Potential selection of antimony and methotrexate cross-resistance in Leishmania infantum circulating strains.

BACKGROUND: Visceral leishmaniasis (VL) resolution depends on a wide range of factors, including the instauration of an effective treatment coupled to a functional host immune system. Patients with a depressed immune system, like the ones receiving methotrexate (MTX), are at higher risk of developing VL and refusing antileishmanial drugs. Moreover, the alarmingly growing levels of antimicrobial resistance, especially in endemic areas, contribute to the increasing the burden of this complex zoonotic disease. PRINCIPAL FINDINGS: To understand the potential links between immunosuppressants and antileishmanial drugs, we have studied the interaction of antimony (Sb) and MTX in a Leishmania infantum reference strain (LiWT) and in two L. infantum clinical strains (LiFS-A and LiFS-B) naturally circulating in non-treated VL dogs in Spain. The LiFS-A strain was isolated before Sb treatment in a case that responded positively to the treatment, while the LiFS-B strain was recovered from a dog before Sb treatment, with the dog later relapsing after the treatment. Our results show that, exposure to Sb or MTX leads to an increase in the production of reactive oxygen species (ROS) in LiWT which correlates with a sensitive phenotype against both drugs in promastigotes and intracellular amastigotes. LiFS-A was sensitive against Sb but resistant against MTX, displaying high levels of protection against ROS when exposed to MTX. LiFS-B was resistant to both drugs. Evaluation of the melting proteomes of the two LiFS, in the presence and absence of Sb and MTX, showed a differential enrichment of direct and indirect targets for both drugs, including common and unique pathways. CONCLUSION: Our results show the potential selection of Sb-MTX cross-resistant parasites in the field, pointing to the possibility to undermine antileishmanial treatment of those patients being treated with immunosuppressant drugs in Leishmania endemic areas.

Leishmania parasites exchange drug-resistance genes through extracellular vesicles.

Leishmania are eukaryotic parasites that have retained the ability to produce extracellular vesicles (EVs) through evolution. To date, it has been unclear if different DNA entities could be associated with Leishmania EVs and whether these could constitute a mechanism of horizontal gene transfer (HGT). Herein, we investigate the DNA content of EVs derived from drug-resistant parasites, as well as the EVs' potential to act as shuttles for DNA transfer. Next-generation sequencing and PCR assays confirm the enrichment of amplicons carrying drug-resistance genes associated with EVs. Transfer assays of drug-resistant EVs highlight a significant impact on the phenotype of recipient parasites induced by the expression of the transferred DNA. Recipient parasites display an enhanced growth and better control of oxidative stress. We provide evidence that eukaryotic EVs function as efficient mediators in HGT, thereby facilitating the transmission of drug-resistance genes and increasing the fitness of cells when encountering stressful environments.

Influence of N-Methylation and Conformation on Almiramide Anti-Leishmanial Activity.

The almiramide N-methylated lipopeptides exhibit promising activity against trypanosomatid parasites. A structure-activity relationship study has been performed to examine the influences of N-methylation and conformation on activity against various strains of leishmaniasis protozoan and on cytotoxicity. The synthesis and biological analysis of twenty-five analogs demonstrated that derivatives with a single methyl group on either the first or fifth residue amide nitrogen exhibited greater activity than the permethylated peptides and relatively high potency against resistant strains. Replacement of amino amide residues in the peptide, by turn inducing α amino γ lactam (Agl) and N-aminoimidazalone (Nai) counterparts, reduced typically anti-parasitic activity; however, peptide amides possessing Agl residues at the second residue retained significant potency in the unmethylated and permethylated series. Systematic study of the effects of methylation and turn geometry on anti-parasitic activity indicated the relevance of an extended conformer about the central residues, and conformational mobility by tertiary amide isomerization and turn geometry at the extremities of the active peptides.

Leishmania infantum infection in a dog imported from Morocco.

A mixed breed dog rescued from Morocco was presented at a Quebec veterinary practice for facial lesions. Leishmaniosis, an exotic disease caused by the zoonotic protozoan Leishmania infantum, was suspected. Genomic DNA extraction from blood samples and polymerase chain reaction (PCR) were used to confirm L. infantum parasitemia. Parasites were successfully cultured from lesion biopsies, and dose-response assays demonstrated susceptibility to miltefosine, a drug that requires importation from Europe. Twenty-eight days of treatment led to the disappearance of lesions, but relapse occurred several months later (consistent with persistent parasitemia on post-treatment analysis). Further treatment would require importation of drugs and significant delays, offering a poor prognosis. Key clinical message: Diagnosis of tropical diseases in Canada will likely become more common in the near future. Having proper diagnostic tools, effective drugs, and stricter control of animal importation are essential to preventing the spread of these dangerous and frequently zoonotic diseases.

Un chien de race croisée réchappé du Maroc fut présenté dans une pratique vétérinaire du Québec pour des lésions faciales. La leishmaniose, une maladie exotique causée par le protozoaire zoonotique Leishmania infantum, fut suspectée. L'extraction d'ADN génomique d'échantillons sanguins et la réaction d'amplification en chaine par la polymérase (PCR) furent utilisées pour confirmer la parasitémie à L. infantum. Les parasites furent cultivés avec succès à partir de biopsies des lésions et des essais dose-réponse ont démontré une sensibilité au miltefosine, un médicament devant être importé d'Europe. Vingt-huit jours de traitement ont mené à la disparition des lésions, mais une rechute se produisit plusieurs mois plus tard (compatible avec une parasitémie persistante lors d'analyses post-traitement). Des traitements supplémentaires nécessiteraient l'importation de médicaments et des délais significatifs, offrant ainsi un pronostic peu optimiste.Message clinique clé :Le diagnostic de maladie tropicale au Canada devrait devenir plus fréquent dans un avenir rapproché. Il est essentiel d'avoir les outils diagnostiques appropriés, des médicaments efficaces et un contrôle plus sévère des importations d'animaux afin de prévenir la propagation de ces dangereuses et fréquentes maladies zoonotiques.(Traduit par Dr Serge Messier).

Unravelling the proteomic signature of extracellular vesicles released by drug-resistant Leishmania infantum parasites.

Leishmaniasis constitutes the 9th largest disease burden among all infectious diseases. Control of this disease is based on a short list of chemotherapeutic agents headed by pentavalent antimonials, followed by miltefosine and amphotericin B; drugs that are far from ideal due to host toxicity, elevated cost, limited access, and high rates of drug resistance. Knowing that the composition of extracellular vesicles (EVs) can vary according to the state of their parental cell, we hypothesized that EVs released by drug-resistant Leishmania infantum parasites could contain unique and differently enriched proteins depending on the drug-resistance mechanisms involved in the survival of their parental cell line. To assess this possibility, we studied EV production, size, morphology, and protein content of three well-characterized drug-resistant L. infantum cell lines and a wild-type strain. Our results are the first to demonstrate that drug-resistance mechanisms can induce changes in the morphology, size, and distribution of L. infantum EVs. In addition, we identified L. infantum's core EV proteome. This proteome is highly conserved among strains, with the exception of a handful of proteins that are enriched differently depending on the drug responsible for induction of antimicrobial resistance. Furthermore, we obtained the first snapshot of proteins enriched in EVs released by antimony-, miltefosine- and amphotericin-resistant parasites. These include several virulence factors, transcription factors, as well as proteins encoded by drug-resistance genes. This detailed study of L. infantum EVs sheds new light on the potential roles of EVs in Leishmania biology, particularly with respect to the parasite's survival in stressful conditions. This work outlines a crucial first step towards the discovery of EV-based profiles capable of predicting response to antileishmanial agents.

MRPA-independent mechanisms of antimony resistance in Leishmania infantum.

Control of both human and canine leishmaniasis is based on a very short list of chemotherapeutic agents, headed by antimonial derivatives (Sb). The utility of these molecules is severely threatened by high rates of drug resistance. The ABC transporter MRPA is one of the few key Sb resistance proteins described to date, whose role in detoxification has been thoroughly studied in Leishmania parasites. Nonetheless, its rapid amplification during drug selection complicates the discovery of other mechanisms potentially involved in Sb resistance. In this study, stepwise drug-resistance selection and next-generation sequencing were combined in the search for novel Sb-resistance mechanisms deployed by parasites when MRPA is abolished by targeted gene disruption. The gene mrpA is not essential in L. infantum, and its disruption leads to an Sb hypersensitive phenotype in both promastigotes and amastigotes. Five independent mrpA-/- mutants were selected for antimony resistance. These mutants displayed major changes in their ploidy, as well as extrachromosomal linear amplifications of the subtelomeric region of chromosome 23, which includes the genes coding for ABCC1 and ABCC2. Overexpression of ABCC2, but not of ABCC1, resulted in increased Sb tolerance in the mrpA-/- mutant. SNP analyses revealed three different heterozygous mutations in the gene coding for a serine acetyltransferase (SAT) involved in de novo cysteine synthesis in Leishmania. Overexpression of satQ390K, satG321R and satG325R variants led to a 2-3.2 -fold increase in Sb resistance in mrpA-/- parasites. Only satG321R and satG325R induced increased Sb resistance in wild-type parasites. These results reinforce and expand knowledge on the complex nature of Sb resistance in Leishmania parasites.

High-throughput identification and quantification of Haemonchus contortus in fecal samples.

Haemonchus contortus are gastrointestinal nematodes of the family Trichostrongylidae that naturally infect small ruminants while grazing, posing a risk to both animal health and farm profitability. Current diagnostics depend on exacting lab techniques, including manual egg counts and larval differentiation, all of which require time, effort, and specialized technicians. The goal of this study was to facilitate and accelerate the identification and quantification of H. contortus in fecal samples through the use of fluorescein-isothiocyanate peanut-agglutinin staining in order to allow automated detection using a 96-well microplate reader. Next, the model was to be validated using samples containing unknown quantities of eggs. Automated analysis of fluorescence emission of known quantities of H. contortus eggs confirmed an almost perfect linear correlation (r = 0.9984, p < 0.0001), indicating that this new approach can satisfactorily be used to quantify H. contortus eggs on a comparative fluorescence scale. As validation, clinical samples containing an unknown quantity of H. contortus eggs were then analyzed by comparing two methods: either Wisconsin Sugar Flotation (WSF) and McMaster counting followed by manual fluorescence microscopy, or WSF coupled with automated microplate reading. Pearson analysis revealed highly significant correlation between manual and automated methods (r = 0.9999, p < 0.0001), while Bland-Altman plots demonstrated excellent agreement between the two (bias = -0.817 ± 9.94 with 95% limits of agreement from -20.31 to 18.67). Overall, these results demonstrate that high-throughput screening fluorescence detection and quantification of H. contortus eggs is both accurate and rapid.