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

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

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 β-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 β-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 β-tubulin sequences, present in the pTB007, were replaced by a Leishmania (Viannia) β-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

BHPR research: qualitative1. Complex reasoning determines patients' perception of outcome following foot surgery in rheumatoid arhtritis

Background: Foot surgery is common in patients with RA but research into surgical outcomes is limited and conceptually flawed as current outcome measures lack face validity: to date no one has asked patients what is important to them. This study aimed to determine which factors are important to patients when evaluating the success of foot surgery in RA Methods: Semi structured interviews of RA patients who had undergone foot surgery were conducted and transcribed verbatim. Thematic analysis of interviews was conducted to explore issues that were important to patients. Results: 11 RA patients (9 ♂, mean age 59, dis dur = 22yrs, mean of 3 yrs post op) with mixed experiences of foot surgery were interviewed. Patients interpreted outcome in respect to a multitude of factors, frequently positive change in one aspect contrasted with negative opinions about another. Overall, four major themes emerged. Function: Functional ability & participation in valued activities were very important to patients. Walking ability was a key concern but patients interpreted levels of activity in light of other aspects of their disease, reflecting on change in functional ability more than overall level. Positive feelings of improved mobility were often moderated by negative self perception ("I mean, I still walk like a waddling duck”). Appearance: Appearance was important to almost all patients but perhaps the most complex theme of all. Physical appearance, foot shape, and footwear were closely interlinked, yet patients saw these as distinct separate concepts. Patients need to legitimize these feelings was clear and they frequently entered into a defensive repertoire ("it's not cosmetic surgery; it's something that's more important than that, you know?”). Clinician opinion: Surgeons' post operative evaluation of the procedure was very influential. The impact of this appraisal continued to affect patients' lasting impression irrespective of how the outcome compared to their initial goals ("when he'd done it ... he said that hasn't worked as good as he'd wanted to ... but the pain has gone”). Pain: Whilst pain was important to almost all patients, it appeared to be less important than the other themes. Pain was predominately raised when it influenced other themes, such as function; many still felt the need to legitimize their foot pain in order for health professionals to take it seriously ("in the end I went to my GP because it had happened a few times and I went to an orthopaedic surgeon who was quite dismissive of it, it was like what are you complaining about”). Conclusions: Patients interpret the outcome of foot surgery using a multitude of interrelated factors, particularly functional ability, appearance and surgeons' appraisal of the procedure. While pain was often noted, this appeared less important than other factors in the overall outcome of the surgery. Future research into foot surgery should incorporate the complexity of how patients determine their outcome Disclosure statement: All authors have declared no conflicts of interes

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

<div><p>Background</p><p><i>Leishmania braziliensis</i> is the most prevalent species isolated from patients displaying cutaneous and muco-cutaneous leishmaniasis in South America. However, there are difficulties for studying <i>L</i>. <i>braziliensis</i> pathogenesis or response to chemotherapy <i>in vivo</i> 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 <i>L</i>. <i>braziliensis</i>. The model was tested using miltefosine.</p><p>Methodology/Principal Findings</p><p>A <i>L</i>. <i>braziliensis</i> 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.</p><p>Conclusions/Significance</p><p>Here we described experimental settings for a late-healing model of cutaneous leishmaniasis upon inoculation of a luciferase-expressing <i>L</i>. <i>braziliensis</i> line that can be applied to drug development projects. These settings allowed the monitoring of the transient efficacy of a short-term miltefosine administration.</p></div

Characterisation of a transgenic line of <i>L</i>. <i>braziliensis</i> expressing luciferase.

<p>(A) Schematic representation of the linear cassette for integration in the SSU rDNA <i>locus</i>. SSU, small subunit; SAS, splice acceptor site; <i>luc2</i>, <i>luc2</i> coding sequence; α-tub IR, intergenic region of the <i>L</i>. <i>enrietii</i> α-tubulin gene; <i>hyg</i>, hygromycin phosphotransferase gene; CPB IR, <i>L</i>. <i>mexicana</i> cysteine protease B gene intergenic region. Position of the primers and size of amplified fragments are indicated by the dotted lines. (B) Size separation of PCR products amplified using primers complementary to the <i>luc2</i> ORF extremities from genomic DNA purified from four independent transfected clones (lanes 1 to 4) and from the wild type strain (lane 5). (C) Agarose gel electrophoresis of amplified products obtained from <i>Lb</i>-WT (lane 1) or <i>Lb</i>-LUC (lane 2) with the pairs of primers S1/luc2<i>R</i> or luc2<i>i</i>/S4. (-) indicates the control reaction without template DNA. (D) Luminescence intensity and number of <i>Lb</i>-LUC promastigotes. Promastigotes were serially diluted and luminescence was measured using a microplate reader, after addition of luciferin. RLU, relative light units.</p

Susceptibility of promastigotes to MF after recovery from BALB/c mice treated with 5 or 15 mg/kg/day.

<p>Susceptibility of promastigotes to MF after recovery from BALB/c mice treated with 5 or 15 mg/kg/day.</p

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

Drug susceptibility in promastigotes and intracellular amastigotes of the wild type and the transgenic line of <i>L</i>. <i>braziliensis</i> H3227.

<p>Drug susceptibility in promastigotes and intracellular amastigotes of the wild type and the transgenic line of <i>L</i>. <i>braziliensis</i> H3227.</p

Parasite burden of left hind footpad of BALB/c mice treated with MF and evaluated by bioluminescence.

<p>Groups of 5 animals untreated (NT), or treated with 5 or 15 mg/kg/day MF (MF 5 and MF 15 respectively) were evaluated at 40, 120 and 160 days (6^th, 17^th and 23^th week respectively) post-inoculation (pi). The bars on the right show a pseudo-colour scale representing light intensities.</p