We investigated the properties of leishmania exosomes with respect to influencing innate and adaptive immune responses. Exosomes from Leishmania donovani modulated human monocyte cytokine responses ...to IFN-γ in a bimodal fashion by promoting IL-10 production and inhibiting that of TNF-α. Moreover, these vesicles were inhibitory with respect to cytokine responses (IL-12p70, TNF-α, and IL-10) by human monocyte-derived dendritic cells. Exosomes from wild-type (WT) L. donovani failed to prime monocyte-derived dendritic cells to drive the differentiation of naive CD4 T cells into IFN-γ-producing Th1 cells. In contrast, vesicles from heat shock protein (HSP)100(-/-) L. donovani showed a gain-of-function and proinflammatory phenotype and promoted the differentiation of naive CD4 lymphocytes into Th1 cells. Proteomic analysis showed that exosomes from WT and HSP100(-/-) leishmania had distinct protein cargo, suggesting that packaging of proteins into exosomes is dependent in part on HSP100. Treatment of C57BL/6 mice with WT L. donovani exosomes prior to challenge with WT organisms exacerbated infection and promoted IL-10 production in the spleen. In contrast, HSP100(-/-) exosomes promoted spleen cell production of IFN-γ and did not adversely affect hepatic parasite burdens. Furthermore, the proparasitic properties of WT exosomes were not species specific because BALB/c mice exposed to Leishmania major exosomes showed increased Th2 polarization and exacerbation of disease in response to infection with L. major. These findings demonstrate that leishmania exosomes are predominantly immunosuppressive. Moreover, to our knowledge, this is the first evidence to suggest that changes in the protein cargo of exosomes may influence the impact of these vesicles on myeloid cell function.
Cognate tRNAs deliver specific amino acids to translating ribosomes according to the standard genetic code, and three codons with no cognate tRNAs serve as stop codons. Some protists have reassigned ...all stop codons as sense codons, neglecting this fundamental principle
. Here we analyse the in-frame stop codons in 7,259 predicted protein-coding genes of a previously undescribed trypanosomatid, Blastocrithidia nonstop. We reveal that in this species in-frame stop codons are underrepresented in genes expressed at high levels and that UAA serves as the only termination codon. Whereas new tRNAs
fully cognate to UAG and UAA evolved to reassign these stop codons, the UGA reassignment followed a different path through shortening the anticodon stem of tRNA
from five to four base pairs (bp). The canonical 5-bp tRNA
recognizes UGG as dictated by the genetic code, whereas its shortened 4-bp variant incorporates tryptophan also into in-frame UGA. Mimicking this evolutionary twist by engineering both variants from B. nonstop, Trypanosoma brucei and Saccharomyces cerevisiae and expressing them in the last two species, we recorded a significantly higher readthrough for all 4-bp variants. Furthermore, a gene encoding B. nonstop release factor 1 acquired a mutation that specifically restricts UGA recognition, robustly potentiating the UGA reassignment. Virtually the same strategy has been adopted by the ciliate Condylostoma magnum. Hence, we describe a previously unknown, universal mechanism that has been exploited in unrelated eukaryotes with reassigned stop codons.
Malleable mitochondrion of Trypanosoma brucei Verner, Zdeněk; Basu, Somsuvro; Benz, Corinna ...
International review of cell and molecular biology,
2015, Letnik:
315
Journal Article
Recenzirano
The importance of mitochondria for a typical aerobic eukaryotic cell is undeniable, as the list of necessary mitochondrial processes is steadily growing. Here, we summarize the current knowledge of ...mitochondrial biology of an early-branching parasitic protist, Trypanosoma brucei, a causative agent of serious human and cattle diseases. We present a comprehensive survey of its mitochondrial pathways including kinetoplast DNA replication and maintenance, gene expression, protein and metabolite import, major metabolic pathways, Fe-S cluster synthesis, ion homeostasis, organellar dynamics, and other processes. As we describe in this chapter, the single mitochondrion of T. brucei is everything but simple and as such rivals mitochondria of multicellular organisms.
Display omitted
•Catalase in Leptomonas seymouri is present in the cytoplasm and a subset of glycosomes.•Cytoplasmic retention of L. seymouri catalase is H2O2-dependent.•The ablation of catalase in ...this parasite is not detrimental in vivo.•Catalase overexpression resulted in a substantially higher parasite load in experimental infection of Dysdercus peruvianus.
Nearly all aerobic organisms are equipped with catalases, powerful enzymes scavenging hydrogen peroxide and facilitating defense against harmful reactive oxygen species. In trypanosomatids, this enzyme was not present in the common ancestor, yet it had been independently acquired by different lineages of monoxenous trypanosomatids from different bacteria at least three times. This observation posited an obvious question: why was catalase so “sought after” if many trypanosomatid groups do just fine without it? In this work, we analyzed subcellular localization and function of catalase in Leptomonas seymouri. We demonstrated that this enzyme is present in the cytoplasm and a subset of glycosomes, and that its cytoplasmic retention is H2O2-dependent. The ablation of catalase in this parasite is not detrimental in vivo, while its overexpression resulted in a substantially higher parasite load in the experimental infection of Dysdercus peruvianus. We propose that the capacity of studied flagellates to modulate the catalase activity in the midgut of its insect host facilitates their development and protects them from oxidative damage at elevated temperatures.
Establishment of the early genetic code likely required strategies to ensure translational accuracy and inevitably involved tRNA post-transcriptional modifications. One such modification, ...wybutosine/wyosine is crucial for translational fidelity in Archaea and Eukarya; yet it does not occur in Bacteria and has never been described in mitochondria. Here, we present genetic, molecular and mass spectromery data demonstrating the first example of wyosine in mitochondria, a situation thus far unique to kinetoplastids. We also show that these modifications are important for mitochondrial function, underscoring their biological significance. This work focuses on TyW1, the enzyme required for the most critical step of wyosine biosynthesis. Based on molecular phylogeny, we suggest that the kinetoplastids pathways evolved via gene duplication and acquisition of an FMN-binding domain now prevalent in TyW1 of most eukaryotes. These findings are discussed in the context of the extensive U-insertion RNA editing in trypanosome mitochondria, which may have provided selective pressure for maintenance of mitochondrial wyosine in this lineage.
The convoluted history of haem biosynthesis Kořený, Luděk; Oborník, Miroslav; Horáková, Eva ...
Biological reviews of the Cambridge Philosophical Society,
February 2022, 2022-02-00, 20220201, Letnik:
97, Številka:
1
Journal Article
Recenzirano
Odprti dostop
ABSTRACT
The capacity of haem to transfer electrons, bind diatomic gases, and catalyse various biochemical reactions makes it one of the essential biomolecules on Earth and one that was likely used ...by the earliest forms of cellular life. Since the description of haem biosynthesis, our understanding of this multi‐step pathway has been almost exclusively derived from a handful of model organisms from narrow taxonomic contexts. Recent advances in genome sequencing and functional studies of diverse and previously neglected groups have led to discoveries of alternative routes of haem biosynthesis that deviate from the ‘classical’ pathway. In this review, we take an evolutionarily broad approach to illuminate the remarkable diversity and adaptability of haem synthesis, from prokaryotes to eukaryotes, showing the range of strategies that organisms employ to obtain and utilise haem. In particular, the complex evolutionary histories of eukaryotes that involve multiple endosymbioses and horizontal gene transfers are reflected in the mosaic origin of numerous metabolic pathways with haem biosynthesis being a striking case. We show how different evolutionary trajectories and distinct life strategies resulted in pronounced tensions and differences in the spatial organisation of the haem biosynthesis pathway, in some cases leading to a complete loss of a haem‐synthesis capacity and, rarely, even loss of a requirement for haem altogether.
Iron-associated biology of Trypanosoma brucei Basu, Somsuvro; Horáková, Eva; Lukeš, Julius
Biochimica et biophysica acta,
February 2016, 2016-Feb, 2016-02-00, Letnik:
1860, Številka:
2
Journal Article
Recenzirano
Every eukaryote requires iron, which is also true for the parasitic protist Trypanosoma brucei, the causative agent of sleeping sickness in humans and nagana in cattle. T. brucei undergoes a complex ...life cycle during which its single mitochondrion is subject to major metabolic and morphological changes.
This review covers what is known about processes associated with iron–sulfur clusters and heme metabolism in T. brucei. We discuss strategies by which iron and heme are acquired and utilized by this model parasite, emphasizing the differences between its two life cycle stages residing in the bloodstream of the mammalian host and gut of the insect vector. Finally, the role of iron in the host–parasite interactions is discussed along with their possible exploitation in fighting these deadly parasites.
The processes associated with acquisition and utilization of iron, distinct in the two life stages of T. brucei, are fine tuned for the dramatically different host environment occupied by them. Although the composition and compartmentalization of the iron–sulfur cluster assembly seem to be conserved, some unique features of the iron acquisition strategies may be exploited for medical interventions against these parasites.
As early-branching protists, trypanosomes and related flagellates are known to harbor an array of unique features, with the acquisition of iron being another peculiarity. Thanks to intense research within the last decade, understanding of iron–sulfur cluster assembly and iron metabolism in T. brucei is among the most advanced of all eukaryotes.
•Trypanosoma brucei sequesters iron in several unique processes unlike its hosts.•T. brucei retains the conserved eukaryotic Fe/S protein biosynthetic machineries.•The heme-auxotroph T. brucei relies exclusively on the host-provided heme.•Iron and heme acquisition-utilization varies in the two life-stages of T. brucei.
Environmental friendly agricultural management has an urgent need for finding a sustainable strategy for the usage of different by-products from bioenergy production. These are either used as soil ...amendments or fertilizers. This study is aimed at evaluating if and how soil organic matter changes after the application of biochar, compost, and digestate. A pot experiment was conducted with Haplic Cambisol (low range arable soil) in Phytotron CLF PlantMaster (Wertingen, Germany). The chemical composition of isolated humic acids (HA) was determined by an inductively coupled plasma-mass spectrometer (ICP-MS). FT-IR spectroscopy and CHNS analysis were used for detailed chemical and optical characterization. Soil magnetic properties - radical concentration, g-parameters of radicals, and iron ions were evaluated by EPR spectroscopy. The results showed that amending arable soil with biochar, digestate and compost results in chemical and structural changes of humic substances. The radicals originated in biochar and digestate are built-in to the structure of the humic acid, which was confirmed by EPR g-parameter values. Despite a relatively high concentration of paramagnetic metal ions Fe and Mn the effect of semiquinone radical quenching was not observed. That suggests a conclusion that metal ions of studied amendments are binding in HA structure and did not disturb natural radical processes in the soil. It was also concluded that the effect of applied material depends mainly on its chemical properties and the soil type.
Resistance to African trypanosomes in humans relies in part on the high affinity targeting of a trypanosome lytic factor 1 (TLF1) to a trypanosome haptoglobin-hemoglobin receptor (HpHbR). While TLF1 ...avoidance by the inactivation of HpHbR contributes to Trypanosoma brucei gambiense human infectivity, the evolutionary trade-off of this adaptation is unknown, as the physiological function of the receptor remains to be elucidated. Here we show that uptake of hemoglobin via HpHbR constitutes the sole heme import pathway in the trypanosome bloodstream stage. T. b. gambiense strains carrying the inactivating mutation in HpHbR, as well as genetically engineered T. b. brucei HpHbR knock-out lines show only trace levels of intracellular heme and lack hemoprotein-based enzymatic activities, thereby providing an uncommon example of aerobic parasitic proliferation in the absence of heme. We further show that HpHbR facilitates the developmental progression from proliferating long slender forms to cell cycle-arrested stumpy forms in T. b. brucei. Accordingly, T. b. gambiense was found to be poorly competent for slender-to-stumpy differentiation unless a functional HpHbR receptor derived from T. b. brucei was genetically restored. Altogether, we identify heme-deficient metabolism and disrupted cellular differentiation as two distinct HpHbR-dependent evolutionary trade-offs for T. b. gambiense human infectivity.
Catalase is a widespread heme‐containing enzyme, which converts hydrogen peroxide (H2O2) to water and molecular oxygen, thereby protecting cells from the toxic effects of H2O2. Trypanosoma brucei is ...an aerobic protist, which conspicuously lacks this potent enzyme, present in virtually all organisms exposed to oxidative stress. To uncover the reasons for its absence in T. brucei, we overexpressed different catalases in procyclic and bloodstream stages of the parasite. The heterologous enzymes originated from the related insect‐confined trypanosomatid Crithidia fasciculata and the human. While the trypanosomatid enzyme (cCAT) operates at low temperatures, its human homolog (hCAT) is adapted to the warm‐blooded environment. Despite the presence of peroxisomal targeting signal in hCAT, both human and C. fasciculata catalases localized to the cytosol of T. brucei. Even though cCAT was efficiently expressed in both life cycle stages, the enzyme was active in the procyclic stage, increasing cell's resistance to the H2O2 stress, yet its activity was suppressed in the cultured bloodstream stage. Surprisingly, following the expression of hCAT, the ability to establish the T. brucei infection in the tsetse fly midgut was compromised. In the mouse model, hCAT attenuated parasitemia and, consequently, increased the host's survival. Hence, we suggest that the activity of catalase in T. brucei is beneficial in vitro, yet it becomes detrimental for parasite's proliferation in both invertebrate and vertebrate hosts, leading to an inability to carry this, otherwise omnipresent, enzyme.
A unique feature of the catalase reaction is the production of molecular oxygen after the decomposition of hydrogen peroxide, which is detectable directly in the form of macroscopic bubbles. We showed that the activity of heterologously expressed Crithidia catalase is similar to human catalase in insect form trypanosomes but significantly lower in their bloodstream forms.
PDF
