Leishmania Ribosomal Protein (RP) paralogous genes compensate each other's expression maintaining protein native levels.

In the protozoan parasite Leishmania, most genes encoding for ribosomal proteins (RPs) are present as two or more copies in the genome. However, their untranslated regions (UTRs) are predominantly divergent and might be associated with a distinct regulation of the expression of paralogous genes. Herein, we investigated the expression profiles of two RPs (S16 and L13a) encoded by duplicated genes in Leishmania major. The genes encoding for the S16 protein possess identical coding sequences (CDSs) and divergent UTRs, whereas the CDSs of L13a diverge by two amino acids and by their UTRs. Using CRISPR/Cas9 genome editing, we generated knockout (Δ) and endogenously tagged transfectants for each paralog of L13a and S16 genes. Combining tagged and Δ cell lines we found evidence of differential expression of both RPS16 and RPL13a isoforms throughout parasite development, with one isoform consistently more abundant than its respective copy. In addition, compensatory expression was observed for each paralog upon deletion of the corresponding isoform, suggesting functional conservation between these proteins. This differential expression pattern relates to post-translational processes, given compensation occurs at the level of the protein, with no alterations detected at transcript level. Ribosomal profiles for RPL13a indicate a standard behavior for these paralogues suggestive of interaction with heavy RNA-protein complexes, as already reported for other RPs in trypanosomatids. We identified paralog-specific bound to their 3'UTRs which may be influential in regulating paralog expression. In support, we identified conserved cis-elements within the 3'UTRs of RPS16 and RPL13a; cis-elements exclusive to the UTR of the more abundant paralog or to the less abundant ones were identified.

Effective Genome Editing in Leishmania (Viannia) braziliensis Stably Expressing Cas9 and T7 RNA Polymerase.

Until 2015, loss-of-function studies to elucidate protein function in Leishmania relied on gene disruption through homologous recombination. Then, the CRISPR/Cas9 revolution reached these protozoan parasites allowing efficient genome editing with one round of transfection. In addition, the development of LeishGEdit, a PCR-based toolkit for generating knockouts and tagged lines using CRISPR/Cas9, allowed a more straightforward and effective genome editing. In this system, the plasmid pTB007 is delivered to Leishmania for episomal expression or integration in the ß-tubulin locus and for the stable expression of T7 RNA polymerase and Cas9. In South America, and especially in Brazil, Leishmania (Viannia) braziliensis is the most frequent etiological agent of tegumentary leishmaniasis. The L. braziliensis ß-tubulin locus presents significant sequence divergence in comparison with Leishmania major, which precludes the efficient integration of pTB007 and the stable expression of Cas9. To overcome this limitation, the L. major ß-tubulin sequences, present in the pTB007, were replaced by a Leishmania (Viannia) ß-tubulin conserved sequence generating the pTB007_Viannia plasmid. This modification allowed the successful integration of the pTB007_Viannia cassette in the L. braziliensis M2903 genome, and in silico predictions suggest that this can also be achieved in other Viannia species. The activity of Cas9 was evaluated by knocking out the flagellar protein PF16, which caused a phenotype of immobility in these transfectants. Endogenous PF16 was also successfully tagged with mNeonGreen, and an in-locus complementation strategy was employed to return a C-terminally tagged copy of the PF16 gene to the original locus, which resulted in the recovery of swimming capacity. The modified plasmid pTB007_Viannia allowed the integration and stable expression of both T7 RNA polymerase and Cas9 in L. braziliensis and provided an important tool for the study of the biology of this parasite.

Full nucleotide sequencing of ribosomal DNA internal transcribed spacer of Leishmania species causing cutaneous leishmaniasis in Brazil and its potential for species typing.

Species-specific diagnosis still represents a challenge in leishmaniasis management, particularly in regions with multiple endemic species. In Brazil, seven species have been recognized as etiological agents of cutaneous leishmaniasis. The disease comprises complex clinical presentation patterns, classified as localized, diffuse, disseminated and mucocutaneous leishmaniasis. In this study, we characterized the full nucleotide sequence of a region comprising the ribosomal DNA internal transcribed spacers 1 and 2 and 5.8 S gene of reference strains of Leishmania (Viannia) species reported as causative agents of human leishmaniasis in Brazil. The analysis of the nucleotide sequence of this region was able to discriminate species in the Leishmania (Viannia) subgenus and to determine intra- and interspecies phylogenetic relationships.

Ros3 (Lem3p/CDC50) Gene Dosage Is Implicated in Miltefosine Susceptibility in Leishmania (Viannia) braziliensis Clinical Isolates and in Leishmania (Leishmania) major.

The Ros3 protein is a component of the MT-Ros3 transporter complex, considered as the main route of miltefosine entry in Leishmania. L. braziliensis clinical isolates presenting differences in miltefosine susceptibility and uptake were previously shown to differentially express ros3. In this work, we showed that the ros3 gene copy number was increased in the isolate presenting the highest rates of miltefosine uptake and, thus, the highest susceptibility to this drug. The role of the ros3 gene dosage in miltefosine susceptibility was then investigated through a modulation of the gene copy number using two distinct approaches: through an overexpression of ros3 in a tolerant L. braziliensis clinical isolate and in L. major and by generating mono- and diallelic knockouts of this gene in L. major using clustered regularly interspaced short palindromic repeats (CRISPR) Cas9 (Cas = CRISPR-associated). Although the levels of ros3 mRNA were increased at least 40-fold in overexpressing clones, no significant reduction in the half-maximal effective concentration (EC50) for miltefosine was observed in these parasites. The partial or complete deletion of ros3 in L. major, in turn, resulted in a significant increase of 3 and 20 times, respectively, in the EC50 to miltefosine. We unequivocally showed that the ros3 copy number is one of the factors involved in the differential susceptibility and uptake of miltefosine.

Investigation of the pathways related to intrinsic miltefosine tolerance in Leishmania (Viannia) braziliensis clinical isolates reveals differences in drug uptake.

In Brazil, cutaneous leishmaniasis is caused predominantly by L. (V.) braziliensis. The few therapeutic drugs available exhibit several limitations, mainly related to drug toxicity and reduced efficacy in some regions. Miltefosine (MF), the only oral drug available for leishmaniasis treatment, is not widely available and has not yet been approved for human use in Brazil. Our group previously reported the existence of differential susceptibility among L. (V.) braziliensis clinical isolates. In this work, we further characterized three of these isolates of L. (V.) braziliensis chosen because they exhibited the lowest and the highest MF half maximal inhibitory concentrations and were therefore considered less tolerant or more tolerant, respectively. Uptake of MF, and also of phosphocholine, were found to be significantly different in more tolerant parasites compared to the less sensitive isolate, which raised the hypothesis of differences in the MF transport complex Miltefosine Transporter (MT)-Ros3. Although some polymorphisms in those genes were found, they did not correlate with the drug susceptibility phenotype. Drug efflux and compartmentalization were similar in the isolates tested, and amphotericin B susceptibility was retained in MF tolerant parasites, suggesting that increased fitness was also not the basis of observed differences. Transcriptomic analysis revealed that Ros3 mRNA levels were upregulated in the sensitive strain compared to the tolerant ones. Increased mRNA abundance in more tolerant isolates was validated by quantitative PCR. Our results suggest that differential gene expression of the MT transporter complex is the basis of the differential susceptibility in these unselected, naturally occurring parasites.

Identification of Leishmania (Viannia) species and clinical isolates of Leishmania (Leishmania) amazonensis from Brazil using PCR-RFLP of the heat-shock protein 70 gene reveals some unexpected observations.

Hsp70 is a cytoplasmic heat-shock protein, encoded by a multicopy tandemly repeated gene that has recently been gaining popularity as a valuable marker for typing Leishmania species. In this study, we used a previously described hsp70 PCR-RFLP method for identifying Brazilian Leishmania isolates. We identified two distinct L. (L.) amazonensis hsp70 alleles that resulted in two different RFLP patterns. Also, we found RFLP polymorphisms amongst L. (Viannia) naiffi strains. The profiles of both L. (V.) shawi and L. (V.) lindenbergi were very similar to those of other L. (Viannia) species. The observations described herein reflect the polymorphism found within species of Leishmania and indicate that results from this hsp70 PCR-RFLP method should be used with caution when typing isolates from clinical cases of leishmaniasis and Leishmania species from Brazil.

Topical tamoxifen in the therapy of cutaneous leishmaniasis.

The aims of the present work were to test the effect of tamoxifen administered topically and the therapeutic efficacy of tamoxifen and pentavalent antimonial combinations in an experimental model of cutaneous leishmaniasis. BALB/c mice infected with a luciferase expressing line of Leishmania amazonensis were treated with topical tamoxifen in two different formulations (ethanol or oil-free cream) as monotherapy or in co-administration with pentavalent antimonial. Treatment efficacy was evaluated by lesion size and parasite burden, quantified through luminescence, at the end of treatment and 4 weeks later. Topical tamoxifen, formulated in ethanol or as a cream, was shown to be effective. The interaction between tamoxifen and pentavalent antimonial was additive in vitro. Treatment with combined schemes containing tamoxifen and pentavalent antimonial was effective in reducing lesion size and parasite burden. Co-administration of tamoxifen and pentavalent antimonial was superior to monotherapy with antimonial.

Susceptibility to Miltefosine in Brazilian Clinical Isolates of Leishmania (Viannia) braziliensis.

Leishmania (Viannia) braziliensis is the main causative species of tegumentary leishmaniasis in Brazil. In this study, we evaluated the susceptibility of 16 clinical isolates of L. (V.) braziliensis from different regions of Brazil to miltefosine in vitro. Half-maximal inhibitory concentrations of miltefosine varied from 22.9 to 144.2 µM against promastigotes and from 0.3 to 4.2 µM against intracellular amastigotes. No significant differences were found between isolates of different geographical origins. A clear correlation between the EC50 against promastigotes and amastigotes within each isolate was found. These findings contribute to the evaluation of miltefosine's potential and limitations for the treatment of tegumentary leishmaniasis in Brazil.

A Luciferase-Expressing Leishmania braziliensis Line That Leads to Sustained Skin Lesions in BALB/c Mice and Allows Monitoring of Miltefosine Treatment Outcome.

BACKGROUND: Leishmania braziliensis is the most prevalent species isolated from patients displaying cutaneous and muco-cutaneous leishmaniasis in South America. However, there are difficulties for studying L. braziliensis pathogenesis or response to chemotherapy in vivo due to the natural resistance of most mouse strains to infection with these parasites. The aim of this work was to develop an experimental set up that could be used to assess drug efficacy against L. braziliensis. The model was tested using miltefosine. METHODOLOGY/PRINCIPAL FINDINGS: A L. braziliensis line, originally isolated from a cutaneous leishmaniasis patient, was passaged repeatedly in laboratory rodents and further genetically manipulated to express luciferase. Once collected from a culture of parasites freshly transformed from amastigotes, 106 wild type or luciferase-expressing stationary phase promastigotes were inoculated subcutaneously in young BALB/c mice or golden hamsters. In both groups, sustained cutaneous lesions developed at the site of inoculation, no spontaneous self- healing being observed 4 months post-inoculation, if left untreated. Compared to the wild type line features, no difference was noted for the luciferase-transgenic line. Infected animals were treated with 5 or 15 mg/kg/day miltefosine orally for 15 days. At the end of treatment, lesions had regressed and parasites were not detected. However, relapses were observed in animals treated with both doses of miltefosine. CONCLUSIONS/SIGNIFICANCE: Here we described experimental settings for a late-healing model of cutaneous leishmaniasis upon inoculation of a luciferase-expressing L. braziliensis line that can be applied to drug development projects. These settings allowed the monitoring of the transient efficacy of a short-term miltefosine administration.