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.

Mild depolarization of the inner mitochondrial membrane is a crucial component of an anti-aging program.

The mitochondria of various tissues from mice, naked mole rats (NMRs), and bats possess two mechanistically similar systems to prevent the generation of mitochondrial reactive oxygen species (mROS): hexokinases I and II and creatine kinase bound to mitochondrial membranes. Both systems operate in a manner such that one of the kinase substrates (mitochondrial ATP) is electrophoretically transported by the ATP/ADP antiporter to the catalytic site of bound hexokinase or bound creatine kinase without ATP dilution in the cytosol. One of the kinase reaction products, ADP, is transported back to the mitochondrial matrix via the antiporter, again through an electrophoretic process without cytosol dilution. The system in question continuously supports H+-ATP synthase with ADP until glucose or creatine is available. Under these conditions, the membrane potential, ∆ψ, is maintained at a lower than maximal level (i.e., mild depolarization of mitochondria). This ∆ψ decrease is sufficient to completely inhibit mROS generation. In 2.5-y-old mice, mild depolarization disappears in the skeletal muscles, diaphragm, heart, spleen, and brain and partially in the lung and kidney. This age-dependent decrease in the levels of bound kinases is not observed in NMRs and bats for many years. As a result, ROS-mediated protein damage, which is substantial during the aging of short-lived mice, is stabilized at low levels during the aging of long-lived NMRs and bats. It is suggested that this mitochondrial mild depolarization is a crucial component of the mitochondrial anti-aging system.

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.

The proteolytic activity of the paracaspase MALT1 is key in T cell activation.

The paracaspase MALT1 is pivotal in antigen receptor-mediated lymphocyte activation and lymphomagenesis. MALT1 contains a caspase-like domain, but it is unknown whether this domain is proteolytically active. Here we report that MALT1 had arginine-directed proteolytic activity that was activated after T cell stimulation, and we identify the signaling protein Bcl-10 as a MALT1 substrate. Processing of Bcl-10 after Arg228 was required for T cell receptor-induced cell adhesion to fibronectin. In contrast, MALT1 activity but not Bcl-10 cleavage was essential for optimal activation of transcription factor NF-kappaB and production of interleukin 2. Thus, the proteolytic activity of MALT1 is central to T cell activation, which suggests a possible target for the development of immunomodulatory or anticancer drugs.

Viruses of protozoan parasites and viral therapy: Is the time now right?

Infections caused by protozoan parasites burden the world with huge costs in terms of human and animal health. Most parasitic diseases caused by protozoans are neglected, particularly those associated with poverty and tropical countries, but the paucity of drug treatments and vaccines combined with increasing problems of drug resistance are becoming major concerns for their control and eradication. In this climate, the discovery/repurposing of new drugs and increasing effort in vaccine development should be supplemented with an exploration of new alternative/synergic treatment strategies. Viruses, either native or engineered, have been employed successfully as highly effective and selective therapeutic approaches to treat cancer (oncolytic viruses) and antibiotic-resistant bacterial diseases (phage therapy). Increasing evidence is accumulating that many protozoan, but also helminth, parasites harbour a range of different classes of viruses that are mostly absent from humans. Although some of these viruses appear to have no effect on their parasite hosts, others either have a clear direct negative impact on the parasite or may, in fact, contribute to the virulence of parasites for humans. This review will focus mainly on the viruses identified in protozoan parasites that are of medical importance. Inspired and informed by the experience gained from the application of oncolytic virus- and phage-therapy, rationally-driven strategies to employ these viruses successfully against parasitic diseases will be presented and discussed in the light of the current knowledge of the virus biology and the complex interplay between the viruses, the parasite hosts and the human host. We also highlight knowledge gaps that should be addressed to advance the potential of virotherapy against parasitic diseases.

Food restriction delays seasonal sexual maturation but does not increase torpor use in male bats.

Balancing energy budgets can be challenging, especially in periods of food shortage, adverse weather conditions and increased energy demand due to reproduction. Bats have particularly high energy demands compared to other mammals and regularly use torpor to save energy. However, while torpor limits energy expenditure, it can also downregulate important processes, such as sperm production. This constraint could result in a trade-off between energy saving and future reproductive capacity. We mimicked harsh conditions by restricting food and tested the effect on changes in body mass, torpor use and seasonal sexual maturation in male parti-coloured bats (Vespertilio murinus). Food-restricted individuals managed to maintain their initial body mass, while in well-fed males, mass increased. Interestingly, despite large differences in food availability, there were only small differences in torpor patterns. However, well-fed males reached sexual maturity up to half a month earlier. Our results thus reveal a complex trade-off in resource allocation; independent of resource availability, males maintain a similar thermoregulation strategy and favour fast sexual maturation, but limited resources and low body mass moderate this latter process.

Antiviral screening identifies adenosine analogs targeting the endogenous dsRNA Leishmania RNA virus 1 (LRV1) pathogenicity factor.

The endogenous double-stranded RNA (dsRNA) virus Leishmaniavirus (LRV1) has been implicated as a pathogenicity factor for leishmaniasis in rodent models and human disease, and associated with drug-treatment failures in Leishmania braziliensis and Leishmania guyanensis infections. Thus, methods targeting LRV1 could have therapeutic benefit. Here we screened a panel of antivirals for parasite and LRV1 inhibition, focusing on nucleoside analogs to capitalize on the highly active salvage pathways of Leishmania, which are purine auxotrophs. Applying a capsid flow cytometry assay, we identified two 2'-C-methyladenosine analogs showing selective inhibition of LRV1. Treatment resulted in loss of LRV1 with first-order kinetics, as expected for random virus segregation, and elimination within six cell doublings, consistent with a measured LRV1 copy number of about 15. Viral loss was specific to antiviral nucleoside treatment and not induced by growth inhibitors, in contrast to fungal dsRNA viruses. Comparisons of drug-treated LRV1+ and LRV1- lines recapitulated LRV1-dependent pathology and parasite replication in mouse infections, and cytokine secretion in macrophage infections. Agents targeting Totiviridae have not been described previously, nor are there many examples of inhibitors acting against dsRNA viruses more generally. The compounds identified here provide a key proof-of-principle in support of further studies identifying efficacious antivirals for use in in vivo studies of LRV1-mediated virulence.

Type I interferons induced by endogenous or exogenous viral infections promote metastasis and relapse of leishmaniasis.

The presence of the endogenous Leishmania RNA virus 1 (LRV1) replicating stably within some parasite species has been associated with the development of more severe forms of leishmaniasis and relapses after drug treatment in humans. Here, we show that the disease-exacerbatory role of LRV1 relies on type I IFN (type I IFNs) production by macrophages and signaling in vivo. Moreover, infecting mice with the LRV1-cured Leishmania guyanensis (LgyLRV1- ) strain of parasites followed by type I IFN treatment increased lesion size and parasite burden, quantitatively reproducing the LRV1-bearing (LgyLRV1+ ) infection phenotype. This finding suggested the possibility that exogenous viral infections could likewise increase pathogenicity, which was tested by coinfecting mice with L. guyanensis and lymphocytic choriomeningitis virus (LCMV), or the sand fly-transmitted arbovirus Toscana virus (TOSV). The type I IFN antiviral response increased the pathology of L. guyanensis infection, accompanied by down-regulation of the IFN-γ receptor normally required for antileishmanial control. Further, LCMV coinfection of IFN-γ-deficient mice promoted parasite dissemination to secondary sites, reproducing the LgyLRV1+ metastatic phenotype. Remarkably, LCMV coinfection of mice that had healed from L. guyanensis infection induced reactivation of disease pathology, overriding the protective adaptive immune response. Our findings establish that type I IFN-dependent responses, arising from endogenous viral elements (dsRNA/LRV1), or exogenous coinfection with IFN-inducing viruses, are able to synergize with New World Leishmania parasites in both primary and relapse infections. Thus, viral infections likely represent a significant risk factor along with parasite and host factors, thereby contributing to the pathological spectrum of human leishmaniasis.

Leishmania amazonensis downregulates macrophage iNOS expression via Histone Deacetylase 1 (HDAC1): a novel parasite evasion mechanism.

The induced expression of nitric oxide synthase (iNOS) controls the intracellular growth of Leishmania in infected macrophages. Histones deacetylases (HDACs) negatively regulate gene expression through the formation of complexes containing transcription factors such as NF-κB p50/50. Herein, we demonstrated the occupancy of p50/p50_HDAC1 to iNOS promoter associated with reduced levels of H3K9Ac. Remarkably, we found increased levels of HDAC1 in L. amazonensis-infected macrophages. HDAC1 upregulation was not found in L. major-infected macrophages. The parasite intracellular load was reduced in HDAC1 knocked-down macrophages, which presented increased nitric oxide levels. HDAC1 silencing led to the occupancy of CBP/p300 to iNOS promoter and the rise of H3K9Ac modification. Importantly, the immunostaining of skin samples from hiporeactive cutaneous leishmaniasis patients infected with L. amazonensis, revealed high levels of HDAC1. In brief, L. amazonensis induces HDAC1 in infected macrophages, which contribute to parasite survival and is associated to hiporeactive stage found in L. amazonensis infected patients.

Modification of sperm fatty acid composition during epididymal maturation in bats.

Biochemical properties of polyunsaturated fatty acids (PUFAs) are fundamental to sperm movements. Amongst all adjustments operated during epididymal maturation, sperm membrane lipid composition is remodelled. Specifically, the proportion of PUFAs usually increases from the caput towards the cauda epididymidis. In mammals, PUFAs are predominantly acquired through the diet, which can consequently impact male fertility. We aimed at analysing to what extent n-6 and n-3 PUFAs are incorporated into sperm in the Seba's short-tailed bat (Carollia perspicillata), and at demonstrating the effect of the sperm fatty acid composition on sperm mobility. We therefore provided food varying in fatty acid composition to males of C. perspicillata and measured the fatty acid composition and mobility traits in spermatozoa collected from the caput and cauda epididymides. We found that n-6 and n-3 PUFAs and saturated fatty acids were significantly related to sperm velocity but not to the proportion of progressive sperm (i.e. motility). Concomitant to an increase in sperm velocity, the level of fatty acid saturation increased from the caput to the cauda epididymidis, while the proportion of PUFAs remained similar along the epididymis. A reduction in n-6 PUFAs counterbalanced an increase in n-3 PUFAs. The food treatments did not affect the sperm fatty acid composition. Our results suggest that a precise endogenous control rather than dietary effects determines sperm fatty acid composition in C. perspicillata.

How to master the host immune system? Leishmania parasites have the solutions!

Infection by protozoan parasites of the genus Leishmania results in the development of leishmaniasis, an increasingly prevalent group of diseases affecting over 12 million people worldwide. Leishmaniasis can have very different outcomes ranging from cutaneous lesions, mucosal lesions to visceralization depending on the species of the infecting parasite and on the immune response developed by the host. As an obligate intracellular parasite, residing within macrophages, Leishmania evolved in strict contact with the host immune system, developing different mechanisms to evade or modulate the immune response. Various types of immune responses are observed during different Leishmania spp. infections, resulting in parasite clearance but also contributing to the pathogenesis, thus increasing the complexity of the course of the disease. Interestingly, depending on the type of leishmaniasis developed, opposite treatment strategies, which either boost or inhibit the inflammatory response, have shown efficacy. In this review, we summarize the contribution of different immune cell types to the development of the anti-leishmanial immune response and the parasite strategies to evade and modulate host immunity. Further, we discuss the involvement of co-infecting pathogens in the determination of the outcome of leishmaniasis and on the effectiveness of treatment and the implication of the immune response for treatment and vaccine development.

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.

Leishmaniavirus-Dependent Metastatic Leishmaniasis Is Prevented by Blocking IL-17A.

Cutaneous leishmaniasis has various outcomes, ranging from self-healing reddened papules to extensive open ulcerations that metastasise to secondary sites and are often resistant to standard therapies. In the case of L. guyanensis (L.g), about 5-10% of all infections result in metastatic complications. We recently showed that a cytoplasmic virus within L.g parasites (LRV1) is able to act as a potent innate immunogen, worsening disease outcome in a murine model. In this study, we investigated the immunophenotype of human patients infected by L.g and found a significant association between the inflammatory cytokine IL-17A, the presence of LRV1 and disease chronicity. Further, IL-17A was inversely correlated to the protective cytokine IFN-γ. These findings were experimentally corroborated in our murine model, where IL-17A produced in LRV1+ L.g infection contributed to parasite virulence and dissemination in the absence of IFN-γ. Additionally, IL-17A inhibition in mice using digoxin or SR1001, showed therapeutic promise in limiting parasite virulence. Thus, this murine model of LRV1-dependent infectious metastasis validated markers of disease chronicity in humans and elucidated the immunologic mechanism for the dissemination of Leishmania parasites to secondary sites. Moreover, it confirms the prognostic value of LRV1 and IL-17A detection to prevent metastatic leishmaniasis in human patients.

Presence of Leishmania RNA Virus 1 in Leishmania guyanensis Increases the Risk of First-Line Treatment Failure and Symptomatic Relapse.

Treatment failure and symptomatic relapse are major concerns in American tegumentary leishmaniasis (TL). Such complications are seen frequently in Leishmania guyanensis infections, in which patients respond variously to first-line antileishmanials and are more prone to develop chronic cutaneous leishmaniasis. The factors underlying this pathology, however, are unknown. Recently, we reported that a double-stranded RNA virus, Leishmania RNA virus 1 (LRV1), nested within L. guyanensis parasites is able to exacerbate experimental murine leishmaniasis by inducing a hyperinflammatory response. This report investigates the prevalence of LRV1 in human L. guyanensis infection and its effect on treatment efficacy, as well as its correlation to symptomatic relapses after the completion of first-line treatment. In our cohort of 75 patients with a diagnosis of primary localized American TL, the prevalence of LRV1-positive L. guyanensis infection was elevated to 58%. All patients infected with LRV1-negative L. guyanensis were cured after 1 dose (22 of 31 [71%]) or 2 doses (31 of 31 [100%]) of pentamidine. In contrast, 12 of 44 LRV1-positive patients (27%) presented with persistent infection and symptomatic relapse that required extended therapy and the use of second-line drugs. Finally, LRV1 presence was associated with a significant increase in levels of intra-lesional inflammatory markers. In conclusion, LRV1 status in L. guyanensis infection is significantly predictive (P = .0009) of first-line treatment failure and symptomatic relapse and has the potential to guide therapeutic choices in American TL.

Modification of sperm quality after sexual abstinence in Seba's short-tailed bat, Carollia perspicillata.

In polygynous mating systems, few males have stable access to sexual mates. With an expected higher copulation rate, harem males may deplete seminal fluids or increase epididymal sperm maturation, generating poor sperm quality. In a first study, we reported a higher sperm quality in sneaker males of Carollia perspicillata To test whether the lower sperm quality observed in harem males was generated by an elevated copulation rate, we temporarily removed males of both social statuses from the colony. We thus assessed status-related changes of sperm quality resulting from sexual abstinence. Moreover, released from territory and female guarding, harem males were expected to show a reduction in somatic costs. On the basis of sperm competition models, we predicted a higher resource investment in the ejaculate with the reduction of pre-copulatory efforts. In line with our predictions, sperm quality of harem males improved significantly in contrast to sneaker males, whose sperm quality did not change. Without an increase in ejaculate lipid peroxidation, our results also provide evidence that the duration of sexual abstinence was not sufficient to generate sperm oxidative damage through senescence. Harem males did not show a reduction in blood lipid peroxidation or in the ratio of oxidized to reduced glutathione. In line with the maintenance of these somatic costs, harem males did not invest more superoxide dismutase to the ejaculate to maintain sperm quality. Our results suggest that a difference in copulation rate rather than an adaptation to sperm competition provides sneaker males with higher sperm quality in C. perspicillata.

Cross-presenting dendritic cells are required for control of Leishmania major infection.

Leishmania major infection induces self-healing cutaneous lesions in C57BL/6 mice. Both IL-12 and IFN-γ are essential for the control of infection. We infected Jun dimerization protein p21SNFT (Batf3(-/-) ) mice (C57BL/6 background) that lack the major IL-12 producing and cross-presenting CD8α(+) and CD103(+) DC subsets. Batf3(-/-) mice displayed enhanced susceptibility with larger lesions and higher parasite burden. Additionally, cells from draining lymph nodes of infected Batf3(-/-) mice secreted less IFN-γ, but more Th2- and Th17-type cytokines, mirrored by increased serum IgE and Leishmania-specific immunoglobulin 1 (Th2 indicating). Importantly, CD8α(+) DCs isolated from lymph nodes of L. major-infected mice induced significantly more IFN-γ secretion by L. major-stimulated immune T cells than CD103(+) DCs. We next developed CD11c-diptheria toxin receptor: Batf3(-/-) mixed bone marrow chimeras to determine when the DCs are important for the control of infection. Mice depleted of Batf-3-dependent DCs from day 17 or wild-type mice depleted of cross-presenting DCs from 17-19 days after infection maintained significantly larger lesions similar to mice whose Batf-3-dependent DCs were depleted from the onset of infection. Thus, we have identified a crucial role for Batf-3-dependent DCs in protection against L. major.

Role of Toll-like receptor 9 signaling in experimental Leishmania braziliensis infection.

Infection with Leishmania braziliensis causes cutaneous or mucocutaneous leishmaniasis in humans. Toll-like receptor 9 (TLR9) expression has been found in granulomas of lesions in L. braziliensis-infected individuals. L. braziliensis inoculation in mice induces very small lesions that are self-healing, whereas deficiency in the TLR adaptor molecule, MyD88, renders mice susceptible to infection. The TLR involved has not been identified, prompting us to investigate if TLR9 triggering by the parasite contributes to the strong resistance to infection observed in L. braziliensis-inoculated mice. The parasites activated wild-type (WT) dendritic cells (DCs) in vitro but not DCs derived from TLR9(-/-) mice. TLR9(-/-) mice inoculated with L. braziliensis exhibited a transient susceptibility characterized by increased lesion size and parasite burden compared to those of WT mice. Surprisingly, elevated levels of gamma interferon (IFN-γ) were measured at the site of infection and in draining lymph node T cells of TLR9(-/-) mice at the peak of susceptibility, suggesting that unlike observations in vitro, the parasite could induce DC activation leading to the development of Th1 cells in the absence of TLR9 expression. Taken together, these data show that TLR9 signaling is important for the early control of lesion development and parasite burden but is dispensable for the differentiation of Th1 cells secreting IFN-γ, and the high levels of this cytokine are not sufficient to control early parasite replication following L. braziliensis infection.

Mating without intromission in a bat.

Copulatory behaviours stand as cornerstones of sexual selection, yet they remain mysterious in many species. Because of their nocturnal and elusive lifestyle, the copulatory behaviours of bats have been mostly overlooked1. Several aspects of bat reproduction differ from other mammals (e.g. prolonged sperm storage2, delayed development3). Here, we show that in serotine bats (Eptesicus serotinus) the penis is used as a 'copulatory arm' rather than an intromittent organ, revealing a novel copulatory behaviour in mammals.

Processing of metacaspase into a cytoplasmic catalytic domain mediating cell death in Leishmania major.

Metacaspases are cysteine peptidases that could play a role similar to caspases in the cell death programme of plants, fungi and protozoa. The human protozoan parasite Leishmania major expresses a single metacaspase (LmjMCA) harbouring a central domain with the catalytic dyad histidine and cysteine as found in caspases. In this study, we investigated the processing sites important for the maturation of LmjMCA catalytic domain, the cellular localization of LmjMCA polypeptides, and the functional role of the catalytic domain in the cell death pathway of Leishmania parasites. Although LmjMCA polypeptide precursor form harbours a functional mitochondrial localization signal (MLS), we determined that LmjMCA polypeptides are mainly localized in the cytoplasm. In stress conditions, LmjMCA precursor forms were extensively processed into soluble forms containing the catalytic domain. This domain was sufficient to enhance sensitivity of parasites to hydrogen peroxide by impairing the mitochondrion. These data provide experimental evidences of the importance of LmjMCA processing into an active catalytic domain and of its role in disrupting mitochondria, which could be relevant in the design of new drugs to fight leishmaniasis and likely other protozoan parasitic diseases.