The SET7 protein of Leishmania donovani moderates the parasite's response to a hostile oxidative environment.

SET domain proteins methylate specific lysines on proteins, triggering stimulation or repression of downstream processes. Twenty-nine SET domain proteins have been identified in Leishmania donovani through sequence annotations. This study initiates the first investigation into these proteins. We find LdSET7 is predominantly cytosolic. Although not essential, set7 deletion slows down promastigote growth and hypersensitizes the parasite to hydroxyurea-induced G1/S arrest. Intriguingly, set7-nulls survive more proficiently than set7+/+ parasites within host macrophages, suggesting that LdSET7 moderates parasite response to the inhospitable intracellular environment. set7-null in vitro promastigote cultures are highly tolerant to hydrogen peroxide (H2O2)-induced stress, reflected in their growth pattern, and no detectable DNA damage at H2O2 concentrations tested. This is linked to reactive oxygen species levels remaining virtually unperturbed in set7-nulls in response to H2O2 exposure, contrasting to increased reactive oxygen species in set7+/+ cells under similar conditions. In analyzing the cell's ability to scavenge hydroperoxides, we find peroxidase activity is not upregulated in response to H2O2 exposure in set7-nulls. Rather, constitutive basal levels of peroxidase activity are significantly higher in these cells, implicating this to be a factor contributing to the parasite's high tolerance to H2O2. Higher levels of peroxidase activity in set7-nulls are coupled to upregulation of tryparedoxin peroxidase transcripts. Rescue experiments using an LdSET7 mutant suggest that LdSET7 methylation activity is critical to the modulation of the cell's response to oxidative environment. Thus, LdSET7 tunes the parasite's behavior within host cells, enabling the establishment and persistence of infection without eradicating the host cell population it needs for survival.

Molecular regulation of virulence in Legionella pneumophila.

Legionella pneumophila is a gram-negative bacteria found in natural and anthropogenic aquatic environments such as evaporative cooling towers, where it reproduces as an intracellular parasite of cohabiting protozoa. If L. pneumophila is aerosolized and inhaled by a susceptible person, bacteria may colonize their alveolar macrophages causing the opportunistic pneumonia Legionnaires' disease. L. pneumophila utilizes an elaborate regulatory network to control virulence processes such as the Dot/Icm Type IV secretion system and effector repertoire, responding to changing nutritional cues as their host becomes depleted. The bacteria subsequently differentiate to a transmissive state that can survive in the environment until a replacement host is encountered and colonized. In this review, we discuss the lifecycle of L. pneumophila and the molecular regulatory network that senses nutritional depletion via the stringent response, a link to stationary phase-like metabolic changes via alternative sigma factors, and two-component systems that are homologous to stress sensors in other pathogens, to regulate differentiation between the intracellular replicative phase and more transmissible states. Together, we highlight how this prototypic intracellular pathogen offers enormous potential in understanding how molecular mechanisms enable intracellular parasitism and pathogenicity.

Trypanosoma cruzi infection reduces the population fitness of Mepraia spinolai, a Chagas disease vector.

The hematophagous insect Mepraia spinolai (Hemiptera: Reduviidae: Triatominae) is naturally infected with the protozoan parasite Trypanosoma cruzi, the agent of Chagas disease in humans. In this study, we compared the demographic parameters of M. spinolai with and without T. cruzi infection. We collected the immature life table data of 479 M. spinolai individuals of control cohort (reared on mice without T. cruzi infection) and 563 M. spinolai individuals of treatment cohort (reared on mice with T. cruzi infection). Nymphs were maintained in individual compartments inside a growth chamber (26°C; 65-75%) until adult emergence; moulting and survival were recorded daily. For the adult life table study of the control, we used 24 pairs of adults from the control cohort. For the adult life table study of T. cruzi-infected cohort, 25 infected females were paired with 25 males from the control cohort. Life table data were analysed using bootstrap-match technique based on the age-stage, two-sex life table. The preadult survival rate (0.5282) of the control cohort was significantly higher than that of the infected cohort (0.2913). However, the mean fecundity of reproductive females (Fr = 22.29 eggs/♀) and net reproductive rate of population (R0 = 5.07 offspring/individual) of the 0.5th percentile bootstrap-match control cohort were not significantly different from those of the infected cohort (Fr = 23.35 eggs/♀, R0 = 3.77 offspring/individual). Due to the shorter total preoviposition period and higher proportion of reproductive female, the intrinsic rate of increase (r = 0.0053 d-1 ) and finite rate of increase (λ = 1.0053 d-1 ) of control cohort of M. spinolai were significantly higher than those of the T. cruzi-infected cohort (r = 0.0035 d-1 , λ = 1.0035 d-1 ). These results suggest that T. cruzi infection reduces the population fitness of the Chagas disease vector M. spinolai.

Inositol phosphorylceramide synthase null Leishmania are viable and virulent in animal infections where salvage of host sphingomyelin predominates.

Many pathogens synthesize inositol phosphorylceramide (IPC) as the major sphingolipid (SL), differing from the mammalian host where sphingomyelin (SM) or more complex SLs predominate. The divergence between IPC synthase and mammalian SL synthases has prompted interest as a potential drug target. However, in the trypanosomatid protozoan Leishmania, cultured insect stage promastigotes lack de novo SL synthesis (Δspt2-) and SLs survive and remain virulent, as infective amastigotes salvage host SLs and continue to produce IPC. To further understand the role of IPC, we generated null IPCS mutants in Leishmania major (Δipcs-). Unexpectedly and unlike fungi where IPCS is essential, Δipcs- was remarkably normal in culture and highly virulent in mouse infections. Both IPCS activity and IPC were absent in Δipcs- promastigotes and amastigotes, arguing against an alternative route of IPC synthesis. Notably, salvaged mammalian SM was highly abundant in purified amastigotes from both WT and Δipcs-, and salvaged SLs could be further metabolized into IPC. SM was about 7-fold more abundant than IPC in WT amastigotes, establishing that SM is the dominant amastigote SL, thereby rendering IPC partially redundant. These data suggest that SM salvage likely plays key roles in the survival and virulence of both WT and Δipcs- parasites in the infected host, confirmation of which will require the development of methods or mutants deficient in host SL/SM uptake in the future. Our findings call into question the suitability of IPCS as a target for chemotherapy, instead suggesting that approaches targeting SM/SL uptake or catabolism may warrant further emphasis.

Metacyclogenesis as the Starting Point of Chagas Disease.

Chagas disease is a neglected infectious disease caused by the protozoan Trypanosoma cruzi, primarily transmitted by triatomine vectors, and it threatens approximately seventy-five million people worldwide. This parasite undergoes a complex life cycle, transitioning between hosts and shifting from extracellular to intracellular stages. To ensure its survival in these diverse environments, T. cruzi undergoes extreme morphological and molecular changes. The metacyclic trypomastigote (MT) form, which arises from the metacyclogenesis (MTG) process in the triatomine hindgut, serves as a crucial link between the insect and human hosts and can be considered the starting point of Chagas disease. This review provides an overview of the current knowledge regarding the parasite's life cycle, molecular pathways, and mechanisms involved in metabolic and morphological adaptations during MTG, enabling the MT to evade the immune system and successfully infect human cells.

Comprehensive characterization of purine and pyrimidine transport activities in Trichomonas vaginalis and functional cloning of a trichomonad nucleoside transporter.

Trichomoniasis is a common and widespread sexually-transmitted infection, caused by the protozoan parasite Trichomonas vaginalis. T. vaginalis lacks the biosynthetic pathways for purines and pyrimidines, making nucleoside metabolism a drug target. Here we report the first comprehensive investigation into purine and pyrimidine uptake by T. vaginalis. Multiple carriers were identified and characterized with regard to substrate selectivity and affinity. For nucleobases, a high-affinity adenine transporter, a possible guanine transporter and a low affinity uracil transporter were found. Nucleoside transporters included two high affinity adenosine/guanosine/uridine/cytidine transporters distinguished by different affinities to inosine, a lower affinity adenosine transporter, and a thymidine transporter. Nine Equilibrative Nucleoside Transporter (ENT) genes were identified in the T. vaginalis genome. All were expressed equally in metronidazole-resistant and -sensitive strains. Only TvagENT2 was significantly upregulated in the presence of extracellular purines; expression was not affected by co-culture with human cervical epithelial cells. All TvagENTs were cloned and separately expressed in Trypanosoma brucei. We identified the main broad specificity nucleoside carrier, with high affinity for uridine and cytidine as well as purine nucleosides including inosine, as TvagENT3. The in-depth characterization of purine and pyrimidine transporters provides a critical foundation for the development of new anti-trichomonal nucleoside analogues.

Covalent Reversible Inhibitors of Cysteine Proteases Containing the Nitrile Warhead: Recent Advancement in the Field of Viral and Parasitic Diseases.

In the field of drug discovery, the nitrile group is well represented among drugs and biologically active compounds. It can form both non-covalent and covalent interactions with diverse biological targets, and it is amenable as an electrophilic warhead for covalent inhibition. The main advantage of the nitrile group as a warhead is mainly due to its milder electrophilic character relative to other more reactive groups (e.g., -CHO), reducing the possibility of unwanted reactions that would hinder the development of safe drugs, coupled to the ease of installation through different synthetic approaches. The covalent inhibition is a well-assessed design approach for serine, threonine, and cysteine protease inhibitors. The mechanism of hydrolysis of these enzymes involves the formation of a covalent acyl intermediate, and this mechanism can be exploited by introducing electrophilic warheads in order to mimic this covalent intermediate. Due to the relevant role played by the cysteine protease in the survival and replication of infective agents, spanning from viruses to protozoan parasites, we will review the most relevant and recent examples of protease inhibitors presenting a nitrile group that have been introduced to form or to facilitate the formation of a covalent bond with the catalytic cysteine active site residue.

Crystal structures of the RNA triphosphatase from Trypanosoma cruzi provide insights into how it recognizes the 5'-end of the RNA substrate.

RNA triphosphatase catalyzes the first step in mRNA cap formation, hydrolysis of the terminal phosphate from the nascent mRNA transcript. The RNA triphosphatase from the protozoan parasite Trypanosoma cruzi, TcCet1, belongs to the family of triphosphate tunnel metalloenzymes (TTMs). TcCet1 is a promising antiprotozoal drug target because the mechanism and structure of the protozoan RNA triphosphatases are completely different from those of the RNA triphosphatases found in mammalian and arthropod hosts. Here, we report several crystal structures of the catalytically active form of TcCet1 complexed with a divalent cation and an inorganic tripolyphosphate in the active-site tunnel at 2.20-2.51 Å resolutions. The structures revealed that the overall structure, the architecture of the tunnel, and the arrangement of the metal-binding site in TcCet1 are similar to those in other TTM proteins. On the basis of the position of three sulfate ions that cocrystallized on the positively charged surface of the protein and results obtained from mutational analysis, we identified an RNA-binding site in TcCet1. We conclude that the 5'-end of the triphosphate RNA substrate enters the active-site tunnel directionally. The structural information reported here provides valuable insight into designing inhibitors that could specifically block the entry of the triphosphate RNA substrate into the TTM-type RNA triphosphatases of T. cruzi and related pathogens.

Adjuvanted vaccines driven protection against visceral infection in BALB/c mice by Leishmania donovani.

Kinteoplastid protozoan parasite of genus Leishmania is the pathogen that causes leishmaniasis. Its prevalence is highest after malaria and visceral leishmaniasis is the most dreaded form of infection. No vaccine is available for the disease management and it relies wholly on a few chemotherapeutic agents which are toxic and besides drug resistance their costs are the limitations. Therefore, development of an effective vaccine is urgently required. In this study, Montanide ISA 201 and AddaVax were assessed for their adjuvant potential along with formalin-inactivated or killed vaccine for the immune induction. Immunological and parasitological studies were conducted to evaluate the efficacy of different vaccine formulations in BALB/c mice before challenge infection as well as 4, 8, and 12 weeks after challenge. The efficacy of vaccines was evidenced with reduced parasite burden, the higher DTH response, Th1 cytokines, and IgG2a isotype antibody in immunized mice. All the vaccines showed their potential against Leishmania donovani infection and vaccine formulated with Montanide ISA 201 exhibited maximum efficacy. Our results suggest the potential of these vaccine formulations in controlling Leishmania infection.

Characterization of the ATP4 ion pump in Toxoplasma gondii.

The Plasmodium falciparum ATPase PfATP4 is the target of a diverse range of antimalarial compounds, including the clinical drug candidate cipargamin. PfATP4 was originally annotated as a Ca2+ transporter, but recent evidence suggests that it is a Na+ efflux pump, extruding Na+ in exchange for H+ Here we demonstrate that ATP4 proteins belong to a clade of P-type ATPases that are restricted to apicomplexans and their closest relatives. We employed a variety of genetic and physiological approaches to investigate the ATP4 protein of the apicomplexan Toxoplasma gondii, TgATP4. We show that TgATP4 is a plasma membrane protein. Knockdown of TgATP4 had no effect on resting pH or Ca2+ but rendered parasites unable to regulate their cytosolic Na+ concentration ([Na+]cyt). PfATP4 inhibitors caused an increase in [Na+]cyt and a cytosolic alkalinization in WT but not TgATP4 knockdown parasites. Parasites in which TgATP4 was knocked down or disrupted exhibited a growth defect, attributable to reduced viability of extracellular parasites. Parasites in which TgATP4 had been disrupted showed reduced virulence in mice. These results provide evidence for ATP4 proteins playing a key conserved role in Na+ regulation in apicomplexan parasites.

The GRA15 protein from Toxoplasma gondii enhances host defense responses by activating the interferon stimulator STING.

Toxoplasma gondii is an important neurotropic pathogen that establishes latent infections in humans that can cause toxoplasmosis in immunocompromised individuals. It replicates inside host cells and has developed several strategies to manipulate host immune responses. However, the cytoplasmic pathogen-sensing pathway that detects T. gondii is not well-characterized. Here, we found that cyclic GMP-AMP synthase (cGAS), a sensor of foreign dsDNA, is required for activation of anti-T. gondii immune signaling in a mouse model. We also found that mice deficient in STING (Stinggt/gt mice) are much more susceptible to T. gondii infection than WT mice. Of note, the induction of inflammatory cytokines, type I IFNs, and interferon-stimulated genes in the spleen from Stinggt/gt mice was significantly impaired. Stinggt/gt mice exhibited more severe symptoms than cGAS-deficient mice after T. gondii infection. Interestingly, we found that the dense granule protein GRA15 from T. gondii is secreted into the host cell cytoplasm and then localizes to the endoplasmic reticulum, mediated by the second transmembrane motif in GRA15, which is essential for activating STING and innate immune responses. Mechanistically, GRA15 promoted STING polyubiquitination at Lys-337 and STING oligomerization in a TRAF protein-dependent manner. Accordingly, GRA15-deficient T. gondii failed to elicit robust innate immune responses compared with WT T. gondii. Consequently, GRA15-/-T. gondii was more virulent and caused higher mortality of WT mice but not Stinggt/gt mice upon infection. Together, T. gondii infection triggers cGAS/STING signaling, which is enhanced by GRA15 in a STING- and TRAF-dependent manner.

Novel Antiparasitic Activity of the Antifungal Lead Occidiofungin.

Novel antiparasitic activity was observed for the antifungal occidiofungin. It efficaciously and irreversibly inhibited the zoonotic enteric parasite Cryptosporidium parvumin vitro with limited cytotoxicity (50% effective concentration [EC50] = 120 nM versus 50% cytotoxic concentration [TC50] = 988 nM), and its application disrupted the parasite morphology. This study expands the spectrum of activity of a glycolipopeptide named occidiofungin. Occidiofungin has poor gastrointestinal tract absorption properties, supporting future investigations into its potential activities on other enteric parasites.

A review of outbreaks of cryptosporidiosis due to unpasteurized milk.

PURPOSE: This review analysed outbreaks of human cryptosporidiosis due to raw milk. The objective of our study was to highlight and identify underestimated and underreported aspects of transmission of the parasite as well as the added value of genotyping Cryptosporidium isolates. METHODS: We conducted a descriptive literature review using the digital archives Pubmed and Embase. All original papers and case reports referring to outbreaks of Cryptosporidium due to unpasteurized milk were reviewed. The cross-references from these publications were also included. RESULTS: Outbreaks have been described in the USA, Australia, and the UK. Laboratory evidence of Cryptosporidium from milk specimens was lacking in the majority of the investigations. However, in most recent reports molecular tests on stool specimens along with epidemiological data supported that the infection was acquired through the consumption of unpasteurized milk. As the incubation period for Cryptosporidium is relatively long (days to weeks) compared with many other foodborne pathogens (hours to days), these reports often lack microbiological confirmation because, by the time the outbreak was identified, the possibly contaminated milk was not available anymore. CONCLUSION: Cryptosporidiosis is generally considered a waterborne intestinal infection, but several reports on foodborne transmission (including through raw milk) have been reported in the literature. Calves are frequently infected with Cryptosporidium spp., which does not multiply in milk. However, Cryptosporidium oocysts can survive if pasteurization fails. Thus, pasteurization is essential to inactivate oocysts. Although cryptosporidiosis cases acquired from raw milk are seldom reported, the risk should not be underestimated and Cryptosporidium should be considered as a potential agent of contamination. Genotyping Cryptosporidium isolates might be a supportive tool to strengthen epidemiologic evidence as well as to estimate the burden of the disease.

The dynamics of ultrastructural changes during Entamoeba invadens encystation.

Entamoeba histolytica infection causes amoebiasis, which is a global public health problem. The major route of infection is oral ingestion of E. histolytica cysts, cysts being the sole form responsible for host-to-host transmission. Cysts are produced by cell differentiation from proliferative trophozoites in a process termed 'encystation'. Therefore, encystation is an important process from a medical as well as a biological perspective. Previous electron microscopy studies have shown the ultrastructure of precysts and mature cysts; however, the dynamics of ultrastructural changes during encystation were ambiguous. Here, we analysed a series of Entamoeba invadens encysting cells by transmission electron microscopy. Entamoeba invadens is a model for encystation and the cells were prepared by short interval time course sampling from in vitro encystation-inducing cultures. We related sampled cells to stage conversion, which was monitored in the overall population by flow cytometry. The present approach revealed the dynamics of ultrastructure changes during E. invadens encystation. Importantly, the results indicate a functional linkage of processes that are crucial in encystation, such as glycogen accumulation and cyst wall formation. Hence, this study provides a reference for studying sequential molecular events during Entamoeba encystation.

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.

Continual renewal and replication of persistent Leishmania major parasites in concomitantly immune hosts.

In most natural infections or after recovery, small numbers of Leishmania parasites remain indefinitely in the host. Persistent parasites play a vital role in protective immunity against disease pathology upon reinfection through the process of concomitant immunity, as well as in transmission and reactivation, yet are poorly understood. A key question is whether persistent parasites undergo replication, and we devised several approaches to probe the small numbers in persistent infections. We find two populations of persistent Leishmania major: one rapidly replicating, similar to parasites in acute infections, and another showing little evidence of replication. Persistent Leishmania were not found in "safe" immunoprivileged cell types, instead residing in macrophages and DCs, ∼60% of which expressed inducible nitric oxide synthase (iNOS). Remarkably, parasites within iNOS+ cells showed normal morphology and genome integrity and labeled comparably with BrdU to parasites within iNOS- cells, suggesting that these parasites may be unexpectedly resistant to NO. Nonetheless, because persistent parasite numbers remain roughly constant over time, their replication implies that ongoing destruction likewise occurs. Similar results were obtained with the attenuated lpg2- mutant, a convenient model that rapidly enters a persistent state without inducing pathology due to loss of the Golgi GDP mannose transporter. These data shed light on Leishmania persistence and concomitant immunity, suggesting a model wherein a parasite reservoir repopulates itself indefinitely, whereas some progeny are terminated in antigen-presenting cells, thereby stimulating immunity. This model may be relevant to understanding immunity to other persistent pathogen infections.

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.

Quantitative proteomic profiling of immune responses to Ichthyophthirius multifiliis in common carp skin mucus.

Ichthyophthirius multifiliis, a ciliated protozoan parasite, causes ichthyophthiriasis and leads to considerable economic losses to the aquaculture industry. Understanding the fish immune response and host-parasite interactions could support developing novel strategies for better disease management and control. Fish skin mucus is the first line of defence against infections through the epidermis. Yet, the common carp, Cyprinus carpio, protein-based defence strategies against infection with I. multifiliis at this barrier remain elusive. The skin mucus proteome of common carp was investigated at 1 day and 9 days post-exposure with I. multifiliis. Using nano-LC ESI MS/MS and statistical analysis, the abundance of 19 immune related and signal transduction proteins was found to be differentially regulated in skin mucus of common carp in response to I. multifiliis. The analysis revealed increased abundance values of epithelial chloride channel protein, galactose-specific lectin nattection, high choriolytic enzyme 1 (nephrosin), lysozyme C, granulin and protein-glutamine gamma-glutamyltransferase 2 in I. multifiliis-exposed carp skin mucus. Multiple lectins and a diverse array of distinct serpins with protease inhibitor activity were identified likely implicated in lectin pathway activation and regulation of proteolysis, indicating that these proteins contribute to the carp innate immune system and the protective properties of skin mucus. The results obtained from this proteomic analysis enables a better understanding of fish host response to parasitic infection and gives insights into the key role skin mucus plays in protecting fish against deleterious effects of I. multifiliis.

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.

Lipid bodies accumulation in Leishmania infantum-infected C57BL/6 macrophages.

Lipid bodies (LBs) are intracellular accumulations of neutral lipids surrounded by a single membrane. These organelles are involved in the production of eicosanoids, which modulate immunity by either promoting or dampening inflammatory responses. Leishmania infantum, the etiological agent of visceral leishmaniasis in Brazil, is an intracellular parasite that causes disease by suppressing macrophage microbicidal responses. C57BL/6 mouse bone marrow-derived macrophages infected with L. infantum strain LcJ had higher numbers of LB+ cells (P<.0001) and total LBs than noninfected cultures. Large (>3 µm) LBs were present inside parasitophorous vacuoles (PVs). These results contrast with those of L. infantum-infected BALB/c macrophages, in which the only LBs are derived from parasite, not macrophage origin. Increased LBs in C57BL/6 macrophages in close association with parasites would position host LBs where they could modulate L. infantum infection. These results imply a potential influence of the host genetics on the role of LBs in host-pathogen interactions. Overall, our data support a model in which the expression, and the role of LBs upon infection, ultimately depends on the specific combination of host-pathogen interactions.