Extracellular Vesicles: Translational Agenda Questions for Three Protozoan Parasites.

The protozoan parasites Plasmodium falciparum, Leishmania spp. and Trypanosoma cruzi continue to exert a significant toll on the disease landscape of the human population in sub-Saharan Africa and Latin America. Control measures have helped reduce the burden of their respective diseases-malaria, leishmaniasis and Chagas disease-in endemic regions. However, the need for new drugs, innovative vaccination strategies and molecular markers of disease severity and outcomes has emerged because of developing antimicrobial drug resistance, comparatively inadequate or absent vaccines, and a lack of trustworthy markers of morbid outcomes. Extracellular vesicles (EVs) have been widely reported to play a role in the biology and pathogenicity of P. falciparum, Leishmania spp. and T. cruzi ever since they were discovered. EVs are secreted by a yet to be fully understood mechanism in protozoans into the extracellular milieu and carry a cargo of diverse molecules that reflect the originator cell's metabolic state. Although our understanding of the biogenesis and function of EVs continues to deepen, the question of how EVs in P. falciparum, Leishmania spp. and T. cruzi can serve as targets for a translational agenda into clinical and public health interventions is yet to be fully explored. Here, as a consortium of protozoan researchers, we outline a plan for future researchers and pose three questions to direct an EV's translational agenda in P. falciparum, Leishmania spp. and T. cruzi. We opine that in the long term, executing this blueprint will help bridge the current unmet needs of these medically important protozoan diseases in sub-Saharan Africa and Latin America.

Revisiting Leishmania GP63 host cell targets reveals a limited spectrum of substrates.

Colonization of host phagocytic cells by Leishmania metacyclic promastigotes involves several parasite effectors, including the zinc-dependent metalloprotease GP63. The major mode of action of this virulence factor entails the cleavage/degradation of host cell proteins. Given the potent proteolytic activity of GP63, identification of its substrates requires the adequate preparation of cell lysates to prevent artefactual degradation during cell processing. In the present study, we re-examined the cleavage/degradation of reported GP63 substrates when GP63 activity was efficiently neutralized during the preparation of cell lysates. To this end, we infected bone marrow-derived macrophages with either wild type, Δgp63, and Δgp63+GP63 L. major metacyclic promastigotes for various time points. We prepared cell lysates in the absence or presence of the zinc-metalloprotease inhibitor 1,10-phenanthroline and examined the levels and integrity of ten previously reported host cell GP63 substrates. Inhibition of GP63 activity with 1,10-phenanthroline during the processing of macrophages prevented the cleavage/degradation of several previously described GP63 targets, including PTP-PEST, mTOR, p65RelA, c-Jun, VAMP3, and NLRP3. Conversely, we confirmed that SHP-1, Synaptotagmin XI, VAMP8, and Syntaxin-5 are bona fide GP63 substrates. These results point to the importance of efficiently inhibiting GP63 activity during the preparation of Leishmania-infected host cell lysates. In addition, our results indicate that the role of GP63 in Leishmania pathogenesis must be re-evaluated.

Translational control of the activation of transcription factor NF-κB and production of type I interferon by phosphorylation of the translation factor eIF4E.

Type I interferon is an integral component of the antiviral response, and its production is tightly controlled at the levels of transcription and translation. The eukaryotic translation-initiation factor eIF4E is a rate-limiting factor whose activity is regulated by phosphorylation of Ser209. Here we found that mice and fibroblasts in which eIF4E cannot be phosphorylated were less susceptible to virus infection. More production of type I interferon, resulting from less translation of Nfkbia mRNA (which encodes the inhibitor IκBα), largely explained this phenotype. The lower abundance of IκBα resulted in enhanced activity of the transcription factor NF-κB, which promoted the production of interferon-ß (IFN-ß). Thus, regulated phosphorylation of eIF4E has a key role in antiviral host defense by selectively controlling the translation of an mRNA that encodes a critical suppressor of the innate antiviral response.

Circadian Regulation of Leishmania Parasite Internalisation in Macrophages and Downstream Cellular Events.

Leishmania spp. parasites use macrophages as a host cell during infection. As a result, macrophages have a dual role: clearing the parasite as well as acting as host cells. Recently, studies have shown that macrophages harbour circadian clocks, which affect many of their functions such as phagocytosis, receptor expression and cytokine release. Interestingly, Leishmania major infection in hosts was also shown to be under circadian control. Therefore, we decided to investigate what underlies the rhythms of L. major infection within macrophages. Using a culture model of infection of bone marrow-derived macrophages with L. major promastigotes, we show that the parasites are internalised into macrophages with a 24-h variation dependent on a functional circadian clock in the cells. This was associated with a variation in the number of parasites per macrophage. The cell surface expression of parasite receptors was not controlled by the cells' circadian clock. In contrast, the expression of the components of the endocytic pathway, EEA1 and LC3b, varied according to the time of infection. This was paralleled by variations in parasite-induced ROS production as well as cytokine tumour necrosis factor α. In summary, we have uncovered a time-dependent regulation of the internalisation of L. major promastigotes in macrophages, controlled by the circadian clock in these cells, as well as subsequent cellular events in the endocytic pathway, intracellular signalling and cytokine production.

Genetic Iron Overload Hampers Development of Cutaneous Leishmaniasis in Mice.

The survival, growth, and virulence of Leishmania spp., a group of protozoan parasites, depends on the proper access and regulation of iron. Macrophages, Leishmania's host cell, may divert iron traffic by reducing uptake or by increasing the efflux of iron via the exporter ferroportin. This parasite has adapted by inhibiting the synthesis and inducing the degradation of ferroportin. To study the role of iron in leishmaniasis, we employed Hjv-/- mice, a model of hemochromatosis. The disruption of hemojuvelin (Hjv) abrogates the expression of the iron hormone hepcidin. This allows unrestricted iron entry into the plasma from ferroportin-expressing intestinal epithelial cells and tissue macrophages, resulting in systemic iron overload. Mice were injected with Leishmania major in hind footpads or intraperitoneally. Compared with wild-type controls, Hjv-/- mice displayed transient delayed growth of L. major in hind footpads, with a significant difference in parasite burden 4 weeks post-infection. Following acute intraperitoneal exposure to L. major, Hjv-/- peritoneal cells manifested increased expression of inflammatory cytokines and chemokines (Il1b, Tnfa, Cxcl2, and Ccl2). In response to infection with L. infantum, the causative agent of visceral leishmaniasis, Hjv-/- and control mice developed similar liver and splenic parasite burden despite vastly different tissue iron content and ferroportin expression. Thus, genetic iron overload due to hemojuvelin deficiency appears to mitigate the early development of only cutaneous leishmaniasis.

Clonal copy-number mosaicism in autoreactive T lymphocytes in diabetic NOD mice.

Concordance for type 1 diabetes (T1D) is far from 100% in monozygotic twins and in inbred nonobese diabetic (NOD) mice, despite genetic identity and shared environment during incidence peak years. This points to stochastic determinants, such as postzygotic mutations (PZMs) in the expanding antigen-specific autoreactive T cell lineages, by analogy to their role in the expanding tumor lineage in cancer. Using comparative genomic hybridization of DNA from pancreatic lymph-node memory CD4+ T cells of 25 diabetic NOD mice, we found lymphocyte-exclusive mosaic somatic copy-number aberrations (CNAs) with highly nonrandom independent involvement of the same gene(s) across different mice, some with an autoimmunity association (e.g., Ilf3 and Dgka). We confirmed genes of interest using the gold standard approach for CNA quantification, multiplex ligation-dependent probe amplification (MLPA), as an independent method. As controls, we examined lymphocytes expanded during normal host defense (17 NOD and BALB/c mice infected with Leishmania major parasite). Here, CNAs found were fewer and significantly smaller compared to those in autoreactive cells (P = 0.0019). We determined a low T cell clonality for our samples suggesting a prethymic formation of these CNAs. In this study, we describe a novel, unexplored phenomenon of a potential causal contribution of PZMs in autoreactive T cells in T1D pathogenesis. We expect that exploration of point mutations and studies in human T cells will enable the further delineation of driver genes to target for functional studies. Our findings challenge the classical notions of autoimmunity and open conceptual avenues toward individualized prevention and therapeutics.

The involvement of host circadian clocks in the regulation of the immune response to parasitic infections in mammals.

Circadian rhythms are recurring variations of physiology with a period of ~24 h, generated by circadian clocks located throughout the body. Studies have shown a circadian regulation of many aspects of immunity. Immune cells have intrinsic clock mechanisms, and innate and adaptive immune responses - such as leukocyte migration, magnitude of inflammation, cytokine production and cell differentiation - are under circadian control. This circadian regulation has consequences for infections including parasitic infections. In the context of Leishmania infection, the circadian clock within host immune cells modulates the magnitude of the infection and the inflammatory response triggered by the parasite. As for malaria, rhythms within the immune system were shown to impact the developmental cycles of Plasmodium parasites within red blood cells. Further, host circadian rhythms impact infections by multicellular parasites; for example, infection with helminth Trichuris muris shows different kinetics of worm expulsion depending on time of day of infection, a variation that depends on the dendritic cell clock. Although the research on the circadian control of immunity in the context of parasitic infections is in its infancy, the research reviewed here suggests a crucial involvement of host circadian rhythms in immunity on the development and progression of parasitic infections.

Influence of N-Methylation and Conformation on Almiramide Anti-Leishmanial Activity.

The almiramide N-methylated lipopeptides exhibit promising activity against trypanosomatid parasites. A structure-activity relationship study has been performed to examine the influences of N-methylation and conformation on activity against various strains of leishmaniasis protozoan and on cytotoxicity. The synthesis and biological analysis of twenty-five analogs demonstrated that derivatives with a single methyl group on either the first or fifth residue amide nitrogen exhibited greater activity than the permethylated peptides and relatively high potency against resistant strains. Replacement of amino amide residues in the peptide, by turn inducing α amino γ lactam (Agl) and N-aminoimidazalone (Nai) counterparts, reduced typically anti-parasitic activity; however, peptide amides possessing Agl residues at the second residue retained significant potency in the unmethylated and permethylated series. Systematic study of the effects of methylation and turn geometry on anti-parasitic activity indicated the relevance of an extended conformer about the central residues, and conformational mobility by tertiary amide isomerization and turn geometry at the extremities of the active peptides.

Cysteine Peptidase B Regulates Leishmania mexicana Virulence through the Modulation of GP63 Expression.

Cysteine peptidases play a central role in the biology of Leishmania. In this work, we sought to further elucidate the mechanism(s) by which the cysteine peptidase CPB contributes to L. mexicana virulence and whether CPB participates in the formation of large communal parasitophorous vacuoles induced by these parasites. We initially examined the impact of L. mexicana infection on the trafficking of VAMP3 and VAMP8, two endocytic SNARE proteins associated with phagolysosome biogenesis and function. Using a CPB-deficient mutant, we found that both VAMP3 and VAMP8 were down-modulated in a CPB-dependent manner. We also discovered that expression of the virulence-associated GPI-anchored metalloprotease GP63 was inhibited in the absence of CPB. Expression of GP63 in the CPB-deficient mutant was sufficient to down-modulate VAMP3 and VAMP8. Similarly, episomal expression of GP63 enabled the CPB-deficient mutant to establish infection in macrophages, induce the formation of large communal parasitophorous vacuoles, and cause lesions in mice. These findings implicate CPB in the regulation of GP63 expression and provide evidence that both GP63 and CPB are key virulence factors in L. mexicana.

Impact of Leishmania infection on host macrophage nuclear physiology and nucleopore complex integrity.

The protease GP63 is an important virulence factor of Leishmania parasites. We previously showed that GP63 reaches the perinuclear area of host macrophages and that it directly modifies nuclear translocation of the transcription factors NF-κB and AP-1. Here we describe for the first time, using molecular biology and in-depth proteomic analyses, that GP63 alters the host macrophage nuclear envelope, and impacts on nuclear processes. Our results suggest that GP63 does not appear to use a classical nuclear localization signal common between Leishmania species for import, but degrades nucleoporins, and is responsible for nuclear transport alterations. In the nucleoplasm, GP63 activity accounts for the degradation and mislocalization of proteins involved amongst others in gene expression and in translation. Collectively, our data indicates that Leishmania infection strongly affects nuclear physiology, suggesting that targeting of nuclear physiology may be a strategy beneficial for virulent Leishmania parasites.

Leishmania exosomes and other virulence factors: Impact on innate immune response and macrophage functions.

Leishmania parasites are the causative agents of the leishmaniases, a collection of vector-borne diseases that range from simple cutaneous to fatal visceral forms. Employing potent immune modulation mechanisms, Leishmania is able to render the host macrophage inactive and persist inside its phagolysosome. In the last few years, the role of exosomes in Leishmania-host interactions has been increasingly investigated. For instance, it was reported that Leishmania exosome release is augmented following temperature shift, a condition mimicking parasite's entry into its mammalian host. Leishmania exosomes were found to strongly affect macrophage cell signaling and functions, similarly to whole parasites. Importantly, these vesicles were shown to be pro-inflammatory, capable to recruit neutrophils at their inoculation site exacerbating the pathology. In this review, we provide the most recent insights on the role of exosomes and other virulence factors, especially the surface protease GP63, in Leishmania-host interactions, deepening our knowledge on leishmaniasis and paving the way for the development of new therapeutics.

Adaptation of Leishmania donovani to cutaneous and visceral environments: in vivo selection and proteomic analysis.

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa. Two main forms are found in the Old World, self-limited cutaneous leishmaniasis and potentially fatal visceral leishmaniasis, with parasite dissemination to liver, bone marrow, and spleen. The Leishmania donovani species complex is the causative agent of visceral leishmaniasis worldwide, but atypical L. donovani strains can cause cutaneous leishmaniasis. We hypothesized that L. donovani can adapt to survive in response to restrictions imposed by the host environment. To assess this, we performed in vivo selection in BALB/c mice with a cutaneous L. donovani clinical isolate to select for parasites with increased capacity to survive in visceral organs. We then performed whole cell proteomic analysis and compared this visceral-selected strain to the original cutaneous clinical isolate and to a visceral leishmaniasis clinical isolate. Overall, there were no major shifts in proteomic profiles; however, translation, biosynthetic processes, antioxidant protection, and signaling were elevated in visceral strains. Conversely, transport and trafficking were elevated in the cutaneous strain. Overall, these results provide new insight into the adaptability of Leishmania parasites to the host environment and on the factors that mediate their ability to survive in different organs.

Plasmodium products contribute to severe malarial anemia by inhibiting erythropoietin-induced proliferation of erythroid precursors.

Low reticulocytosis, indicating reduced red blood cell (RBC) output, is an important feature of severe malarial anemia. Evidence supports a role for Plasmodium products, especially hemozoin (Hz), in suppressed erythropoiesis during malaria, but the mechanism(s) involved remains unclear. Here, we demonstrated that low reticulocytosis and suppressed erythropoietin (Epo)-induced erythropoiesis are features of malarial anemia in Plasmodium yoelii- and Plasmodium berghei ANKA-infected mice, similar to our previous observations in Plasmodium chabaudi AS-infected mice. The magnitude of decreases in RBC was a reflection of parasitemia level, but low reticulocytosis was evident despite differences in parasitemia, clinical manifestation, and infection outcome. Schizont extracts and Hz from P. falciparum and P. yoelii and synthetic Hz suppressed Epo-induced proliferation of erythroid precursors in vitro but did not inhibit RBC maturation. To determine whether Hz contributes to malarial anemia, P. yoelii-derived or synthetic Hz was administered to naive mice, and the development of anemia, reticulocytosis, and RBC turnover was determined. Parasite-derived Hz induced significant decreases in RBC and increased RBC turnover with compensatory reticulocytosis, but anemia was not as severe as that in infected mice. Our findings suggest that parasite factors, including Hz, contribute to severe malarial anemia by suppressing Epo-induced proliferation of erythroid precursors.

The protein tyrosine phosphatase SHP-1 regulates phagolysosome biogenesis.

The process of phagocytosis and phagosome maturation involves the recruitment of effector proteins that participate in phagosome formation and in the acidification and/or fusion with various endocytic vesicles. In the current study, we investigated the role of the Src homology region 2 domain-containing phosphatase 1 (SHP-1) in phagolysosome biogenesis. To this end, we used immortalized bone marrow macrophages derived from SHP-1-deficient motheaten mice and their wild-type littermates. We found that SHP-1 is recruited early and remains present on phagosomes for up to 4 h postphagocytosis. Using confocal immunofluorescence microscopy and Western blot analyses on purified phagosome extracts, we observed an impaired recruitment of lysosomal-associated membrane protein 1 in SHP-1-deficient macrophages. Moreover, Western blot analyses revealed that whereas the 51-kDa procathepsin D is recruited to phagosomes, it is not processed into the 46-kDa cathepsin D in the absence of SHP-1, suggesting a defect in acidification. Using the lysosomotropic agent LysoTracker as an indicator of phagosomal pH, we obtained evidence that in the absence of SHP-1, phagosome acidification was impaired. Taken together, these results are consistent with a role for SHP-1 in the regulation of signaling or membrane fusion events involved in phagolysosome biogenesis.

Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species.

The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved to an average 16-fold mean coverage by next-generation DNA sequencing technologies. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described thus providing an opportunity for comparison with the completed genomes of pathogenic Leishmania species. A high synteny was observed between all sequenced Leishmania species. A limited number of chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic to L. major. Globally, >90% of the L. tarentolae gene content was shared with the other Leishmania species. We identified 95 predicted coding sequences unique to L. tarentolae and 250 genes that were absent from L. tarentolae. Interestingly, many of the latter genes were expressed in the intracellular amastigote stage of pathogenic species. In addition, genes coding for products involved in antioxidant defence or participating in vesicular-mediated protein transport were underrepresented in L. tarentolae. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the zinc metallo-peptidase surface glycoprotein GP63 and the promastigote surface antigen PSA31C. Overall, L. tarentolae's gene content appears better adapted to the promastigote insect stage rather than the amastigote mammalian stage.

Time of day and circadian disruption influence host response and parasite growth in a mouse model of cerebral malaria.

Malaria is a disease caused by infection with parasite Plasmodium spp. We studied the circadian regulation of host responses to the parasite, in a mouse model of cerebral malaria. The course of the disease was markedly affected by time of infection, with decreased parasitemia and increased inflammation upon infection in the middle of the night. At this time, there were fewer reticulocytes, which are target cells of the parasites. We next investigated the effects of desynchronization of host clocks on the infection: after 10 weeks of recurrent jet lags, mice showed decreased parasite growth and lack of parasite load rhythmicity, paralleled by a loss of glucose rhythm. Accordingly, disrupting host metabolic rhythms impacted parasite load rhythmicity. In summary, our findings of a circadian modulation of malaria parasite growth and infection shed light on aspects of the disease relevant to human malaria and could contribute to new therapeutic or prophylactic measures.

Clockwork intruders: Do parasites manipulate their hosts' circadian rhythms?

Most organisms have developed circadian clocks to adapt to 24-hour cycles in the environment. These clocks have become crucial for modulating and synchronizing complex behavioral and biological processes. A number of parasites seem to have evolved to take advantage of their hosts' circadian rhythms to favor their own infection and survival. Some species, such as Microphallus sp. and Trypanosoma cruzi, can alter the patterns of locomotor behavior of infected intermediate hosts, which can promote transmission to a subsequent primary host. Some fungi of the genera Ophiocordyceps and Entomophthora, as well as hairworms (Nematomorpha), elicit complex behaviors that promote their host's death at a time and place that optimizes continuation of the parasite's life-cycle. At least in some cases, a proposed mechanism might involve a change in the expression of clock-controlled genes. Lastly, some disease-causing protozoan parasites of the genera Trypanosoma, Plasmodium, and Leishmania induce changes in the circadian rhythms of their primary hosts upon infection. Some of these changes may be attributed to circadian alterations resulting from the host's inflammatory response to the infection or other unexplored responses or adaptations to the illness. Thus, a distinction must be made between manipulation of the parasite and response of the host when studying these alterations in the future. Parasitic manipulation of circadian rhythms, which vastly modulates behavior and physiology, is an essential issue that has been relatively understudied. A deeper understanding of this phenomenon could lead to the development of novel therapeutic approaches for the diseases that these parasites convey.

Identification of potential novel proteomic markers of Leishmania spp.-derived exosomes.

Introduction: Extracellular vesicles (EVs) are heterogenous cell-derived membrane-bound structures which can be subdivided into three distinct classes according to distinct morphological characteristics, cellular origins, and functions. Small EVs, or exosomes, can be produced by the protozoan parasite Leishmania through the evolutionarily conserved ESCRT pathway, and act as effectors of virulence and drivers of pathogenesis within mammalian hosts. Techniques for the identification of EVs of non-mammalian origin, however, remain inaccurate in comparison to their well-characterized mammalian counterparts. Thus, we still lack reliable and specific markers for Leishmania-derived exosomes, which poses a significant challenge to the field. Methods: Herein, we utilized serial differential ultracentrifugation to separate Leishmania-derived EV populations into three distinct fractions. Nanoparticle tracking analysis and transmission electron microscopy were used to validate their morphological characteristics, and bioinformatic analysis of LC-MS/MS proteomics corroborated cellular origins and function. Discussion: Proteomic data indicated potential novel proteic markers of Leishmania-derived exosomes, including proteins involved in endosomal machinery and the ESCRT pathway, as well as the parasitic phosphatase PRL-1. Further investigation is required to determine the specificity and sensitivity of these markers.

Circadian Control of the Response of Macrophages to Plasmodium Spp.-Infected Red Blood Cells.

Malaria is a serious vector-borne disease characterized by periodic episodes of high fever and strong immune responses that are coordinated with the daily synchronized parasite replication cycle inside RBCs. As immune cells harbor an autonomous circadian clock that controls various aspects of the immune response, we sought to determine whether the intensity of the immune response to Plasmodium spp., the parasite causing malaria, depends on time of infection. To do this, we developed a culture model in which mouse bone marrow-derived macrophages are stimulated with RBCs infected with Plasmodium berghei ANKA (iRBCs). Lysed iRBCs, but not intact iRBCs or uninfected RBCs, triggered an inflammatory immune response in bone marrow-derived macrophages. By stimulating at four different circadian time points (16, 22, 28, or 34 h postsynchronization of the cells' clock), 24-h rhythms in reactive oxygen species and cytokines/chemokines were found. Furthermore, the analysis of the macrophage proteome and phosphoproteome revealed global changes in response to iRBCs that varied according to circadian time. This included many proteins and signaling pathways known to be involved in the response to Plasmodium infection. In summary, our findings show that the circadian clock within macrophages determines the magnitude of the inflammatory response upon stimulation with ruptured iRBCs, along with changes of the cell proteome and phosphoproteome.