The antioxidant response favors Leishmania parasites survival, limits inflammation and reprograms the host cell metabolism.

The oxidative burst generated by the host immune system can restrict intracellular parasite entry and growth. While this burst leads to the induction of antioxidative enzymes, the molecular mechanisms and the consequences of this counter-response on the life of intracellular human parasites are largely unknown. The transcription factor NF-E2-related factor (NRF2) could be a key mediator of antioxidant signaling during infection due to the entry of parasites. Here, we showed that NRF2 was strongly upregulated in infection with the human Leishmania protozoan parasites, its activation was dependent on a NADPH oxidase 2 (NOX2) and SRC family of protein tyrosine kinases (SFKs) signaling pathway and it reprogrammed host cell metabolism. In inflammatory leishmaniasis caused by a viral endosymbiont inducing TNF-α in chronic leishmaniasis, NRF2 activation promoted parasite persistence but limited TNF-α production and tissue destruction. These data provided evidence of the dual role of NRF2 in protecting both the invading pathogen from reactive oxygen species and the host from an excess of the TNF-α destructive pro-inflammatory cytokine.

Tilting the balance between RNA interference and replication eradicates Leishmania RNA virus 1 and mitigates the inflammatory response.

Many Leishmania (Viannia) parasites harbor the double-stranded RNA virus Leishmania RNA virus 1 (LRV1), which has been associated with increased disease severity in animal models and humans and with drug treatment failures in humans. Remarkably, LRV1 survives in the presence of an active RNAi pathway, which in many organisms controls RNA viruses. We found significant levels (0.4 to 2.5%) of small RNAs derived from LRV1 in both Leishmania braziliensis and Leishmania guyanensis, mapping across both strands and with properties consistent with Dicer-mediated cleavage of the dsRNA genome. LRV1 lacks cis- or trans-acting RNAi inhibitory activities, suggesting that virus retention must be maintained by a balance between RNAi activity and LRV1 replication. To tilt this balance toward elimination, we targeted LRV1 using long-hairpin/stem-loop constructs similar to those effective against chromosomal genes. LRV1 was completely eliminated, at high efficiency, accompanied by a massive overproduction of LRV1-specific siRNAs, representing as much as 87% of the total. For both L. braziliensis and L. guyanensis, RNAi-derived LRV1-negative lines were no longer able to induce a Toll-like receptor 3-dependent hyperinflammatory cytokine response in infected macrophages. We demonstrate in vitro a role for LRV1 in virulence of L. braziliensis, the Leishmania species responsible for the vast majority of mucocutaneous leishmaniasis cases. These findings establish a targeted method for elimination of LRV1, and potentially of other Leishmania viruses, which will facilitate mechanistic dissection of the role of LRV1-mediated virulence. Moreover, our data establish a third paradigm for RNAi-viral relationships in evolution: one of balance rather than elimination.

Immunomodulatory and Antileishmanial Activity of Phenylpropanoid Dimers Isolated from Nectandra leucantha.

Three phenylpropanoid dimers (1-3) including two new metabolites were isolated from the extract of the twigs of Nectandra leucantha using antileishmanial bioassay-guided fractionation. The in vitro antiparasitic activity of the isolated compounds against Leishmania donovani parasites and mammalian cytotoxicity and immunomodulatory effects were evaluated. Compounds 1-3 were effective against the intracellular amastigotes within macrophages, with IC50 values of 26.7, 17.8, and 101.9 µM, respectively. The mammalian cytotoxicity, given by the 50% cytotoxic concentration (CC50), was evaluated against peritoneal macrophages. Compounds 1 and 3 were not toxic up to 290 µM, whereas compound 2 demonstrated a CC50 value of 111.2 µM. Compounds 1-3 also suppressed production of disease exacerbatory cytokines IL-6 and IL-10 but had minimal effect on nitric oxide production in L. donovani-infected macrophages, indicating that antileishmanial activity of these compounds is mediated via an NO-independent mechanism. Therefore, these new natural products could represent promising scaffolds for drug design studies for leishmaniasis.

Regulated expression of the Leishmania major surface virulence factor lipophosphoglycan using conditionally destabilized fusion proteins.

Surface glycoconjugates play important roles in the infectious cycle of Leishmania major, including the abundant lipophosphoglycan (LPG) implicated in parasite survival in the sand fly vector and the initial stages of establishment in the mammalian host macrophage. We describe a system for inducible expression of LPG, applying a novel protein-based system that allows controlled degradation of a key LPG biosynthetic enzyme, UDP-galactopyranose mutase (UGM). This methodology relies on a mutated FK506-binding protein (FKBP) destabilizing domain (dd) fused to the protein of interest; in the absence of rapamycin analogs, such as Shld1, the dd domain is destabilized, leading to proteasomal degradation, whereas drug treatment confers stabilization. Tests in L. major using dd fusions to a panel of reporters and cellular proteins confirmed its functionality, with a high degree of regulation and low background, and we established the kinetics of protein activation and/or loss. Two inexpensive and widely available ligands, FK506 and rapamycin, functioned similarly to Shld1, without effect on Leishmania growth or differentiation. We generated parasites lacking UGM through deletion of the GLF gene and substitution with a ddGLF fusion construct, either as chromosomal knockins or through episomal complementation; these showed little or no LPG expression in the absence of inducer, whereas in its presence, high levels of LPG were attained rapidly. Complement lysis tests confirmed the correct integrity of the Leishmania LPG coat. These data suggest that the dd approach has great promise in the study of LPG and other pathways relevant to parasite survival and virulence.

An RNA Interference (RNAi) Toolkit and Its Utility for Functional Genetic Analysis of Leishmania (Viannia).

RNA interference (RNAi) is a powerful tool whose efficacy against a broad range of targets enables functional genetic tests individually or systematically. However, the RNAi pathway has been lost in evolution by a variety of eukaryotes including most Leishmania sp. RNAi was retained in species of the Leishmania subgenus Viannia, and here we describe the development, optimization, and application of RNAi tools to the study of L. (Viannia) braziliensis (Lbr). We developed vectors facilitating generation of long-hairpin or "stem-loop" (StL) RNAi knockdown constructs, using GatewayTM site-specific recombinase technology. A survey of applications of RNAi in L. braziliensis included genes interspersed within multigene tandem arrays such as quinonoid dihydropteridine reductase (QDPR), a potential target or modulator of antifolate sensitivity. Other tests include genes involved in cell differentiation and amastigote proliferation (A600), and essential genes of the intraflagellar transport (IFT) pathway. We tested a range of stem lengths targeting the L. braziliensis hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and reporter firefly luciferase (LUC) genes and found that the efficacy of RNAi increased with stem length, and fell off greatly below about 128 nt. We used the StL length dependency to establish a useful 'hypomorphic' approach not possible with other gene ablation strategies, with shorter IFT140 stems yielding viable cells with compromised flagellar morphology. We showed that co-selection for RNAi against adenine phosphoryl transferase (APRT1) using 4-aminopyrazolpyrimidine (APP) could increase the efficacy of RNAi against reporter constructs, a finding that may facilitate improvements in future work. Thus, for many genes, RNAi provides a useful tool for studying Leishmania gene function with some unique advantages.

In and out: Leishmania metastasis by hijacking lymphatic system and migrating immune cells.

The lymphatic system plays a crucial role in mounting immune response against intracellular pathogens, and recent studies have documented its role in facilitating tumor dissemination linked largely with cancer cells. However, in mucocutaneous leishmaniasis (MCL) caused by Leishmania Viannia subgenus showing infectious metastasis and resulting in severe distant secondary lesions, the route of escape of these parasites to secondary sites has not yet been investigated in detail. Our results demonstrated that when infection was associated with inflammation and additionally exacerbated by the presence of dsRNA viral endosymbiont (LRV1), lymphatic vessels could serve as efficient routes for infected cells to egress from the primary site and colonize distant organs. We challenged this hypothesis by using the intracellular Leishmania protozoan parasites Leishmania guyanensis (Lgy) associated with or without a dsRNA viral endosymbiont, exacerbating the infection and responsible for a strong inflammatory response, and favoring metastasis of the infection. We analyzed possible cargo cells and the routes of dissemination through flow cytometry, histological analysis, and in vivo imaging in our metastatic model to show that parasites disseminated not only intracellularly but also as free extracellular parasites using migrating immune cells, lymph nodes (LNs), and lymph vessels, and followed intricate connections of draining and non-draining lymph node to finally end up in the blood and in distant skin, causing new lesions.

Importance of polyphosphate in the Leishmania life cycle.

Protozoan parasites contain negatively charged polymers of a few up to several hundreds of phosphate residues. In other organisms, these poly-phosphate (polyP) chains serve as an energy source and phosphate reservoir, and have been implicated in adaptation to stress and virulence of pathogenic organisms. In this study, we confirmed first that the polyP polymerase vacuolar transporter chaperone 4 (VTC4) is responsible for polyP synthesis in Leishmania parasites. During Leishmaniain vitro culture, polyP is accumulated in logarithmic growth phase and subsequently consumed once stationary phase is reached. However, polyP is not essential since VTC4-deficient (vtc4- ) Leishmania proliferated normally in culture and differentiated into infective metacyclic parasites and into intracellular and axenic amastigotes. In in vivo mouse infections, L. majorVTC4 knockout showed a delay in lesion formation but ultimately gave rise to strong pathology, although we were unable to restore virulence by complementation to confirm this phenotype. Knockdown of VTC4 did not alter the course of L. guyanensis infections in mice, suggesting that polyP was not required for infection, or that very low levels of it suffice for lesion development. At higher temperatures, Leishmania promastigotes highly consumed polyP, and both knockdown or deletion of VTC4 diminished parasite survival. Thus, although polyP was not essential in the life cycle of the parasite, our data suggests a role for polyP in increasing parasite survival at higher temperatures, a situation faced by the parasite when transmitted to humans.

Eukaryotic UDP-galactopyranose mutase (GLF gene) in microbial and metazoal pathogens.

Galactofuranose (Gal(f)) is a novel sugar absent in mammals but present in a variety of pathogenic microbes, often within glycoconjugates that play critical roles in cell surface formation and the infectious cycle. In prokaryotes, Gal(f) is synthesized as the nucleotide sugar UDP-Gal(f) by UDP-galactopyranose mutase (UGM) (gene GLF). Here we used a combinatorial bioinformatics screen to identify a family of candidate eukaryotic GLFs that had previously escaped detection. GLFs from three pathogens, two protozoa (Leishmania major and Trypanosoma cruzi) and one fungus (Cryptococcus neoformans), had UGM activity when expressed in Escherichia coli and assayed in vivo and/or in vitro. Eukaryotic GLFs are closely related to each other but distantly related to prokaryotic GLFs, showing limited conservation of core residues around the substrate-binding site and flavin adenine dinucleotide binding domain. Several eukaryotes not previously investigated for Gal(f) synthesis also showed strong GLF homologs with conservation of key residues. These included other fungi, the alga Chlamydomonas and the algal phleovirus Feldmannia irregularis, parasitic nematodes (Brugia, Onchocerca, and Strongyloides) and Caenorhabditis elegans, and the urochordates Halocynthia and Cionia. The C. elegans open reading frame was shown to encode UGM activity. The GLF phylogenetic distribution suggests that Gal(f) synthesis may occur more broadly in eukaryotes than previously supposed. Overall, GLF/Gal(f) synthesis in eukaryotes appears to occur with a disjunct distribution and often in pathogenic species, similar to what is seen in prokaryotes. Thus, UGM inhibition may provide an attractive drug target in those eukaryotes where Gal(f) plays critical roles in cellular viability and virulence.