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.

Administration of Bevacizumab and the Risk of Chronic Kidney Disease Development in Taiwan Residents: A Population-Based Retrospective Cohort Study.

Vascular endothelial growth factor (VEGF) plays a significant role as a pro-angiogenic and pro-permeability factor within the kidney. Bevacizumab is a pharmaceutical monoclonal anti-VEGF antibody that inhibits the growth of new blood vessels, which blocks blood supply and thereby restricts tumor growth. Thus, we conducted a nationwide study to explore the risk of chronic kidney disease (CKD) development in Taiwan residents after bevacizumab therapy. We drew data from the extensive National Health Insurance Research Database (NHIRD), which encompasses data from >99% of Taiwan's population from 1995 onwards. Individuals who received bevacizumab between 2012-2018 were identified as the bevacizumab cohort, with the index date set at the first usage. We randomly selected dates within the study period for the control group to serve as index dates. We excluded patients with a history of CKD prior to the index date or those <20 years old. In both cohorts, patients' propensity scores matched in a 1:1 ratio based on sex, age, index year, income, urbanization level, comorbidities, and medications. We found patients treated with bevacizumab had a significantly higher risk of contracting CKD than patients without bevacizumab (adjusted hazard ratio = 1.35, 95% confidence interval = 1.35-1.73). The risk of CKD was 1.35-fold higher in participants with bevacizumab treatment than those in the control group. These findings suggest that close monitoring of CKD development after bevacizumab administration is needed.

Viral discovery and diversity in trypanosomatid protozoa with a focus on relatives of the human parasite Leishmania.

Knowledge of viral diversity is expanding greatly, but many lineages remain underexplored. We surveyed RNA viruses in 52 cultured monoxenous relatives of the human parasite Leishmania (Crithidia and Leptomonas), as well as plant-infecting PhytomonasLeptomonas pyrrhocoris was a hotbed for viral discovery, carrying a virus (Leptomonas pyrrhocoris ostravirus 1) with a highly divergent RNA-dependent RNA polymerase missed by conventional BLAST searches, an emergent clade of tombus-like viruses, and an example of viral endogenization. A deep-branching clade of trypanosomatid narnaviruses was found, notable as Leptomonas seymouri bearing Narna-like virus 1 (LepseyNLV1) have been reported in cultures recovered from patients with visceral leishmaniasis. A deep-branching trypanosomatid viral lineage showing strong affinities to bunyaviruses was termed "Leishbunyavirus" (LBV) and judged sufficiently distinct to warrant assignment within a proposed family termed "Leishbunyaviridae" Numerous relatives of trypanosomatid viruses were found in insect metatranscriptomic surveys, which likely arise from trypanosomatid microbiota. Despite extensive sampling we found no relatives of the totivirus Leishmaniavirus (LRV1/2), implying that it was acquired at about the same time the Leishmania became able to parasitize vertebrates. As viruses were found in over a quarter of isolates tested, many more are likely to be found in the >600 unsurveyed trypanosomatid species. Viral loss was occasionally observed in culture, providing potentially isogenic virus-free lines enabling studies probing the biological role of trypanosomatid viruses. These data shed important insights on the emergence of viruses within an important trypanosomatid clade relevant to human disease.

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.

Association of the Endobiont Double-Stranded RNA Virus LRV1 With Treatment Failure for Human Leishmaniasis Caused by Leishmania braziliensis in Peru and Bolivia.

Cutaneous and mucosal leishmaniasis, caused in South America by Leishmania braziliensis, is difficult to cure by chemotherapy (primarily pentavalent antimonials [Sb(V)]). Treatment failure does not correlate well with resistance in vitro, and the factors responsible for treatment failure in patients are not well understood. Many isolates of L. braziliensis (>25%) contain a double-stranded RNA virus named Leishmaniavirus 1 (LRV1), which has also been reported in Leishmania guyanensis, for which an association with increased pathology, metastasis, and parasite replication was found in murine models. Here we probed the relationship of LRV1 to drug treatment success and disease in 97 L. braziliensis-infected patients from Peru and Bolivia. In vitro cultures were established, parasites were typed as L. braziliensis, and the presence of LRV1 was determined by reverse transcription-polymerase chain reaction, followed by sequence analysis. LRV1 was associated significantly with an increased risk of treatment failure (odds ratio, 3.99; P = .04). There was no significant association with intrinsic Sb(V) resistance among parasites, suggesting that treatment failure arises from LRV1-mediated effects on host metabolism and/or parasite survival. The association of LRV1 with clinical drug treatment failure could serve to guide more-effective treatment of tegumentary disease caused by L. braziliensis.

The structure and repertoire of small interfering RNAs in Leishmania (Viannia) braziliensis reveal diversification in the trypanosomatid RNAi pathway.

Among trypanosomatid protozoa the mechanism of RNA interference (RNAi) has been investigated in Trypanosoma brucei and to a lesser extent in Leishmania braziliensis. Although these two parasitic organisms belong to the same family, they are evolutionarily distantly related raising questions about the conservation of the RNAi pathway. Here we carried out an in-depth analysis of small interfering RNAs (siRNAs) associated with L. braziliensis Argonaute1 (LbrAGO1). In contrast to T. brucei, Leishmania siRNAs are sensitive to 3' end oxidation, indicating the absence of blocking groups, and the Leishmania genome does not code for a HEN1 RNA 2'-O-methyltransferase, which modifies small RNA 3' ends. Consistent with this observation, ~20% of siRNA 3' ends carry non-templated uridines. Thus siRNA biogenesis, and most likely their metabolism, is different in these organisms. Similarly to T. brucei, putative mobile elements and repeats constitute the major Leishmania siRNA-producing loci and AGO1 ablation leads to accumulation of long transcripts derived from putative mobile elements. However, contrary to T. brucei, no siRNAs were detected from other genomic regions with the potential to form double-stranded RNA, namely sites of convergent transcription and inverted repeats. Thus, our results indicate that organism-specific diversification has occurred in the RNAi pathway during evolution of the trypanosomatid lineage.

'Transient' genetic suppression facilitates generation of hexose transporter null mutants in Leishmania mexicana.

The genome of Leishmania mexicana encompasses a cluster of three glucose transporter genes designated LmxGT1, LmxGT2 and LmxGT3. Functional and genetic studies of a cluster null mutant (Δlmxgt1-3) have dissected the roles of these proteins in Leishmania metabolism and virulence. However, null mutants were recovered at very low frequency, and comparative genome hybridizations revealed that Δlmxgt1-3 mutants contained a linear extrachromosomal 40 kb amplification of a region on chromosome 29 not amplified in wild type parasites. These data suggested a model where this 29-40k amplicon encoded a second site suppressor contributing to parasite survival in the absence of GT1-3 function. To test this, we quantified the frequency of recovery of knockouts in the presence of individual overexpressed open reading frames covering the 29-40k amplicon. The data mapped the suppressor activity to PIFTC3, encoding a component of the intraflagellar transport pathway. We discuss possible models by which PIFTC3 might act to facilitate loss of GTs specifically. Surprisingly, by plasmid segregation we showed that continued PIFTC3 overexpression was not required for Δlmxgt1-3 viability. These studies provide the first evidence that genetic suppression can occur by providing critical biological functions transiently. This novel form of genetic suppression may extend to other genes, pathways and organisms.

The Oncolytic Avian Reovirus p17 Protein Inhibits Invadopodia Formation in Murine Melanoma Cancer Cells by Suppressing the FAK/Src Pathway and the Formation of theTKs5/NCK1 Complex.

To explore whether the p17 protein of oncolytic avian reovirus (ARV) mediates cell migration and invadopodia formation, we applied several molecular biological approaches for studying the involved cellular factors and signal pathways. We found that ARV p17 activates the p53/phosphatase and tensin homolog (PTEN) pathway to suppress the focal adhesion kinase (FAK)/Src signaling and downstream signal molecules, thus inhibiting cell migration and the formation of invadopodia in murine melanoma cancer cell line (B16-F10). Importantly, p17-induced formation of invadopodia could be reversed in cells transfected with the mutant PTENC124A. p17 protein was found to significantly reduce the expression levels of tyrosine kinase substrate 5 (TKs5), Rab40b, non-catalytic region of tyrosine kinase adaptor protein 1 (NCK1), and matrix metalloproteinases (MMP9), suggesting that TKs5 and Rab40b were transcriptionally downregulated by p17. Furthermore, we found that p17 suppresses the formation of the TKs5/NCK1 complex. Coexpression of TKs5 and Rab40b in B16-F10 cancer cells reversed p17-modulated suppression of the formation of invadopodia. This work provides new insights into p17-modulated suppression of invadopodia formation by activating the p53/PTEN pathway, suppressing the FAK/Src pathway, and inhibiting the formation of the TKs5/NCK1 complex.

Parasite hybridization promotes spreading of endosymbiotic viruses.

Viruses are the most abundant biological entities on Earth and play a significant role in the evolution of many organisms and ecosystems. In pathogenic protozoa, the presence of endosymbiotic viruses has been linked to an increased risk of treatment failure and severe clinical outcome. Here, we studied the molecular epidemiology of the zoonotic disease cutaneous leishmaniasis in Peru and Bolivia through a joint evolutionary analysis of Leishmania braziliensis parasites and their endosymbiotic Leishmania RNA virus. We show that parasite populations circulate in isolated pockets of suitable habitat and are associated with single viral lineages that appear in low prevalence. In contrast, groups of hybrid parasites were geographically and ecologically dispersed, and commonly infected from a pool of genetically diverse viruses. Our results suggest that parasite hybridization, likely due to increased human migration and ecological perturbations, increased the frequency of endosymbiotic interactions known to play a key role in disease severity.

Diversity and dissemination of viruses in pathogenic protozoa.

Viruses are the most abundant biological entities on Earth and play a significant role in the evolution of many organisms and ecosystems. In pathogenic protozoa, the presence of viruses has been linked to an increased risk of treatment failure and severe clinical outcome. Here, we studied the molecular epidemiology of the zoonotic disease cutaneous leishmaniasis in Peru and Bolivia through a joint evolutionary analysis of Leishmania braziliensis and their dsRNA Leishmania virus 1. We show that parasite populations circulate in tropical rainforests and are associated with single viral lineages that appear in low prevalence. In contrast, groups of hybrid parasites are geographically and ecologically more dispersed and associated with an increased prevalence, diversity and spread of viruses. Our results suggest that parasite gene flow and hybridization increased the frequency of parasite-virus symbioses, a process that may change the epidemiology of leishmaniasis in the region.

Retention and loss of RNA interference pathways in trypanosomatid protozoans.

RNA interference (RNAi) pathways are widespread in metaozoans but the genes required show variable occurrence or activity in eukaryotic microbes, including many pathogens. While some Leishmania lack RNAi activity and Argonaute or Dicer genes, we show that Leishmania braziliensis and other species within the Leishmania subgenus Viannia elaborate active RNAi machinery. Strong attenuation of expression from a variety of reporter and endogenous genes was seen. As expected, RNAi knockdowns of the sole Argonaute gene implicated this protein in RNAi. The potential for functional genetics was established by testing RNAi knockdown lines lacking the paraflagellar rod, a key component of the parasite flagellum. This sets the stage for the systematic manipulation of gene expression through RNAi in these predominantly diploid asexual organisms, and may also allow selective RNAi-based chemotherapy. Functional evolutionary surveys of RNAi genes established that RNAi activity was lost after the separation of the Leishmania subgenus Viannia from the remaining Leishmania species, a divergence associated with profound changes in the parasite infectious cycle and virulence. The genus Leishmania therefore offers an accessible system for testing hypothesis about forces that may select for the loss of RNAi during evolution, such as invasion by viruses, changes in genome plasticity mediated by transposable elements and gene amplification (including those mediating drug resistance), and/or alterations in parasite 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.

Leishmania guyanensis suppressed inducible nitric oxide synthase provoked by its viral endosymbiont.

Inducible nitric oxide synthase (iNOS) is essential to the production of nitric oxide (NO), an efficient effector molecule against intracellular human pathogens such as Leishmania protozoan parasites. Some strains of Leishmania are known to bear a viral endosymbiont termed Leishmania RNA virus 1 (LRV1). Recognition of LRV1 by the innate immune sensor Toll-like receptor-3 (TLR3) leads to conditions worsening the disease severity in mice. This process is governed by type I interferon (type I IFNs) arising downstream of TLR3 stimulation and favoring the formation of secondary metastatic lesions. The formation of these lesions is mediated by the inflammatory cytokine IL-17A and occurs in the absence, or low level of, protective cytokine IFN-γ. Here, we described that the presence of LRV1 led to the initial expression of iNOS and low production of NO that failed to control infection. We subsequently showed that LRV1-triggered type I IFN was essential but insufficient to induce robust iNOS induction, which requires strong activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Leishmania guyanensis carrying LRV1 (LgyLRV1+) parasites mitigated strong iNOS production by limiting NF-kB activation via the induction of tumor necrosis factor-alpha-induced protein 3 (TNFAIP3), also known as A20. Moreover, our data suggested that production of LRV1-induced iNOS could be correlated with parasite dissemination and metastasis via elevated secretion of IL-17A in the draining lymph nodes. Our findings support an additional strategy by which LRV1-bearing Leishmania guyanensis evaded killing by nitric oxide and suggest that low levels of LRV1-induced NO might contribute to parasite metastasis.

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.

Genome Assemblies across the Diverse Evolutionary Spectrum of Leishmania Protozoan Parasites.

We report the high-quality draft assemblies and gene annotations for 13 species and/or strains of the protozoan parasite genera Leishmania, Endotrypanum, and Crithidia, which span the phylogenetic diversity of the subfamily Leishmaniinae within the kinetoplastid order of the phylum Euglenazoa. These resources will support studies on the origins of parasitism.

Spontaneous excision and facilitated recovery as a control for phenotypes arising from RNA interference and other dominant transgenes.

An essential control for genetic manipulation of microbes is the regeneration of the wild-type state and phenotype to validate that any mutant phenotypes are 'on target'. For Leishmania gene knockouts, this is often done by re-expression of the target gene from episomal vectors, often bearing counter-selectable markers. Methods for similarly validating the outcomes from dominant mutations such as those arising from RNA interference (RNAi) are needed. We present here such an approach, relying on facilitated recovery after spontaneous excision - or 'popouts' - of dominant transgenes stably inserted into the ribosomal RNA array, utilizing GFP as a marker and single cell sorting to recover regenerated WT controls. We validate its utility using RNA interference knockdowns of the paraflagellar rod gene PFR2 of L. (Viannia) braziliensis. The method yields stably modified lines suitable for long term studies of Leishmania virulence, relies solely on host rather than introduced genetic machinery, and is thus readily applied in many species and circumstances including functional genetic testing.

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.

Leishmania guyanensis parasites block the activation of the inflammasome by inhibiting maturation of IL-1ß.

The various symptomatic outcomes of cutaneous leishmaniasis relates to the type and potency of its underlying inflammatory responses. Presence of the cytoplasmic Leishmania RNA virus-1 (LRV1) within Leishmania guyanensis, worsens lesional inflammation and parasite burden, as the viral dsRNA genome acts as a potent innate immunogen stimulating Toll-Like-Receptor-3 (TLR3). Here we investigated other innate pattern recognition receptors capable of reacting to dsRNA and potentially contributing to LRV1-mediated inflammatory pathology. We included the cytoplasmic dsRNA sensors, namely, the RIG-like receptors (RLRs) and the inflammasome-dependent and -independent Nod-like-receptors (NLRs). Our study found no role for RLRs or inflammasome-dependent NLRs in the pathology of L. guyanensis infection irrespective of its LRV1-status. Further, neither LRV1-bearing L. guyanensis (LgyLRV1+) nor LRV1-negative L. guyanensis (LgyLRV1-) activated the inflammasome in vitro. Interestingly, similarly to L. donovani, L. guyanensis infection induced the up-regulation of the A20 protein, known to be involved in the evasion of inflammasome activation. Moreover, we observed that LgyLRV1+ promoted the transcription of inflammasome-independent NLRC2 (also called NOD2) and NLRC5. However, only NLRC2 showed some contribution to LRV1-dependent pathology. These data confirmed that the endosomal TLR3 pathway is the dominant route of LRV1-dependent signalling, thus excluding the cytosolic and inflammasome pathways. We postulate that avoidance of the inflammasome pathways is likely an important mechanism of virulence in Leishmania infection irrespective of the LRV1-status.

Development of a semi-automated image-based high-throughput drug screening system.

We previously reported that the innate sensing of the endosymbiont Leishmania RNA virus 1 (LRV1) within Leishmania (Viannia) guyanensis through Toll-like receptor 3, worsens the pathogenesis of parasite infection in mice. The presence of LRV1 has been associated with the failure of first-line treatment in patients infected with LRV1 containing -L. guyanensis and -L. braziliensis parasites. Here, we established a semi-automated image-based high-throughput drug screening (HTDS) protocol to measure parasiticidal activity of the Prestwick chemical library in primary murine macrophages infected with LRV1-containing L. guyanensis. The two-independent screens generated 14 hit compounds with over sixty-nine percent reduction in parasite growth compared to control, at a single dose in both screens. Our screening strategy offers great potential in the search for new drugs and accelerates the discovery rate in the field of drug repurposing against Leishmania. Moreover, this technique allows the concomitant assessment of the effect of drug toxicity on host cell number.

A Narnavirus-Like Element from the Trypanosomatid Protozoan Parasite Leptomonas seymouri.

Genome sequences were determined for a novel RNA virus, Leptomonas seymouri Narna-like virus 1 (LepseyNLV1). A 2.9-kb segment encodes an RNA-dependent RNA polymerase (RdRp), while a smaller 1.5-kb segment showed no database search matches. This is the first report of bisegmented Narnaviridae from insect trypanosomatids.