Structure-Activity Relationship Studies of 9-Alkylamino-1,2,3,4-tetrahydroacridines against Leishmania (Leishmania) infantum Promastigotes.

Leishmaniasis is one of the most neglected diseases in modern times, mainly affecting people from developing countries of the tropics, subtropics and the Mediterranean basin, with approximately 350 million people considered at risk of developing this disease. The incidence of human leishmaniasis has increased over the past decades due to failing prevention and therapeutic measures-there are no vaccines and chemotherapy, which is problematic. Acridine derivatives constitute an interesting group of nitrogen-containing heterocyclic compounds associated with numerous bioactivities, with emphasis to their antileishmanial potential. The present work builds on computational studies focusing on a specific enzyme of the parasite, S-adenosylmethionine decarboxylase (AdoMet DC), with several 1,2,3,4-tetrahydro-acridines emerging as potential inhibitors, evidencing this scaffold as a promising building block for novel antileishmanial pharmaceuticals. Thus, several 1,2,3,4-tetrahydroacridine derivatives have been synthesized, their activity against Leishmania (Leishmania) infantum promastigotes evaluated and a structure-activity relationship (SAR) study was developed based on the results obtained. Even though the majority of the 1,2,3,4-tetrahydroacridines evaluated presented high levels of toxicity, the structural information gathered in this work allowed its application with another scaffold (quinoline), leading to the obtention of N1,N12-bis(7-chloroquinolin-4-yl)dodecane-1,12-diamine (12) as a promising novel antileishmanial agent (IC50 = 0.60 ± 0.11 µM, EC50 = 11.69 ± 3.96 µM and TI = 19.48).

Covalent Conjugation of Amphotericin B to Hyaluronic Acid: An Injectable Water-Soluble Conjugate with Reduced Toxicity and Anti-Leishmanial Potential.

Amphotericin B (AmB) is a highly hydrophobic drug with significant leishmanicidal activity whose use is limited by its poor water solubility and adverse effects. Polymer-drug conjugates are proposed as a delivery system designed to overcome those limitations while improving drug bioavailability, safety, and activity. Here, AmB was covalently linked to periodate-oxidized hyaluronic acid (HA) (oxidation degree of 30.1 ± 5.6%) via a Schiff base (HA-AmB imine). The conjugate presents high water solubility and self-assembles into particles with a mean size of 88.2 ± 17.6 nm, a negative charge (-28.3 ± 0.9 mV), and a drug content of 17.8 ± 1.4%. Spectroscopic studies revealed the presence of AmB in aggregate and super-aggregated forms in the conjugate, which could explain the significant reduction of the in vitro cytotoxicity and hemolytic activity. The formulation showed not only in vitro anti-leishmanial activity against L. infantum-infected macrophages (IC50 = 0.023 µM) but also against an in vivo infected mouse model, promoting a 1.32- and a 4.98-log10 suppression of the L. infantum burden in the spleens and liver, respectively, without toxic effects. In summary, this study describes the safe and effective use of water-soluble HA-AmB imine conjugates for leishmaniasis treatment.

Leishmania type II dehydrogenase is essential for parasite viability irrespective of the presence of an active complex I.

Type II NADH dehydrogenases (NDH2) are monotopic enzymes present in the external or internal face of the mitochondrial inner membrane that contribute to NADH/NAD+ balance by conveying electrons from NADH to ubiquinone without coupled proton translocation. Herein, we characterize the product of a gene present in all species of the human protozoan parasite Leishmania as a bona fide, matrix-oriented, type II NADH dehydrogenase. Within mitochondria, this respiratory activity concurs with that of type I NADH dehydrogenase (complex I) in some Leishmania species but not others. To query the significance of NDH2 in parasite physiology, we attempted its genetic disruption in two parasite species, exhibiting a silent (Leishmania infantum, Li) and a fully operational (Leishmania major, Lm) complex I. Strikingly, this analysis revealed that NDH2 abrogation is not tolerated by Leishmania, not even by complex I-expressing Lm species. Conversely, complex I is dispensable in both species, provided that NDH2 is sufficiently expressed. That a type II dehydrogenase is essential even in the presence of an active complex I places Leishmania NADH metabolism into an entirely unique perspective and suggests unexplored functions for NDH2 that span beyond its complex I-overlapping activities. Notably, by showing that the essential character of NDH2 extends to the disease-causing stage of Leishmania, we genetically validate NDH2-an enzyme without a counterpart in mammals-as a candidate target for leishmanicidal drugs.

Mechanistic Insights into the Leishmanicidal and Bactericidal Activities of Batroxicidin, a Cathelicidin-Related Peptide from a South American Viper (Bothrops atrox).

Snake venoms are important sources of bioactive molecules, including those with antiparasitic activity. Cathelicidins form a class of such molecules, which are produced by a variety of organisms. Batroxicidin (BatxC) is a cathelicidin found in the venom of the common lancehead (Bothrops atrox). In the present work, BatxC and two synthetic analogues, BatxC(C-2.15Phe) and BatxC(C-2.14Phe)des-Phe1, were assessed for their microbicidal activity. All three peptides showed a broad-spectrum activity on Gram-positive and -negative bacteria, as well as promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. Circular dichroism (CD) and nuclear magnetic resonance (NMR) data indicated that the three peptides changed their structure upon interaction with membranes. Biomimetic membrane model studies demonstrated that the peptides exert a permeabilization effect in prokaryotic membranes, leading to cell morphology distortion, which was confirmed by atomic force microscopy (AFM). The molecules considered in this work exhibited bactericidal and leishmanicidal activity at low concentrations, with the AFM data suggesting membrane pore formation as their mechanism of action. These peptides stand as valuable prototype drugs to be further investigated and eventually used to treat bacterial and protozoal infections.

Epigallocathechin-O-3-Gallate Inhibits Trypanothione Reductase of Leishmania infantum, Causing Alterations in Redox Balance and Leading to Parasite Death.

Leishmania infantum is a protozoan parasite that causes a vector borne infectious disease in humans known as visceral leishmaniasis (VL). This pathology, also caused by L. donovani, presently impacts the health of 500,000 people worldwide, and is treated with outdated anti-parasitic drugs that suffer from poor treatment regimens, severe side effects, high cost and/or emergence of resistant parasites. In previous works we have disclosed the anti-Leishmania activity of (-)-Epigallocatechin 3-O-gallate (EGCG), a flavonoid compound present in green tea leaves. To date, the mechanism of action of EGCG against Leishmania remains unknown. This work aims to shed new light into the leishmanicidal mode of action of EGCG. Towards this goal, we first confirmed that EGCG inhibits L. infantum promastigote proliferation in a concentration-dependent manner. Second, we established that the leishmanicidal effect of EGCG was associated with i) mitochondria depolarization and ii) decreased concentration of intracellular ATP, and iii) increased concentration of intracellular H2O2. Third, we found that the leishmanicidal effect and the elevated H2O2 levels induced by of EGCG can be abolished by PEG-catalase, strongly suggesting that this flavonoid kills L. infantum promastigotes by disturbing their intracellular redox balance. Finally, we gathered in silico and in vitro evidence that EGCG binds to trypanothione reductase (TR), a central enzyme of the redox homeostasis of Leishmania, acting as a competitive inhibitor of its trypanothione substrate.

Biological Evaluation and Mechanistic Studies of Quinolin-(1H)-Imines as a New Chemotype against Leishmaniasis.

Leishmaniasis is one of the most challenging neglected tropical diseases and remains a global threat to public health. Currently available therapies for leishmaniases present significant drawbacks and are rendered increasingly inefficient due to parasite resistance, making the need for more effective, safer, and less expensive drugs an urgent one. In our efforts to identify novel chemical scaffolds for the development of antileishmanial agents, we have screened in-house antiplasmodial libraries against axenic and intracellular forms of Leishmania infantum, Leishmania amazonensis, and Leishmania major. Several of the screened compounds showed half-maximal inhibitory concentrations (IC50s) against intracellular L. infantum parasites in the submicromolar range (compounds 1h, IC50 = 0.9 µM, and 1n, IC50 = 0.7 µM) and selectivity indexes of 11 and 9.7, respectively. Compounds also displayed activity against L. amazonensis and L. major parasites, albeit in the low micromolar range. Mechanistic studies revealed that compound 1n efficiently inhibits oxygen consumption and significantly decreases the mitochondrial membrane potential in L. infantum axenic amastigotes, suggesting that this chemotype acts, at least in part, by interfering with mitochondrial function. Structure-activity analysis suggests that compound 1n is a promising antileishmanial lead and emphasizes the potential of the quinoline-(1H)-imine chemotype for the future development of new antileishmanial agents.

Synthesis and evaluation of novel chromanone and quinolinone analogues of uniflorol as anti-leishmanial agents.

Within this work, we describe the design and synthesis of a range of novel chromanones and quinolinones, based on natural products reported to possess anti-leishmanial action. The core heterocycles were obtained either via classical or ionic liquid mediated Kabbe condensation in the case of chromanones, or aqueous Sonogashira based alkynylation followed by acid-catalysed cyclisation in the case of quinolinones. Upon testing in promastigotes, axenic amastigotes and Leishmania-infected macrophages, compound 13c was identified as displaying interesting activity, inhibiting axenic amastigotes and intracellular amastigotes with IC50s of 25.3 and 24.6µM respectively.

Chaperone activation and client binding of a 2-cysteine peroxiredoxin.

Many 2-Cys-peroxiredoxins (2-Cys-Prxs) are dual-function proteins, either acting as peroxidases under non-stress conditions or as chaperones during stress. The mechanism by which 2-Cys-Prxs switch functions remains to be defined. Our work focuses on Leishmania infantum mitochondrial 2-Cys-Prx, whose reduced, decameric subpopulation adopts chaperone function during heat shock, an activity that facilitates the transition from insects to warm-blooded host environments. Here, we have solved the cryo-EM structure of mTXNPx in complex with a thermally unfolded client protein, and revealed that the flexible N-termini of mTXNPx form a well-resolved central belt that contacts and encapsulates the unstructured client protein in the center of the decamer ring. In vivo and in vitro cross-linking studies provide further support for these interactions, and demonstrate that mTXNPx decamers undergo temperature-dependent structural rearrangements specifically at the dimer-dimer interfaces. These structural changes appear crucial for exposing chaperone-client binding sites that are buried in the peroxidase-active protein.

Development of an automated image analysis protocol for quantification of intracellular forms of Leishmania spp.

Leishmania parasites cause a set of neglected tropical diseases with considerable public health impact, the leishmaniases, which are often fatal if left untreated. Since current treatments for the leishmaniases exhibit high toxicity, low efficacy and prohibitive prices, many laboratories throughout the world are engaged in research for the discovery of novel chemotherapeutics. This entails the necessity of screening large numbers of compounds against the clinically relevant form of the parasite, the obligatory intracellular amastigote, a procedure that in many laboratories is still carried out by manual inspection. To overcome this well-known bottleneck in Leishmania drug development, several studies have recently attempted to automate this process. Here we implemented an image-based high content triage assay for Leishmania which has the added advantages of using primary macrophages instead of macrophage cell lines and of enabling identification of active compounds against parasite species developing both in small individual phagolysosomes (such as L. infantum) and in large communal vacuoles (such as L. amazonensis). The automated image analysis protocol is made available for IN Cell Analyzer systems, and, importantly, also for the open-source CellProfiler software, in this way extending its implementation to any laboratory involved in drug development as well as in other aspects of Leishmania research requiring analysis of in vitro infected macrophages.

In Silico Discovery of a Substituted 6-Methoxy-quinalidine with Leishmanicidal Activity in Leishmania infantum.

There is an urgent need for the discovery of new antileishmanial drugs with a new mechanism of action. Type 2 NADH dehydrogenase from Leishmania infantum (LiNDH2) is an enzyme of the parasite's respiratory system, which catalyzes the electron transfer from NADH to ubiquinone without coupled proton pumping. In previous studies of the related NADH: ubiquinone oxidoreductase crystal structure from Saccharomyces cerevisiae, two ubiquinone-binding sites (UQI and UQII) were identified and shown to play an important role in the NDH-2-catalyzed oxidoreduction reaction. Based on the available structural data, we developed a three-dimensional structural model of LiNDH2 using homology detection methods and performed an in silico virtual screening campaign to search for potential inhibitors targeting the LiNDH2 ubiquinone-binding site 1-UQI. Selected compounds displaying favorable properties in the computational screening experiments were assayed for inhibitory activity in the structurally similar recombinant NDH-2 from S. aureus and leishmanicidal activity was determined in the wild-type axenic amastigotes and promastigotes of L. infantum. The identified compound, a substituted 6-methoxy-quinalidine, showed promising nanomolar leishmanicidal activity on wild-type axenic promastigotes and amastigotes of L. infantum and the potential for further development.

Calcium and magnesium ions modulate the oligomeric state and function of mitochondrial 2-Cys peroxiredoxins in Leishmania parasites.

Leishmania parasites have evolved a number of strategies to cope with the harsh environmental changes during mammalian infection. One of these mechanisms involves the functional gain that allows mitochondrial 2-Cys peroxiredoxins to act as molecular chaperones when forming decamers. This function is critical for parasite infectivity in mammals, and its activation has been considered to be controlled exclusively by the enzyme redox state under physiological conditions. Herein, we have revealed that magnesium and calcium ions play a major role in modulating the ability of these enzymes to act as molecular chaperones, surpassing the redox effect. These ions are directly involved in mitochondrial metabolism and participate in a novel mechanism to stabilize the decameric form of 2-Cys peroxiredoxins in Leishmania mitochondria. Moreover, we have demonstrated that a constitutively dimeric Prx1m mutant impairs the survival of Leishmania under heat stress, supporting the central role of the chaperone function of Prx1m for Leishmania parasites during the transition from insect to mammalian hosts.

Thaulin-1: The first antimicrobial peptide isolated from the skin of a Patagonian frog Pleurodema thaul (Anura: Leptodactylidae: Leiuperinae) with activity against Escherichia coli.

Patagonia's biodiversity has been explored from many points of view, however, skin secretions of native amphibians have not been evaluated for antimicrobial peptide research until now. In this sense, Pleurodema thaul is the first amphibian specie to be studied from this large region of South America. Analysis of cDNA-encoding peptide in skin samples allowed identification of four new antimicrobial peptides. The predicted mature peptides were synthesized and all of them showed weak or null antimicrobial activity against Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli with the exception of thaulin-1, a cationic 26-residue linear, amphipathic, Gly- and Leu-rich peptide with moderate antimicrobial activity against E. coli (MIC of 24.7µM). AFM and SPR studies suggested a preferential interaction between these peptides and bacterial membranes. Cytotoxicity assays showed that thaulin peptides had minimal effects at MIC concentrations towards human and animal cells. These are the first peptides described for amphibians of the Pleurodema genus. These findings highlight the potential of the Patagonian region's unexplored biodiversity as a source for new molecule discovery.

Ocellatin-PT antimicrobial peptides: High-resolution microscopy studies in antileishmania models and interactions with mimetic membrane systems.

Although the mechanism of action of antimicrobial peptides (AMPs) is not clear, they can interact electrostatically with the cell membranes of microorganisms. New ocellatin-PT peptides were recently isolated from the skin secretion of Leptodactylus pustulatus. The secondary structure of these AMPs and their effect on Leishmania infantum cells, and on different lipid surface models was characterized in this work. The results showed that all ocellatin-PT peptides have an α-helix structure and five of them (PT3, PT4, PT6 to PT8) have leishmanicidal activity; PT1 and PT2 affected the cellular morphology of the parasites and showed greater affinity for leishmania and bacteria-mimicking lipid membranes than for those of mammals. The results show selectivity of ocellatin-PTs to the membranes of microorganisms and the applicability of biophysical methods to clarify the interaction of AMPs with cell membranes.

Decreased antimony uptake and overexpression of genes of thiol metabolism are associated with drug resistance in a canine isolate of Leishmania infantum.

Visceral leishmaniasis (VL) caused by the protozoan parasite Leishmania infantum, is one of the most important zoonotic diseases affecting dogs and humans in the Mediterranean area. The presence of infected dogs as the main reservoir host of L. infantum is regarded as the most significant risk for potential human infection. We have studied the susceptibility profile to antimony and other anti-leishmania drugs (amphotericin B, miltefosine, paromomycin) in Leishmania infantum isolates extracted from a dog before and after two therapeutic interventions with meglumine antimoniate (subcutaneous Glucantime(®), 100 mg/kg/day for 28 days). After the therapeutic intervention, these parasites were significantly less susceptible to antimony than pretreatment isolate, presenting a resistance index of 6-fold to Sb(III) for promastigotes and >3-fold to Sb(III) and 3-fold to Sb(V) for intracellular amastigotes. The susceptibility profile of this resistant L. infantum line is related to a decreased antimony uptake due to lower aquaglyceroporin-1 expression levels. Additionally, other mechanisms including an increase in thiols and overexpression of enzymes involved in thiol metabolism, such as ornithine decarboxylase, trypanothione reductase, mitochondrial tryparedoxin and mitochondrial tryparedoxin peroxidase, could contribute to the resistance as antimony detoxification mechanisms. A major contribution of this study in a canine L. infantum isolate is to find an antimony-resistant mechanism similar to that previously described in other human clinical isolates.

Studies in the mouse model identify strain variability as a major determinant of disease outcome in Leishmania infantum infection.

BACKGROUND: Visceral leishmaniasis is a severe and potentially fatal disease caused by protozoa of the genus Leishmania, transmitted by phlebotomine sandflies. In Europe and the Mediterranean region, L. infantum is the commonest agent of visceral leishmaniasis, causing a wide spectrum of clinical manifestations, including asymptomatic carriage, cutaneous lesions and severe visceral disease. Visceral leishmaniasis is more frequent in immunocompromised individuals and data obtained in experimental models of infection have highlighted the importance of the host immune response, namely the efficient activation of host's macrophages, in determining infection outcome. Conversely, few studies have addressed a possible contribution of parasite variability to this outcome. METHODS: In this study, we compared three isolates of L. infantum regarding their capacity to grow in the organs of mice, the way they activate the host's macrophages and other components of the immune response and also their capacity to cope with host's antimicrobial mechanisms, namely reactive oxygen and nitrogen species. RESULTS: We found that the three parasite strains significantly differed regarding the degree to which they induced nitric oxide synthase (NOS2) and arginase expression in infected macrophages and the pattern of cytokine production they induced in the host, resulting in different degrees of inflammatory response in infected livers. Additionally, the three strains also significantly differed in their in vitro susceptibility to reactive oxygen and nitrogen species. This variability was reflected in the capacity of each strain to persist and proliferate in the organs of wild-type as well as NOS2- and phagocyte oxidase- deficient mice. CONCLUSIONS: The results obtained in this study show that parasite strain variability is an important determinant of disease outcome in L. infantum visceral leishmaniasis, with relevant implications for studies on host-pathogen interaction and also for leishmanicidal drug development.

Targeted delivery of paromomycin in murine infectious diseases through association to nano lipid systems.

Treatment of intracellular infections such as those caused by Mycobacterium spp. and Leishmania spp. is often hampered by limited access of drugs to infected cells. This is the case of paromomycin (PRM), an antibiotic with broad spectrum in vitro activity against protozoa and mycobacteria. Association of chemotherapeutics to liposomes is a worthy strategy to circumvent poor drug accessibility. Six different PRM liposomal formulations were produced, physicochemically characterized and biologically evaluated in a macrophagic cell line confirming their adequacy for in vivo studies. Biodistribution profiles of PRM liposomes revealed preferential targeting of the antibiotic to the liver, spleen and lungs, relative to free PRM, which translated into an enhanced therapeutic effect in murine models infected with Mycobacterium avium and Leishmania infantum and an absence of toxic effects. Our findings demonstrate the advantages of associating PRM to liposomes indicating their potential as an alternative therapeutic strategy for mycobacterial and parasite infections. FROM THE CLINICAL EDITOR: Infections caused by intracellular organisms such as Mycobacterium and Leishmania remain a significant problem worldwide. Although effective drugs are available, their actions are limited by access into the intracellular compartment. In this article, the authors developed different liposomal formulations as drug carriers of paromomycin and investigated their efficacy in a mouse model. The positive should provide another treatment option for these organisms in the near future.

New insights into neutrophil and Leishmania infantum in vitro immune interactions.

The interaction between polymorphonuclear leukocytes (PMN) or neutrophils and Leishmania became an interesting focus of research, since PMN turn out to be essential cells in transiently hosting the parasites. This study aims to evaluate whether L. infantum, the etiological agent of zoonotic visceral leishmaniasis, influences the in vitro functional activity of murine neutrophils. Phagocytosis, chemotaxis, oxidative burst, degranulation and apoptosis assays were performed. Cytokines, chemokines and toll-like receptors gene expression were evaluated by Real-time PCR. Results indicate that some of the innate features of PMN immunity were activated when in contact with L. infantum. However, parasites might negatively interfere with PMN defense mechanisms compromising the link between innate and acquired immunity. This work provides additional insights on the inflammatory immune interactions between neutrophils and L. infantum highlighting the role of PMN in Leishmania infection.

Mitochondrial peroxiredoxin functions as crucial chaperone reservoir in Leishmania infantum.

Cytosolic eukaryotic 2-Cys-peroxiredoxins have been widely reported to act as dual-function proteins, either detoxifying reactive oxygen species or acting as chaperones to prevent protein aggregation. Several stimuli, including peroxide-mediated sulfinic acid formation at the active site cysteine, have been proposed to trigger the chaperone activity. However, the mechanism underlying this activation and the extent to which the chaperone function is crucial under physiological conditions in vivo remained unknown. Here we demonstrate that in the vector-borne protozoan parasite Leishmania infantum, mitochondrial peroxiredoxin (Prx) exerts intrinsic ATP-independent chaperone activity, protecting a wide variety of different proteins against heat stress-mediated unfolding in vitro and in vivo. Activation of the chaperone function appears to be induced by temperature-mediated restructuring of the reduced decamers, promoting binding of unfolding client proteins in the center of Prx's ringlike structure. Client proteins are maintained in a folding-competent conformation until restoration of nonstress conditions, upon which they are released and transferred to ATP-dependent chaperones for refolding. Interference with client binding impairs parasite infectivity, providing compelling evidence for the in vivo importance of Prx's chaperone function. Our results suggest that reduced Prx provides a mitochondrial chaperone reservoir, which allows L. infantum to deal successfully with protein unfolding conditions during the transition from insect to the mammalian hosts and to generate viable parasites capable of perpetuating infection.

LiZIP3 is a cellular zinc transporter that mediates the tightly regulated import of zinc in Leishmania infantum parasites.

Cellular zinc homeostasis ensures that the intracellular concentration of this element is kept within limits that enable its participation in critical physiological processes without exerting toxic effects. We report here the identification and characterization of the first mediator of zinc homeostasis in Leishmania infantum, LiZIP3, a member of the ZIP family of divalent metal-ion transporters. The zinc transporter activity of LiZIP3 was first disclosed by its capacity to rescue the growth of Saccharomyces cerevisiae strains deficient in zinc acquisition. Subsequent expression of LiZIP3 in Xenopus laevis oocytes was shown to stimulate the uptake of a broad range of metal ions, among which Zn(2+) was the preferred LiZIP3 substrate (K0.5 ≈ 0.1 µM). Evidence that LiZIP3 functions as a zinc importer in L. infantum came from the observations that the protein locates to the cell membrane and that its overexpression leads to augmented zinc internalization. Importantly, expression and cell-surface location of LiZIP3 are lost when parasites face high zinc bioavailability. LiZIP3 decline in response to zinc is regulated at the mRNA level in a process involving (a) short-lived protein(s). Collectively, our data reveal that LiZIP3 enables L. infantum to acquire zinc in a highly regulated manner, hence contributing to zinc homeostasis.

The mitochondrial complex I of trypanosomatids--an overview of current knowledge.

The contribution of trypanosomatid mitochondrial complex I for energy transduction has long been debated. Herein, we summarize current knowledge on the composition and relevance of this enzyme. Bioinformatic and proteomic analyses allowed the identification of many conserved and trypanosomatid-specific subunits of NADH:ubiquinone oxidoreductase, revealing a multifunctional enzyme capable of performing bioenergetic activities and possibly, also of functioning in fatty acid metabolism. A multimeric structure organized in 5 domains of more than 2 MDa is predicted, in contrast to the 1 MDa described for mammalian complex I. The relevance of mitochondrial complex I within the Trypanosomatidae family is quite diverse with its NADH oxidation activity being dispensable for both procyclic and bloodstream Trypanosoma brucei, whereas in Phytomonas serpens the enzyme is the only respiratory complex able to sustain membrane potential. Aside from complex I, trypanosomatid mitochondria contain a type II NADH dehydrogenase and a NADH-dependent fumarate reductase as alternative electron entry points into the respiratory chain and thus, some trypanosomatids may have bypassed the need for complex I. The involvement of each of these enzymes in the maintenance of the mitochondrial redox balance in trypanosomatids is still an open question and requires further investigation.