

Over a 6-month span, three patients under 5 years old with cutaneous leishmaniasis presented to the Pediatric Infectious Diseases Clinic at the University of Texas Southwestern Medical Center/Children's Health Dallas. None had traveled outside of northern Texas/southern Oklahoma; all had Leishmania mexicana infections confirmed by PCR. We provide case descriptions and images to increase the awareness of this disease among United States (US) physicians and scientists. Two patients responded to fluconazole, but the youngest required topical paromomycin. Combining these cases with guidelines and our literature review, we suggest that (i) higher doses (10-12 mg/kg/day) of fluconazole should be considered in young children to maximize likelihood and rapidity of response and (ii) patients should transition to alternate agents if they do not respond to high-dose fluconazole within 6 weeks. Furthermore, and of particular interest to the broad microbiology community, we used samples from these cases as a proof of concept to propose a mechanism to strain-type US-endemic L. mexicana. For our analysis, we sequenced three housekeeping genes and the internal transcribed sequence 2 of the ribosomal RNA gene. We identified genetic changes that not only allow us to distinguish US-based L. mexicana strains from strains found in other areas of the Americas but also establish polymorphisms that differ between US isolates. These techniques will allow documentation of genetic changes in this parasite as its range expands. Hence, our cases of cutaneous leishmaniasis provide significant evolutionary, treatment, and public health implications as climate change increases exposure to formerly tropical diseases in previously non-endemic areas. IMPORTANCE: Leishmaniasis is a parasitic disease that typically affects tropical regions worldwide. However, the vector that carries Leishmania is spreading northward into the United States (US). Within a 6-month period, three young cutaneous leishmaniasis patients were seen at the Pediatric Infectious Diseases Clinic at the University of Texas Southwestern Medical Center/Children's Health Dallas. None had traveled outside of northern Texas and southern Oklahoma. We document their presentations, treatments, and outcomes and compare their management to clinical practice guidelines for leishmaniasis. We also analyzed the sequences of three critical genes in Leishmania mexicana isolated from these patients. We found changes that not only distinguish US-based strains from strains found elsewhere but also differ between US isolates. Monitoring these sequences will allow tracking of genetic changes in parasites over time. Our findings have significant US public health implications as people are increasingly likely to be exposed to what were once tropical diseases.

Whole cell reconstructions of Leishmania mexicana through the cell cycle.

The unicellular parasite Leishmania has a precisely defined cell architecture that is inherited by each subsequent generation, requiring a highly coordinated pattern of duplication and segregation of organelles and cytoskeletal structures. A framework of nuclear division and morphological changes is known from light microscopy, yet this has limited resolution and the intrinsic organisation of organelles within the cell body and their manner of duplication and inheritance is unknown. Using volume electron microscopy approaches, we have produced three-dimensional reconstructions of different promastigote cell cycle stages to give a spatial and quantitative overview of organelle positioning, division and inheritance. The first morphological indications seen in our dataset that a new cell cycle had begun were the assembly of a new flagellum, the duplication of the contractile vacuole and the increase in volume of the nucleus and kinetoplast. We showed that the progression of the cytokinesis furrow created a specific pattern of membrane indentations, while our analysis of sub-pellicular microtubule organisation indicated that there is likely a preferred site of new microtubule insertion. The daughter cells retained these indentations in their cell body for a period post-abscission. By comparing cultured and sand fly derived promastigotes, we found an increase in the number and overall volume of lipid droplets in the promastigotes from the sand fly, reflecting a change in their metabolism to ensure transmissibility to the mammalian host. Our insights into the cell cycle mechanics of Leishmania will support future molecular cell biology analyses of these parasites.

Complete assembly, annotation of virulence genes and CRISPR editing of the genome of Leishmania amazonensis PH8 strain.

We report the sequencing and assembly of the PH8 strain of Leishmania amazonensis one of the etiological agents of leishmaniasis. After combining data from long Pacbio reads, short Illumina reads and synteny with the Leishmania mexicana genome, the sequence of 34 chromosomes with 8317 annotated genes was generated. Multigene families encoding three virulence factors, A2, amastins and the GP63 metalloproteases, were identified and compared to their annotation in other Leishmania species. As they have been recently recognized as virulence factors essential for disease establishment and progression of the infection, we also identified 14 genes encoding proteins involved in parasite iron and heme metabolism and compared to genes from other Trypanosomatids. To follow these studies with a genetic approach to address the role of virulence factors, we tested two CRISPR-Cas9 protocols to generate L. amazonensis knockout cell lines, using the Miltefosine transporter gene as a proof of concept.

Transgenic overexpression of heat shock protein (HSP83) enhances protein kinase A activity, disrupts GP63 surface protease expression and alters promastigote morphology in Leishmania amazonensis.

Leishmania parasites undergo morphological changes during their infectious life cycle, including developmental transitions within the sandfly vector, culminating in metacyclic stages that are pre-adapted for infection. Upon entering vertebrate host phagocytes, Leishmania differentiate into intracellular amastigotes, the form that is ultimately transmitted back to the vector to complete the life cycle. Although environmental conditions that induce these cellular transitions are well-established, molecular mechanisms governing Leishmania morphologic differentiation in response to these cues remain largely uncharacterized. Previous studies indicate a key role for HSP83 in both promastigote metacyclogenesis and amastigote differentiation. To further elucidate HSP83 functions in the Leishmania lifecycle, we examined the biological impact of experimentally elevating HSP83 gene expression in Leishmania. Significantly, HSP83 overexpression was associated with altered metacyclic morphology, increased protein kinase A (PKA) activity and decreased expression of the Leishmania major surface protease, GP63. Corroborating these findings, overexpression of the L. amazonensis PKA catalytic subunit resulted in a largely similar phenotype. Our findings demonstrate for the first time in Leishmania, a functional link between HSP83 and PKA in the control of Leishmania gene expression, replication and morphogenesis.

Detection of parasite-derived tRNA and rRNA fragments in the peripheral blood of mice experimentally infected with Leishmania donovani and Leishmania amazonensis using next-generation sequencing analysis.

All prokaryotes and eukaryotes, including parasites, release extracellular vesicles or exosomes that contain selected proteins, lipids, nucleic acids, glycoconjugates, and metabolites. Leishmania exosomes are highly enriched in small RNAs derived from the rRNAs and tRNAs of the protozoan parasite species. Here, using plasma exosomes isolated by a kit and next-generation sequencing, we report the detection of fragments of parasite-derived rRNAs and tRNAs in the peripheral plasma samples of mice experimentally infected with Leishmania donovani and Leishmania amazonensis, the causative agents of Old World visceral leishmaniasis and New World disseminated cutaneous leishmaniasis, respectively. Detected RNA molecules of 28S rRNA, 5.8S rRNA, tRNA-Glu, and tRNA-Thr were common to both plasma samples of mice inoculated with L. donovani and L. amazonensis, whereas tRNA-Ile and tRNA-Trp were only detected in L. amazonensis-infected mice. The detected rRNAs and tRNA isotypes were matched with the exosomal components reported in a previous key study. Our preliminary results suggested that parasite-derived small RNAs were circulating in the blood of mice infected with Leishmania species, providing a better understanding of the roles of exosomal components in leishmaniasis and also new insights into exosome-based biomarkers for Leishmania infection.

Molecular Detection of Leishmania (Leishmania) mexicana in Sandflies from the State of Yucatan, Mexico.

Background: Leishmaniases are a group of vector-borne zoonotic diseases of public health relevance within the tropical and subtropical regions of the world. The state of Yucatan is a vulnerable and receptive area to localized cutaneous leishmaniasis (LCL) due to its proximity to the high-transmission endemic states of Campeche and Quintana Roo. Autochthonous cases of LCL caused by Leishmania (Leishmania) mexicana have been documented in the state, showing a geographical expansion of the disease. Materials and Methods: Using CO2-supplemented Centers for Disease Control and Prevention light traps and Shannon traps, we captured anthropophilic sandflies in the surroundings of a locality with recent records of autochthonous cases of LCL. Sandflies carrying Leishmania DNA were evidenced using PCR. Results: A total of 140 Phlebotominae (Diptera: Psychodidae) females of four species were captured: Lutzomyia (Tricholateralis) cruciata (Coquillett), Psathyromyia (Psathyromyia) shannoni (Dyar), Lutzomyia (Lutzomyia) longipalpis (Lutz and Neiva), and Dampfomyia (Coromyia) deleoni (Fairchild and Hertig). Molecular results showed that 6.1% (95% confidence interval [CI] = 2.3-12.9%) of Lu. cruciata and 43.8% (95% CI = 19.8-70.1%) of Pa. shannoni showed evidence of carrying L. (L.) mexicana DNA. Conclusion: We provide evidence of anthropophilic sandflies carrying L. mexicana DNA in a municipality with recorded autochthonous cases of LCL caused by this parasite species in the state of Yucatan, suggesting the emergence of new focus of LCL in Mexico.

Expression Profile of Genes Related to the Th17 Pathway in Macrophages Infected by Leishmania major and Leishmania amazonensis: The Use of Gene Regulatory Networks in Modeling This Pathway.

Leishmania amazonensis and Leishmania major are the causative agents of cutaneous and mucocutaneous diseases. The infections' outcome depends on host-parasite interactions and Th1/Th2 response, and in cutaneous form, regulation of Th17 cytokines has been reported to maintain inflammation in lesions. Despite that, the Th17 regulatory scenario remains unclear. With the aim to gain a better understanding of the transcription factors (TFs) and genes involved in Th17 induction, in this study, the role of inducing factors of the Th17 pathway in Leishmania-macrophage infection was addressed through computational modeling of gene regulatory networks (GRNs). The Th17 GRN modeling integrated experimentally validated data available in the literature and gene expression data from a time-series RNA-seq experiment (4, 24, 48, and 72 h post-infection). The generated model comprises a total of 10 TFs, 22 coding genes, and 16 cytokines related to the Th17 immune modulation. Addressing the Th17 induction in infected and uninfected macrophages, an increase of 2- to 3-fold in 4-24 h was observed in the former. However, there was a decrease in basal levels at 48-72 h for both groups. In order to evaluate the possible outcomes triggered by GRN component modulation in the Th17 pathway. The generated GRN models promoted an integrative and dynamic view of Leishmania-macrophage interaction over time that extends beyond the analysis of single-gene expression.

Nucleoside Transport and Nucleobase Uptake Null Mutants in Leishmania mexicana for the Routine Expression and Characterization of Purine and Pyrimidine Transporters.

The study of transporters is highly challenging, as they cannot be isolated or studied in suspension, requiring a cellular or vesicular system, and, when mediated by more than one carrier, difficult to interpret. Nucleoside analogues are important drug candidates, and all protozoan pathogens express multiple equilibrative nucleoside transporter (ENT) genes. We have therefore developed a system for the routine expression of nucleoside transporters, using CRISPR/cas9 to delete both copies of all three nucleoside transporters from Leishmania mexicana (ΔNT1.1/1.2/2 (SUPKO)). SUPKO grew at the same rate as the parental strain and displayed no apparent deficiencies, owing to the cells' ability to synthesize pyrimidines, and the expression of the LmexNT3 purine nucleobase transporter. Nucleoside transport was barely measurable in SUPKO, but reintroduction of L. mexicana NT1.1, NT1.2, and NT2 restored uptake. Thus, SUPKO provides an ideal null background for the expression and characterization of single ENT transporter genes in isolation. Similarly, an LmexNT3-KO strain provides a null background for transport of purine nucleobases and was used for the functional characterization of T. cruzi NB2, which was determined to be adenine-specific. A 5-fluorouracil-resistant strain (Lmex5FURes) displayed null transport for uracil and 5FU, and was used to express the Aspergillus nidulans uracil transporter FurD.

Report of autochthonous cases of localized cutaneous leishmaniasis caused by Leishmania (Leishmania) mexicana in vulnerable, susceptible areas of Southeastern Mexico.

Localized cutaneous leishmaniasis (LCL) is an endemic disease in several Mexican States with the main endemic areas located in the South-Southeast region of the country, where 90% of Leishmania (Leishmania) mexicana cases are registered. The Southeast region is located in the Yucatan Peninsula, including Campeche, Quintana Roo and Yucatan States. Campeche and Quintana Roo register more than 60% of the cases in the country each year, while in Yucatan the reports are of imported cases due to residents traveling to endemic areas. However, since 2015, autochthonous cases have been diagnosed by health authorities in municipalities with no previous transmission records. We aimed to identify Leishmania parasite species involved in autochthonous cases by means of the PCR technique. The present study included 13 autochthonous cases of LCL with clinical and parasitological diagnoses during 2018 and 2019 by health authorities, without specific identification of the causal agent. Tissue samples were taken by scraping the margins of active lesions and then they were spotted onto an FTATM Elute Microcard. Next, DNA was eluted and used for PCR amplification of specific Leishmania genus and L. (L.) mexicana species-specific fragments. Molecular analysis showed evidence that L. (L.) mexicana was the causal agent of LCL in 12 of the 13 patients; in one patient, PCR was not performed due to the patient's refusal to participate in the study. Identifying Leishmania species that cause LCL is necessary to define efficient treatment schemes and control strategies for the disease in vulnerable and susceptible areas of the Yucatan State's municipalities.

Transcriptome-Wide Identification of Coding and Noncoding RNA-Binding Proteins Defines the Comprehensive RNA Interactome of Leishmania mexicana.

Proteomic profiling of RNA-binding proteins in Leishmania is currently limited to polyadenylated mRNA-binding proteins, leaving proteins that interact with nonadenylated RNAs, including noncoding RNAs and pre-mRNAs, unidentified. Using a combination of unbiased orthogonal organic phase separation methodology and tandem mass tag-labeling-based high resolution quantitative proteomic mass spectrometry, we robustly identified 2,417 RNA-binding proteins, including 1289 putative novel non-poly(A)-RNA-binding proteins across the two main Leishmania life cycle stages. Eight out of 20 Leishmania deubiquitinases, including the recently characterized L. mexicana DUB2 with an elaborate RNA-binding protein interactome were exclusively identified in the non-poly(A)-RNA-interactome. Additionally, an increased representation of WD40 repeat domains were observed in the Leishmania non-poly(A)-RNA-interactome, thus uncovering potential involvement of this protein domain in RNA-protein interactions in Leishmania. We also characterize the protein-bound RNAs using RNA-sequencing and show that in addition to protein coding transcripts ncRNAs are also enriched in the protein-RNA interactome. Differential gene expression analysis revealed enrichment of 142 out of 195 total L. mexicana protein kinase genes in the protein-RNA-interactome, suggesting important role of protein-RNA interactions in the regulation of the Leishmania protein kinome. Additionally, we characterize the quantitative changes in RNA-protein interactions in hundreds of Leishmania proteins following inhibition of heat shock protein 90 (Hsp90). Our results show that the Hsp90 inhibition in Leishmania causes widespread disruption of RNA-protein interactions in ribosomal proteins, proteasomal proteins and translation factors in both life cycle stages, suggesting downstream effect of the inhibition on protein synthesis and degradation pathways in Leishmania. This study defines the comprehensive RNA interactome of Leishmania and provides in-depth insight into the widespread involvement of RNA-protein interactions in Leishmania biology. IMPORTANCE Advances in proteomics and mass spectrometry have revealed the mRNA-binding proteins in many eukaryotic organisms, including the protozoan parasites Leishmania spp., the causative agents of leishmaniasis, a major infectious disease in over 90 tropical and subtropical countries. However, in addition to mRNAs, which constitute only 2 to 5% of the total transcripts, many types of non-coding RNAs participate in crucial biological processes. In Leishmania, RNA-binding proteins serve as primary gene regulators. Therefore, transcriptome-wide identification of RNA-binding proteins is necessary for deciphering the distinctive posttranscriptional mechanisms of gene regulation in Leishmania. Using a combination of highly efficient orthogonal organic phase separation method and tandem mass tag-labeling-based quantitative proteomic mass spectrometry, we provide unprecedented comprehensive molecular definition of the total RNA interactome across the two main Leishmania life cycle stages. In addition, we characterize for the first time the quantitative changes in RNA-protein interactions in Leishmania following inhibition of heat shock protein 90, shedding light into hitherto unknown large-scale downstream molecular effect of the protein inhibition in the parasite. This work provides insight into the importance of total RNA-protein interactions in Leishmania, thus significantly expanding our knowledge of the emergence of RNA-protein interactions in Leishmania biology.

Ku80 is involved in telomere maintenance but dispensable for genomic stability in Leishmania mexicana.

BACKGROUND: Telomeres are indispensable for genome stability maintenance. They are maintained by the telomere-associated protein complex, which include Ku proteins and a telomerase among others. Here, we investigated a role of Ku80 in Leishmania mexicana. Leishmania is a genus of parasitic protists of the family Trypanosomatidae causing a vector-born disease called leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: We used the previously established CRISPR/Cas9 system to mediate ablation of Ku80- and Ku70-encoding genes in L. mexicana. Complete knock-outs of both genes were confirmed by Southern blotting, whole-genome Illumina sequencing, and RT-qPCR. Resulting telomeric phenotypes were subsequently investigated using Southern blotting detection of terminal restriction fragments. The genome integrity in the Ku80- deficient cells was further investigated by whole-genome sequencing. Our work revealed that telomeres in the ΔKu80 L. mexicana are elongated compared to those of the wild type. This is a surprising finding considering that in another model trypanosomatid, Trypanosoma brucei, they are shortened upon ablation of the same gene. A telomere elongation phenotype has been documented in other species and associated with a presence of telomerase-independent alternative telomere lengthening pathway. Our results also showed that Ku80 appears to be not involved in genome stability maintenance in L. mexicana. CONCLUSION/SIGNIFICANCE: Ablation of the Ku proteins in L. mexicana triggers telomere elongation, but does not have an adverse impact on genome integrity.

Micro-CT visualization of a promastigote secretory gel (PSG) and parasite plug in the digestive tract of the sand fly Lutzomyia longipalpis infected with Leishmania mexicana.

Leishmaniasis is a debilitating disease of the tropics, subtropics and southern Europe caused by Leishmania parasites that are transmitted during blood feeding by phlebotomine sand flies (Diptera: Psychodidae). Using non-invasive micro-computed tomography, we were able to visualize the impact of the laboratory model infection of Lutzomyia longipalpis with Leishmania mexicana and its response to a second blood meal. For the first time we were able to show in 3D the plug of promastigote secretory gel (PSG) and parasites in the distended midgut of whole infected sand flies and measure its volume in relation to that of the midgut. We were also able to measure the degree of opening of the stomodeal valve and demonstrate the extension of the PSG and parasites into the pharynx. Although our pilot study could only examine a few flies, it supports the hypothesis that a second, non-infected, blood meal enhances parasite transmission as we showed that the thoracic PSG-parasite plug in infected flies after a second blood meal was, on average, more than twice the volume of the plug in infected flies that did not have a second blood meal.

Differential regulation of E-NTPdases during Leishmania amazonensis lifecycle and effect of their overexpression on parasite infectivity and virulence.

Infections caused by Leishmania amazonensis are characterized by a persistent parasitemia due to the ability of the parasite to modulate the immune response of macrophages. It has been proposed that ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDases) could be able to suppress the host immune defense by reducing the ATP and ADP levels. The AMP generated from E-NTPDase activity can be subsequently hydrolyzed by ecto-nucleotidases, increasing the levels of adenosine, which can reduce the inflammatory response. In the present work, we provide new information about the role of E-NTPDases on infectivity and virulence of L. amazonensis. Our data demonstrate that not only the E-NTPDase activity is differentially regulated during the parasite development but also the expression of the genes ntpd1 and ntpd2. E-NTPDase activity increases significantly in axenic amastigotes and metacyclic promastigotes, both infective forms in mammalian host. A similar profile was found for mRNA levels of the ntpd1 and ntpd2 genes. Using parasites overexpressing the genes ntpd1 and ntpd2, we could demonstrate that L. amazonensis promastigotes overexpressing ntpd2 gene show a remarkable increase in their ability to interact with macrophages compared to controls. In addition, both ntpd1 and ntpd2-overexpressing parasites were more infective to macrophages than controls. The kinetics of lesion formation by transfected parasites were similar to controls until the second week. However, twenty days post-infection, mice infected with ntpd1 and ntpd2-overexpressing parasites presented significantly reduced lesions compared to controls. Interestingly, parasite load reached similar levels among the different experimental groups. Thus, our data show a non-linear relationship between higher E-NTPDase activity and lesion formation. Previous studies have correlated increased ecto-NTPDase activity with virulence and infectivity of Leishmania parasites. Based in our results, we are suggesting that the induced overexpression of E-NTPDases in L. amazonensis could increase extracellular adenosine levels, interfering with the balance of the immune response to promote the pathogen clearance and maintain the host protection.

Divergent Cytochrome c Maturation System in Kinetoplastid Protists.

In eukaryotes, heme attachment through two thioether bonds to mitochondrial cytochromes c and c1 is catalyzed by either multisubunit cytochrome c maturation system I or holocytochrome c synthetase (HCCS). The former was inherited from the alphaproteobacterial progenitor of mitochondria; the latter is a eukaryotic innovation for which prokaryotic ancestry is not evident. HCCS provides one of a few exemplars of de novo protein innovation in eukaryotes, but structure-function insight of HCCS is limited. Uniquely, euglenozoan protists, which include medically relevant kinetoplastids Trypanosoma and Leishmania parasites, attach heme to mitochondrial c-type cytochromes by a single thioether linkage. Yet the mechanism is unknown, as genes encoding proteins with detectable similarity to any proteins involved in cytochrome c maturation in other taxa are absent. Here, a bioinformatics search for proteins conserved in all hemoprotein-containing kinetoplastids identified kinetoplastid cytochrome c synthetase (KCCS), which we reveal as essential and mitochondrial and catalyzes heme attachment to trypanosome cytochrome c KCCS has no sequence identity to other proteins, apart from a slight resemblance within four short motifs suggesting relatedness to HCCS. Thus, KCCS provides a novel resource for studying eukaryotic cytochrome c maturation, possibly with wider relevance, since mutations in human HCCS leads to disease. Moreover, many examples of mitochondrial biochemistry are different in euglenozoans compared to many other eukaryotes; identification of KCCS thus provides another exemplar of extreme, unusual mitochondrial biochemistry in an evolutionarily divergent group of protists.IMPORTANCE Cytochromes c are essential proteins for respiratory and photosynthetic electron transfer. They are posttranslationally modified by covalent attachment of a heme cofactor. Kinetoplastids include important tropical disease-causing parasites; many aspects of their biology differ from other organisms, including their mammalian or plant hosts. Uniquely, kinetoplastids produce cytochromes c with a type of heme attachment not seen elsewhere in nature and were the only cytochrome c-bearing taxa without evidence of protein machinery to attach heme to the apocytochrome. Using bioinformatics, biochemistry, and molecular genetics, we report how kinetoplastids make their cytochromes c Unexpectedly, they use a highly diverged version of an enzyme used for heme-protein attachment in many eukaryotes. Mutations in the human enzyme lead to genetic disease. Identification of kinetoplastid cytochrome c synthetase, thus, solves an evolutionary unknown, provides a possible target for antiparasite drug development, and an unanticipated resource for studying the mechanistic basis of a human genetic disease.

Catalase impairs Leishmania mexicana development and virulence.

Catalase is one of the most abundant enzymes on Earth. It decomposes hydrogen peroxide, thus protecting cells from dangerous reactive oxygen species. The catalase-encoding gene is conspicuously absent from the genome of most representatives of the family Trypanosomatidae. Here, we expressed this protein from the Leishmania mexicana Β-TUBULIN locus using a novel bicistronic expression system, which relies on the 2A peptide of Teschovirus A. We demonstrated that catalase-expressing parasites are severely compromised in their ability to develop in insects, to be transmitted and to infect mice, and to cause clinical manifestation in their mammalian host. Taken together, our data support the hypothesis that the presence of catalase is not compatible with the dixenous life cycle of Leishmania, resulting in loss of this gene from the genome during the evolution of these parasites.

Diverse telomeres in trypanosomatids.

Telomeres are the ends of linear eukaryotic chromosomes facilitating the resolution of the 'end replication and protection' problems, associated with linearity. At the nucleotide level, telomeres typically represent stretches of tandemly arranged telomeric repeats, which vary in length and sequence among different groups of organisms. Recently, a composition of the telomere-associated protein complex has been scrutinized in Trypanosoma brucei. In this work, we subjected proteins from that list to a more detailed bioinformatic analysis and delineated a core set of 20 conserved proteins putatively associated with telomeres in trypanosomatids. Out of these, two proteins (Ku70 and Ku80) are conspicuously missing in representatives of the genus Blastocrithidia, yet telomeres in these species do not appear to be affected. In this work, based on the analysis of a large set of trypanosomatids widely different in their phylogenetic position and life strategies, we demonstrated that telomeres of trypanosomatids are diverse in length, even within groups of closely related species. Our analysis showed that the expression of two proteins predicted to be associated with telomeres (those encoding telomerase and telomere-associated hypothetical protein orthologous to Tb927.6.4330) may directly affect and account for the differences in telomere length within the species of the Leishmania mexicana complex.

Characterization of a protein with unknown function (LinJ.30.3360) in Leishmania amazonensis and Leishmania infantum.

Leishmaniasis is a disease caused by trypanosomatid protozoa of the genus Leishmania. In the Americas, the species Leishmania amazonensis is predominantly associated with American cutaneous leishmaniasis (ACL) while L. infantum is an agent of visceral leishmaniasis (VL). The genome sequences of Leishmania spp. have shown that each genome can contain about 8000 genes encoding proteins, more than half of which have an unknown function (''hypotheticals") at the time of publication. To understand the biology and genome of the organisms, it is important to discover the function of these "hypothetical" proteins; however, few studies have focused on their characterizations. Previously, LinJ.30.3360 (a protein with unknown function) was identified as immunogenic to canine serum with VL and a good antigen to diagnose the visceral form in dogs. Here, we show that the LinJ.30.3360 protein is conserved in L. infantum, L. tarantolae, L. donovani, L. major, L. mexicana, L. braziliensis, L. panamensis, Leptomonas pyrrhocoris, and Leptomonas seymouri. It has been annotated as a MORN (Membrane Occupation and Recognition Nexus) domain protein. However, since the function of this motif is unknown, functional inferences based on the primary sequence are not possible. The protein has a folded ß-leaf secondary structure, and phosphorylation was the only post-translational modification (PTM) found using prediction approach. Experiments have shown that it is located close to the flagellar pocket and presents similar abundance in both L. amazonensis and L. infantum. Furthermore, because it is a conserved protein in trypanosomatids but not in mammals and also because of its antigenicity, LinJ.30.3360 may constitute a potential drug target and/or vaccine for leishmaniasis.

Molecular diagnosis and therapy for cutaneous leishmaniasis of a returned traveler from Mexico.

Leishmaniasis is prevalent in Southern Europe, the Middle East, India, Africa, and Central and South America. Cutaneous leishmaniasis may spontaneously heal over time without treatment; however, risk of visceral dissemination and the impact of cosmetic defect are important concerns. We report a Case of cutaneous leishmaniasis in a patient who ever traveled to Mexico before the onset of a deteriorating wound around the swollen left eyebrow. A diagnosis of infection with Leishmania mexicana was made based on histopathological examination and molecular identification. Systemic treatment with liposomal amphotericin B and ketoconazole were administered with gradual healing of the lesion. Also, this traveler case implicates that the spread of endemic parasitic diseases may be a concealed risk on the public health for Taiwan underlying globalization.

Study on the Occurrence of Genetic Exchange Among Parasites of the Leishmania mexicana Complex.

In Leishmania, genetic exchange has been experimentally demonstrated to occur in the sand fly vector and in promastigote axenic cultures through a meiotic-like process. No evidence of genetic exchange in mammalian hosts have been reported so far, possibly due to the fact that the Leishmania species used in previous studies replicate within individual parasitophorous vacuoles. In the present work, we explored the possibility that residing in communal vacuoles may provide conditions favorable for genetic exchange for L. mexicana and L. amazonensis. Using promastigote lines of both species harboring integrated or episomal drug-resistance markers, we assessed whether genetic exchange can occur in axenic cultures, in infected macrophages as well as in infected mice. We obtained evidence of genetic exchange for L. amazonensis in both axenic promastigote cultures and infected macrophages. However, the resulting products of those putative genetic events were unstable as they did not sustain growth in subsequent sub-cultures, precluding further characterization.

Two phylogenetically divergent isocitrate dehydrogenases are encoded in Leishmania parasites. Molecular and functional characterization of Leishmania mexicana isoenzymes with specificity towards NAD+ and NADP.

Leishmania parasites are of great relevance to public health because they are the causative agents of various long-term and health-threatening diseases in humans. Dependent on the manifestation, drugs either require difficult and lengthy administration, are toxic, expensive, not very effective or have lost efficacy due to the resistance developed by these pathogens against clinical treatments. The intermediary metabolism of Leishmania parasites is characterized by several unusual features, among which whether the Krebs cycle operates in a cyclic and/or in a non-cyclic mode is included. Our survey of the genomes of Leishmania species and monoxenous parasites such as those of the genera Crithidia and Leptomonas (http://www.tritrypdb.org) revealed that two genes encoding putative isocitrate dehydrogenases (IDHs) -with distantly related sequences- are strictly conserved among these parasites. Thus, in this study, we aimed to functionally characterize the two leishmanial IDH isoenzymes, for which we selected the genes LmxM10.0290 (Lmex_IDH-90) and LmxM32.2550 (Lmex_IDH-50) from L. mexicana. Phylogenetic analysis showed that Lmex_IDH-50 clustered with members of Subfamily I, which contains mainly archaeal and bacterial IDHs, and that Lmex_IDH-90 was a close relative of eukaryotic enzymes comprised within Subfamily II IDHs. 3-D homology modeling predicted that both IDHs exhibited the typical folding motifs recognized as canonical for prokaryotic and eukaryotic counterparts, respectively. Both IDH isoforms displayed dual subcellular localization, in the cytosol and the mitochondrion. Kinetic studies showed that Lmex_IDH-50 exclusively catalyzed the reduction of NAD+, while Lmex_IDH-90 solely used NADP+ as coenzyme. Besides, Lmex_IDH-50 differed from Lmex_IDH-90 by exhibiting a nearly 20-fold lower apparent Km value towards isocitrate (2.0 µM vs 43 µM). Our findings showed, for the first time, that the genus Leishmania differentiates not only from other trypanosomatids such as Trypanosoma cruzi and Trypanosoma brucei, but also from most living organisms, by exhibiting two functional homo-dimeric IDHs, highly specific towards NAD+ and NADP+, respectively. It is tempting to argue that any or both types of IDHs might be directly or indirectly linked to the Krebs cycle and/or to the de novo synthesis of glutamate. Our results about the biochemical and structural features of leishmanial IDHs show the relevance of deepening our knowledge of the metabolic processes in these pathogenic parasites to potentially identify new therapeutic targets.