Trypanosomatid parasites of the genus Leishmania are the causative agents of leishmaniasis, a neglected tropical disease with several clinical manifestations. Leishmania major is the causative agent of cutaneous leishmaniasis (CL), which is largely characterized by ulcerative lesions appearing on the skin. Current treatments of leishmaniasis include pentavalent antimonials and amphotericin B, however, the toxic side effects of these drugs and difficulty with distribution makes these options less than ideal. Miltefosine (MIL) is the first oral treatment available for leishmaniasis. Originally developed for cancer chemotherapy, the mechanism of action of MIL in Leishmania spp. is largely unknown. While treatment with MIL has proven effective, higher tolerance to the drug has been observed, and resistance is easily developed in an in vitro environment. Utilizing stepwise selection we generated MIL-resistant cultures of L. major and characterized the fitness of MIL-resistant L. major. Resistant parasites proliferate at a comparable rate to the wild-type (WT) and exhibit similar apoptotic responses. As expected, MIL-resistant parasites demonstrate decreased susceptibility to MIL, which reduces after the drug is withdrawn from culture. Our data demonstrate metacyclogenesis is elevated in MIL-resistant L. major, albeit these parasites display attenuated in vitro and in vivo virulence and standard survival rates in the natural sandfly vector, indicating that development of experimental resistance to miltefosine does not lead to an increased competitive fitness in L. major.

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HePC]), an antitumoral drug, is the only successful oral treatment for VL. In the current study, we describe the phenotypic traits of L. donovani clonal lines that have acquired resistance to HePC. We performed whole-genome and RNA sequencing of these resistant lines to provide an inclusive overview of the multifactorial acquisition of experimental HePC resistance, circumventing the challenge of identifying changes in membrane-bound proteins faced by proteomics. This analysis was complemented by assessment of the in vitro infectivity of HePC-resistant parasites. Our work underscores the importance of complementary "omics" to acquire the most comprehensive insight for multifaceted processes, such as HePC resistance.

Transgenic Leishmania major and Leishmania donovani axenic promastigotes constitutively expressing mCherry were used for in vitro antileishmanial drug screening. This method requires minimal sample manipulation and can be easily adapted to automatic drug tests, allowing primary high-throughput screenings without the need for expensive and sophisticated instruments.P rotozoan parasites of the genus Leishmania are the causative agents of a wide spectrum of human and animal diseases. The clinical manifestations of Leishmania infections range from lesions of the skin and mucous membranes to lethality, the latter caused by visceral species, including Leishmania donovani (1). Leishmania species cause morbidity and mortality throughout large areas of the Old and New World. Leishmaniasis is an emerging disease with an annual incidence of 2 million cases, and more than 12 million people are infected in over 80 countries where the disease is endemic (2), causing 80,000 deaths per year, and 350 million people are at risk of infection and disease. The increased prevalence of Leishmania-human immunodeficiency virus (HIV) coinfection is the major reason for the recent emergence of leishmaniasis in the Western world (3).Since their discovery in the 1940s, the highly toxic pentavalent antimonials [Sb(V)] have been the primary first-line treatment for all types of leishmaniasis in most parts of the world. However, the increasing frequency of relapse in leishmaniasis patients is forcing the use of other chemotherapeutic agents, including amphotericin B, isethionate pentamidine, paromomycin, and miltefosine (4, 5). Unfortunately, the majority of these antiparasitic drugs present severe side effects, there is no guarantee of cure, and some of them are frequently accompanied by emergence of drug resistance (6-8).Alternative treatments are needed; however, advances in drug discovery approaches have not been satisfactory. Although the use of axenic promastigote cultures of Leishmania as a primary screening method has being validated (7-11), the traditional approaches employed to determine the drug effect in culture present several shortcomings. Microscopic counting is prone to error, time-consuming, and requires trained personnel; the use of tetrazolium salts such as MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] to measure cell viability is labor-intensive, and results are not always reliable (12). The more sensitive and less labor-intensive colorimetric resazurin-based method may present variable metabolic behavior under different cell culture conditions. Furthermore, it is not appropriate to perform kinetic experiments due to the significant cytotoxicity that results from prolonged exposure to the reagent (13-15). Other disadvantages of these metabolic assays are the requirement to incubate the substrate with viable cells at 37°C for a sufficient time to generate a measurable signal. This may lead to undesirable artifacts resulting from interactions between the reagent, the tested drug, and the biochemistry ...

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BackgroundProtozoan parasites of the genus Leishmania are responsible for leishmaniasis, a neglected tropical disease affecting millions worldwide. Visceral leishmaniasis (VL), caused by Leishmania donovani, is the most severe form of leishmaniasis with high rates of mortality if left untreated. Current treatments include pentavalent antimonials and amphotericin B. However, high toxicity and emergence of resistance hinder the success of these options. Miltefosine (HePC) is the first oral treatment available for leishmaniasis. While treatment with HePC has proven effective, higher tolerance to the drug has been observed, and experimental resistance is easily developed in an in vitro environment. Several studies, including ours, have revealed that HePC resistance has a multi-factorial origin and this work aims to shed light on this complex mechanism.Methods2D-DIGE quantitative proteomics comparing the soluble proteomes of sensitive and HePC resistant L. donovani lines identified a protein of interest tentatively involved in drug resistance. To test this link, we employed a gain-of-function approach followed by mutagenesis analysis. Functional studies were complemented with flow cytometry to measure HePC incorporation and cell death.ResultsWe identified a mitochondrial HSP70 (HSPA9B) downregulated in HePC-resistant L. donovani promastigotes. The overexpression of HSPA9B in WT lines confers an increased sensitivity to HePC, regardless of whether the expression is ectopic or integrative. Moreover, the increased sensitivity to HePC is specific to the HSPA9B overexpression since dominant negative mutant lines were able to restore HePC susceptibility to WT values. Interestingly, the augmented susceptibility to HePC did not correlate with an increased HePC uptake. Leishmania donovani promastigotes overexpressing HSPA9B were subjected to different environmental stimuli. Our data suggest that HSPA9B is capable of protecting cells from stressful conditions such as low pH and high temperature. This phenotype was further corroborated in axenic amastigotes overexpressing HSPA9B.ConclusionsThe results from this study provide evidence to support the involvement of a mitochondrial HSP70 (HSPA9B) in experimental HePC resistance, a mechanism that is not yet fully understood, and reveal potential fundamental roles of HSPA9B in the biology of Leishmania. Overall, our findings are relevant for current and future antileishmanial chemotherapy strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1904-8) contains supplementary material, which is available to authorized users.

We have identified LmaPA2G4, a homolog of the human proliferation-associated 2G4 protein (also termed Ebp1), in a phosphoproteomic screening. Multiple sequence alignment and cluster analysis revealed that LmaPA2G4 is a non-peptidase member of the M24 family of metallopeptidases. This pseudoenzyme is structurally related to methionine aminopeptidases. A null mutant system based on negative selection allowed us to demonstrate that LmaPA2G4 is an essential gene in Leishmania major. Over-expression of LmaPA2G4 did not alter cell morphology or the ability to differentiate into metacyclic and amastigote stages. Interestingly, the over-expression affected cell proliferation and virulence in mouse footpad analysis. LmaPA2G4 binds a synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [poly(I∶C)] as shown in an electrophoretic mobility shift assay (EMSA). Quantitative proteomics revealed that the over-expression of LmaPA2G4 led to accumulation of factors involved in translation initiation and elongation. Significantly, we found a strong reduction of de novo protein biosynthesis in transgenic parasites using a non-radioactive metabolic labeling assay. In conclusion, LmaPA2G4 is an essential gene and is potentially implicated in fundamental biological mechanisms, such as translation, making it an attractive target for therapeutic intervention.

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