Multiple Mutations in Heterogeneous Miltefosine-Resistant Leishmania major Population as Determined by Whole Genome Sequencing

Leishmania spp. are parasitic protozoa responsible for a spectrum of diseases known as leishmaniasis. There are few drugs available for the treatment of these diseases, and miltefosine is the first oral drug used in treatment of visceral leishmaniasis, a form of the disease that can be lethal if not treated. In this study, we seek to understand the mechanism of action and identify targets of the drug by generating promastigote mutants highly resistant to miltefosine. Two independent mutants were submitted to short read whole genome sequencing. Genome analysis of these mutants has permitted us to identify point mutations in three genes (P-type ATPase, pyridoxal kinase and α-adaptin like protein) that were also present in other independent miltefosine resistant mutants. Some of the new genes identified here could be useful as potential markers for miltefosine resistance in Leishmania. Moreover, our approach has permitted us to highlight that resistance can be highly heterogeneous at the population level with individual clones derived from this population differing both in terms of genotypes but also susceptibility phenotypes. This may have practical applications while studying resistance

The C-terminal region of Trypanosoma cruzi MASPs is antigenic and secreted via exovesicles.

Trypanosoma cruzi is the etiological agent of Chagas disease, a neglected and emerging tropical disease, endemic to South America and present in non-endemic regions due to human migration. The MASP multigene family is specific to T. cruzi, accounting for 6% of the parasite's genome and plays a key role in immune evasion. A common feature of MASPs is the presence of two conserved regions: an N-terminal region codifying for signal peptide and a C-terminal (C-term) region, which potentially acts as GPI-addition signal peptide. Our aim was the analysis of the presence of an immune response against the MASP C-term region. We found that this region is highly conserved, released via exovesicles (EVs) and has an associated immune response as revealed by epitope affinity mapping, IFA and inhibition of the complement lysis assays. We also demonstrate the presence of a fast IgM response in Balb/c mice infected with T. cruzi. Our results reveal the presence of non-canonical secreted peptides in EVs, which can subsequently be exposed to the immune system with a potential role in evading immune system targets in the parasite

Drug Resistance in Eukaryotic Microorganisms

Eukaryotic microbial pathogens are major contributors to illness and death globally. Although much of their impact can be controlled by drug therapy as with prokaryotic microorganisms, the emergence of drug resistance has threatened these treatment efforts. Here, we discuss the challenges posed by eukaryotic microbial pathogens and how these are similar to, or differ from, the challenges of prokaryotic antibiotic resistance. The therapies used for several major eukaryotic microorganisms are then detailed, and the mechanisms that they have evolved to overcome these therapies are described. The rapid emergence of resistance and the restricted pipeline of new drug therapies pose considerable risks to global health and are particularly acute in the developing world. Nonetheless, we detail how the integration of new technology, biological understanding, epidemiology and evolutionary analysis can help sustain existing therapies, anticipate the emergence of resistance or optimize the deployment of new therapies

Molecular mechanism of drug resistance in parasites

El mecanismo de resistencia a fármacos es un proceso multifactorial, en el que pueden coexistir varios mecanismos, entre ellos: modificaciones en la entrada del fármaco, inactivación del fármaco, amplificación de genes que codifican para las proteínas blanco de acción o proteínas implicadas en la eliminación del fármaco al exterior celular, mutaciones en la proteína blanco de acción, y reparación del daño celular. En los últimos años, nuestro grupo ha estudiado sobre los protozoos parásitos Leishmania (L. tropica y L. infantum ) y Trypanosoma cruzi, algunos de estos mecanismos, con especial interés en las proteínas transportadoras pertenecientes a la superfamilia ABC (ATP-hinding kasette), conocidas como Glicoproteínas-P, implicadas en la eliminación de fármacos y sustancias tóxicas. El estudio molecular y funcional de estos mecanismos, facilitará el desarrollo de estrategias alternativas de tratamiento a emplear en casos de resistencia a fármacos en parásitos.Drug resistance is a multifactorial process involving different coexistent mechanisms, among them: modifications of drug entry, intracellular inactivation of drug, amplification of genes coding for target proteins or proteins involved in drug efflux, mutations in target proteins and increased repair of cell damage. Last years, our research group has been involved in the study of drug resistant mechanisms in the protozoan parasites Leishmania (L. tropica y L. infantum) and Trypanosoma cruzi. Our focus has been related with the study of transporters inc1uded in the superfamily ABC (ATP-binding cassette), specifically the P-glycoproteins involved in efflux of drugs and xenobiotics. Molecular and functional studies of these mechanisms, will help significantly the development of treatment alternative strategies to use in case of drug resistance in parasites

Mecanismo molecular de resistencia a fármacos en parásitos

El mecanismo de resistencia a fármacos es un proceso multifactorial, en el que pueden coexistir varios mecanismos, entre ellos: modificaciones en la entrada del fármaco, inactivación del fármaco, amplificación de genes que codifican para las proteínas blanco de acción o proteínas implicadas en la eliminación del fármaco al exterior celular, mutaciones en la proteína blanco de acción, y reparación del daño celular. En los últimos años, nuestro grupo ha estudiado sobre los protozoos parásitos Leishmania (L. tropica y L. infantum ) y Trypanosoma cruzi, algunos de estos mecanismos, con especial interés en las proteínas transportadoras pertenecientes a la superfamilia ABC (ATP-hinding kasette), conocidas como Glicoproteínas-P, implicadas en la eliminación de fármacos y sustancias tóxicas. El estudio molecular y funcional de estos mecanismos, facilitará el desarrollo de estrategias alternativas de tratamiento a emplear en casos de resistencia a fármacos en parásitos.Drug resistance is a multifactorial process involving different coexistent mechanisms, among them: modifications of drug entry, intracellular inactivation of drug, amplification of genes coding for target proteins or proteins involved in drug efflux, mutations in target proteins and increased repair of cell damage. Last years, our research group has been involved in the study of drug resistant mechanisms in the protozoan parasites Leishmania (L. tropica y L. infantum) and Trypanosoma cruzi. Our focus has been related with the study of transporters inc1uded in the superfamily ABC (ATP-binding cassette), specifically the P-glycoproteins involved in efflux of drugs and xenobiotics. Molecular and functional studies of these mechanisms, will help significantly the development of treatment alternative strategies to use in case of drug resistance in parasites

Leishmania LABCG1 and LABCG2 transporters are involved in virulence and oxidative stress: Functional linkage with autophagy

Background: The G subfamily of ABC (ATP-binding cassette) transporters of Leishmania include 6 genes (ABCG1-G6), some with relevant biological functions associated with drug resistance and phospholipid transport. Several studies have shown that Leishmania LABCG2 transporter plays a role in the exposure of phosphatidylserine (PS), in virulence and in resistance to antimonials. However, the involvement of this transporter in other key biological processes has not been studied. Methods: To better understand the biological function of LABCG2 and its nearly identical tandem-repeated transporter LABCG1, we have generated Leishmania major null mutant parasites for both genes (ΔLABCG1-2). NBD-PS uptake, infectivity, metacyclogenesis, autophagy and thiols were measured. Results: Leishmania major ΔLABCG1-2 parasites present a reduction in NBD-PS uptake, infectivity and virulence. In addition, we have shown that ΔLABCG1-2 parasites in stationary phase growth underwent less metacyclogenesis and presented differences in the plasma membrane's lipophosphoglycan composition. Considering that autophagy is an important process in terms of parasite virulence and cell differentiation, we have shown an autophagy defect in ΔLABCG1-2 parasites, detected by monitoring expression of the autophagosome marker RFP-ATG8. This defect correlates with increased levels of reactive oxygen species and higher non-protein thiol content in ΔLABCG1-2 parasites. HPLC analysis revealed that trypanothione and glutathione were the main molecules accumulated in these ΔLABCG1-2 parasites. The decrease in non-protein thiol levels due to preincubation with buthionine sulphoximide (a γ-glutamylcysteine synthetase inhibitor) restored the autophagy process in ΔLABCG1-2 parasites, indicating a relationship between autophagy and thiol content. Conclusions: LABCG1-2 transporters from Leishmania could be considered as phosphatidylserine and non-protein thiol transporters. They probably accomplish transportation in conjunction with other molecules that are involved in oxidative stress, autophagy, metacyclogenesis and infectivity processes. The overall conclusion is that LABCG1-2 transporters could play a key role in Leishmania cell survival and infectivity.This work was supported by the Spanish Grants SAF2015-68042-R (to S.C. and F.G.), SAF2012-34267 (to F.G.), by the Proyecto de Excelencia, Junta de Andalucia, Ref. CTS-7282 (to F.G.) and by FEDER funds from the EU to S.C. and F.G. A. Perea was a student of the PhD program “Biochemistry and Molecular Biology” of the University of Granada (Spain) and was supported by a fellowship for predoctoral contracts for PhD training from the Ministerio de Economia y Competitividad (in charge of Project SAF2012-34267)

Functional role of highly conserved residues of the N-terminal tail and first transmembrane segment of a P4-ATPase

The P4 family of P-type ATPases (P4-ATPases) plays an important role in maintaining phospholipid asymmetry in eukaryotic cell membranes. Leishmania miltefosine transporter (LMT) is a plasma membrane (PM) P4-ATPase that catalyses translocation into the parasite of the leishmanicidal drug miltefosine as well as phosphatidylcholine and phosphatidylethanolamine analogues. In the present study, we analysed the role, in LMT, of a series of highly conserved amino acids previously undescribed in the N-terminal region of P4-ATPases. Seven residues were identified and, according to an LMT structural model, five were located in the cytosolic N-terminal tail (Asn58, Ile60, Lys64, Tyr65 and Phe70) and the other two (Pro72 and Phe79) in the first transmembrane segment (TM1). Alanine-scanning mutagenesis analysis showed that N58A, Y65A and F79A mutations caused a considerable reduction in the LMT translocase activity. These mutations did not affect protein expression levels. We generated additional mutations in these three residues to assess the influence of the conservation degree on LMT translocase activity. Some of these mutations reduced expression levels without affecting the interaction between LMT and its CDC50 subunit, LRos3. Conserved and non-conserved mutations in the invariant residue Asn58 drastically reduced the translocase activity. Consequently, Asn58 may be necessary to achieve optimal catalytic LMT activity as previously described for the potentially equivalent Asn39 of the sarco/endoplasmic reticulum Ca-ATPase isoform 1a (SERCA1a). Additionally, conservation of a hydrophobic residue at position 79 is crucial for LMT stability

Effet des enzymes protéolytiques sur la pénétration du

Dans ce travail nous étudions les modifications produites par trois enzymes protéolytiques, sur la pénétration des formes métacycliques de Trypanosoma cruzi de culture dans des macrophages péritonéaux non stimulés de souris. Les enzymes protéolytiques employées sont : trypsine, papaïne et collagenase. Quand les formes métacycliques ou les macrophages sont prétraités avec les enzymes protéolytiques, le taux de pénétration diminue, mais le phénomène n’est pas complètement inhibé. De plus, nous observons que le nombre des parasites par cellule demeure stable au cours des expériences

Leishmania LABCG2 transporter is involved in ATP-dependent transport of thiols

The Leishmania LABCG2 transporter has a key role in the redox metabolism of these protozoan parasites. Recently, the involvement of LABCG2 in virulence, autophagy and oxidative stress has been described. Null mutant parasites for LABCG2 present an increase in the intracellular levels of glutathione (GSH) and trypanothione [T(SH)]. On the other hand, parasites overexpressing LABCG2 transporter export non-protein thiols to the extracellular medium. To explore if LABCG2 may mediate an active transport of non-protein thiols, the effect of these molecules on ATPase activity of LABCG2 as well as the ability of LABCG2 to transport them was determined using a baculovirus-Sf9 insect cell system. Our results indicate that all thiols tested [GSH, T(SH)] as well as their oxidized forms GSSG and TS (trypanothione disulfide) stimulate LABCG2-ATPase basal activity. We have measured the transport of [H]-GSH in inside-out Sf9 cell membrane vesicles expressing LABCG2-GFP (green fluorescence protein), finding that LABCG2 was able to mediate a rapid and concentration-dependent uptake of [H]-GSH in the presence of ATP. Finally, we have analyzed the ability of different thiol species to compete for this uptake, T(SH) and TS being the best competitors. The IC value for [H]-GSH uptake in the presence of increasing concentrations of T(SH) was less than 100 μM, highlighting the affinity of this thiol for LABCG2. These results provide the first direct evidence that LABCG2 is an ABC transporter of reduced and oxidized non-protein thiols in Leishmania, suggesting that this transporter can play a role in the redox metabolism and related processes in this protozoan parasite