Endosymbiosis in trypanosomatids: The bacterium division depends on microtubule dynamism.

Microtubules are components of the cytoskeleton that perform essential functions in eukaryotes, such as those related to shape change, motility and cell division. In this context some characteristics of these filaments are essential, such as polarity and dynamic instability. In trypanosomatids, microtubules are integral to ultrastructure organization, intracellular transport and mitotic processes. Some species of trypanosomatids co-evolve with a symbiotic bacterium in a mutualistic association that is marked by extensive metabolic exchanges and a coordinated division of the symbiont with other cellular structures, such as the nucleus and the kinetoplast. It is already established that the bacterium division is microtubule-dependent, so in this work, it was investigated whether the dynamism and remodeling of these filaments is capable of affecting the prokaryote division. To this purpose, Angomonas deanei was treated with Trichostatin A (TSA), a deacetylase inhibitor, and mutant cells for histone deacetylase 6 (HDAC6) were obtained by CRISPR-Cas9. A decrease in proliferation, an enhancement in tubulin acetylation, as well as morphological and ultrastructural changes, were observed in TSA-treated protozoa and mutant cells. In both cases, symbiont filamentation occurred, indicating that prokaryote cell division is dependent on microtubule dynamism.

Microorganisms as a Potential Source of Molecules to Control Trypanosomatid Diseases.

Trypanosomatids are the causative agents of leishmaniasis and trypanosomiasis, which affect about 20 million people in the world's poorest countries, leading to 95,000 deaths per year. They are often associated with malnutrition, weak immune systems, low quality housing, and population migration. They are generally recognized as neglected tropical diseases. New drugs against these parasitic protozoa are urgently needed to counteract drug resistance, toxicity, and the high cost of commercially available drugs. Microbial bioprospecting for new molecules may play a crucial role in developing a new generation of antiparasitic drugs. This article reviews the current state of the available literature on chemically defined metabolites of microbial origin that have demonstrated antitrypanosomatid activity. In this review, bacterial and fungal metabolites are presented; they originate from a range of microorganisms, including cyanobacteria, heterotrophic bacteria, and filamentous fungi. We hope to provide a useful overview for future research to identify hits that may become the lead compounds needed to accelerate the discovery of new drugs against trypanosomatids.

UDP-glycosyltransferase genes in trypanosomatid genomes have diversified independently to meet the distinct developmental needs of parasite adaptations.

BACKGROUND: Trypanosomatid parasites such as Trypanosoma spp. and Leishmania spp. are a major source of infectious disease in humans and domestic animals worldwide. Fundamental to the host-parasite interactions of these potent pathogens are their cell surfaces, which are highly decorated with glycosylated proteins and other macromolecules. Trypanosomatid genomes contain large multi-copy gene families encoding UDP-dependent glycosyltransferases (UGTs), the primary role of which is cell-surface decoration. Here we report a phylogenetic analysis of UGTs from diverse trypanosomatid genomes, the aim of which was to understand the origin and evolution of their diversity. RESULTS: By combining phylogenetics with analyses of recombination, and selection, we compared UGT repertoire, genomic context and sequence evolution across 19 trypanosomatids. We identified a UGT lineage present in stercorarian trypanosomes and a free-living kinetoplastid Bodo saltans that likely represents the ancestral state of this gene family. The phylogeny of parasite-specific genes shows that UGTs repertoire in Leishmaniinae and salivarian trypanosomes has expanded independently and with distinct evolutionary dynamics. In the former, the ancestral UGT repertoire was organised in a tandem array from which sporadic transpositions to telomeric regions occurred, allowing expansion most likely through telomeric exchange. In the latter, the ancestral UGT repertoire was comprised of seven subtelomeric lineages, two of which have greatly expanded potentially by gene transposition between these dynamic regions of the genome. CONCLUSIONS: The phylogeny of UGTs confirms that they represent a substantial parasite-specific innovation, which has diversified independently in the distinct trypanosomatid lineages. Nonetheless, developmental regulation has been a strong driver of UGTs diversification in both African trypanosomes and Leishmania.

Quantitative PCR assessment of Lotmaria passim in Apis mellifera colonies co-infected naturally with Nosema ceranae.

A recently described trypanosomatid species Lotmaria passim and the microsporidium Nosema ceranae infect the honey bee (Apis mellifera), but the interspecific dynamic of these two common gut parasites is unknown. In this study, a real-time qPCR assay was developed to enable the specific detection and quantification of L. passim. The annual dynamics of N. ceranae and L. passim infections were evaluated in ten A. mellifera colonies naturally infected with both parasites at one apiary in Serbia from March 2016 to March 2017. Ten samples (60 bees abdomens) were taken from each colony on 8 sampling occasions. L. passim infection level was evaluated with qPCR, while N. ceranae infection was measured by spore counts. N. ceranae infection level was significantly higher in comparison with that of L. passim (spore or cell equivalents/bee, respectively). Significant positive correlation between infection levels of the parasite species indicates their similar annual dynamics, whilst the differences in the levels of infection between particular months point to a seasonal pattern in the incidence of both parasites. The assay which has been developed and validated creates opportunity for detailed study of L. passim infection kinetics and the improvement in the management practices in beekeeping related to these two parasites.

Phytomonas: trypanosomatids adapted to plant environments.

Over 100 years after trypanosomatids were first discovered in plant tissues, Phytomonas parasites have now been isolated across the globe from members of 24 different plant families. Most identified species have not been associated with any plant pathology and to date only two species are definitively known to cause plant disease. These diseases (wilt of palm and coffee phloem necrosis) are problematic in areas of South America where they threaten the economies of developing countries. In contrast to their mammalian infective relatives, our knowledge of the biology of Phytomonas parasites and how they interact with their plant hosts is limited. This review draws together a century of research into plant trypanosomatids, from the first isolations and experimental infections to the recent publication of the first Phytomonas genomes. The availability of genomic data for these plant parasites opens a new avenue for comparative investigations into trypanosomatid biology and provides fresh insight into how this important group of parasites have adapted to survive in a spectrum of hosts from crocodiles to coconuts.

Nectar chemistry mediates the behavior of parasitized bees: consequences for plant fitness.

Plants produce an array of secondary metabolites that play important ecological roles as anti-herbivore and anti-pathogen defenses. Many herbivores experience physiological costs when they consume secondary metabolites, yet some also benefit, for example when these chemicals confer resistance to parasites and predators. Secondary metabolites are often present in nectar and pollen, which is paradoxical given that floral rewards are important in the attraction of mutualists rather than deterrence of antagonists. Motivated by studies of interactions among plants, herbivores, and parasites, as well as research showing that secondary metabolites can reduce bee disease, we characterized the occurrence of two iridoid glycosides, aucubin and catalpol, in floral rewards and other tissues of the bee pollinated plant, Chelone glabra. We then experimentally investigated effects of nectar iridoid glycoside concentrations on the foraging behavior of bumble bee pollinators naturally afflicted by a parasitoid fly and a protozoan intestinal parasite, and subsequent effects on an estimate of plant reproduction. We found that floral nectar had lower iridoid glycoside concentrations than leaves, pollen, and corollas, and that, compared to those plant parts, the relative ratio of the two primary iridoid glycosides, aucubin and catalpol, was reversed in nectar. Whether bees carried parasitoid fly larvae did not affect their response to nectar chemistry; however, there was a significant interaction between protozoan parasite infection and nectar treatment, with infected bees foraging longer at flowers with high compared to low nectar iridoid glycoside concentrations. Parasitized bees were also more likely to return to inflorescences with high iridoid glycoside nectar. Consequently, flowers in the high iridoid glycoside nectar treatment donated significantly more pollen to conspecific stigmas than did flowers in the low iridoid glycoside treatment, suggesting an increase in male plant fitness. Taken together, these results demonstrate that nectar secondary metabolites can mediate the behavior of pollinators with subsequent benefits for estimates of plant reproduction.

Parasites in bloom: flowers aid dispersal and transmission of pollinator parasites within and between bee species.

The dispersal of parasites is critical for epidemiology, and the interspecific vectoring of parasites when species share resources may play an underappreciated role in parasite dispersal. One of the best examples of such a situation is the shared use of flowers by pollinators, but the importance of flowers and interspecific vectoring in the dispersal of pollinator parasites is poorly understood and frequently overlooked. Here, we use an experimental approach to show that during even short foraging periods of 3 h, three bumblebee parasites and two honeybee parasites were dispersed effectively onto flowers by their hosts, and then vectored readily between flowers by non-host pollinator species. The results suggest that flowers are likely to be hotspots for the transmission of pollinator parasites and that considering potential vector, as well as host, species will be of general importance for understanding the distribution and transmission of parasites in the environment and between pollinators.

Symbiont transmission entails the risk of parasite infection.

Like many animals, firebugs (Hemiptera, Pyrrhocoridae) rely on behavioural adaptations to successfully endow their offspring with microbial mutualists. To transmit the nutritionally beneficial Coriobacteriaceae symbionts, female firebugs smear egg surfaces with symbiont-containing faecal droplets that are subsequently ingested by newly hatched nymphs through active probing to initiate infection. Alternatively, the symbionts can be acquired horizontally through contact with faeces of infected conspecifics. Here, we report that these adaptations ensuring successful transmission of bacterial symbionts among firebugs are exploited by the specialized trypanosomatid parasite Leptomonas pyrrhocoris. Using comparative transcriptomics, fluorescence in situ hybridization (FISH) and controlled bioassays, we demonstrate that the transmission cycle of L. pyrrhocoris mirrors that of the bacterial mutualists, with high efficiency for both vertical and horizontal transmission. This indicates that the parasite capitalizes on pre-existing behavioural adaptations (egg smearing and probing) to facilitate its own transfer within host populations, adaptations that likely evolved to initiate and maintain an association with beneficial gut symbionts. Thus, the transmission of mutualistic microbes across host generations can entail a significant risk of co-transmitting pathogens or parasites, thereby exerting selective pressures on the host to evolve more specific mechanisms of transfer.

Mitochondrial respiration and genomic analysis provide insight into the influence of the symbiotic bacterium on host trypanosomatid oxygen consumption.

Certain trypanosomatids co-evolve with an endosymbiotic bacterium in a mutualistic relationship that is characterized by intense metabolic exchanges. Symbionts were able to respire for up to 4 h after isolation from Angomonas deanei. FCCP (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) similarly increased respiration in wild-type and aposymbiotic protozoa, though a higher maximal O2 consumption capacity was observed in the symbiont-containing cells. Rotenone, a complex I inhibitor, did not affect A. deanei respiration, whereas TTFA (thenoyltrifluoroacetone), a complex II activity inhibitor, completely blocked respiration in both strains. Antimycin A and cyanide, inhibitors of complexes III and IV, respectively, abolished O2 consumption, but the aposymbiotic protozoa were more sensitive to both compounds. Oligomycin did not affect cell respiration, whereas carboxyatractyloside (CAT), an inhibitor of the ADP-ATP translocator, slightly reduced O2 consumption. In the A. deanei genome, sequences encoding most proteins of the respiratory chain are present. The symbiont genome lost part of the electron transport system (ETS), but complex I, a cytochrome d oxidase, and FoF1-ATP synthase remain. In conclusion, this work suggests that the symbiont influences the mitochondrial respiration of the host protozoan.

Auxin production by the plant trypanosomatid Phytomonas serpens and auxin homoeostasis in infected tomato fruits.

Previously we have characterized the complete gene encoding a pyruvate decarboxylase (PDC)/indolepyruvate decarboxylase (IPDC) of Phytomonas serpens, a trypanosomatid highly abundant in tomato fruits. Phylogenetic analyses indicated that the clade that contains the trypanosomatid protein behaves as a sister group of IPDCs of γ-proteobacteria. Since IPDCs are key enzymes in the biosynthesis of the plant hormone indole-3-acetic acid (IAA), the ability for IAA production by P. serpens was investigated. Similar to many microorganisms, the production of IAA and related indolic compounds, quantified by high performance liquid chromatography, increased in P. serpens media in response to amounts of tryptophan. The auxin functionality was confirmed in the hypocotyl elongation assay. In tomato fruits inoculated with P. serpens the concentration of free IAA had no significant variation, whereas increased levels of IAA-amide and IAA-ester conjugates were observed. The data suggest that the auxin produced by the flagellate is converted to IAA conjugates, keeping unaltered the concentration of free IAA. Ethanol also accumulated in P. serpens-conditioned media, as the result of a PDC activity. In the article we discuss the hypothesis of the bifunctionality of P. serpens PDC/IPDC and provide a three-dimensional model of the enzyme.

Structural characterization of the cell division cycle in Strigomonas culicis, an endosymbiont-bearing trypanosomatid.

Strigomonas culicis (previously referred to as Blastocrithidia culicis) is a monoxenic trypanosomatid harboring a symbiotic bacterium, which maintains an obligatory relationship with the host protozoan. Investigations of the cell cycle in symbiont harboring trypanosomatids suggest that the bacterium divides in coordination with other host cell structures, particularly the nucleus. In this study we used light and electron microscopy followed by three-dimensional reconstruction to characterize the symbiont division during the cell cycle of S. culicis. We observed that during this process, the symbiotic bacterium presents different forms and is found at different positions in relationship to the host cell structures. At the G1/S phase of the protozoan cell cycle, the endosymbiont exhibits a constricted form that appears to elongate, resulting in the bacterium division, which occurs before kinetoplast and nucleus segregation. During cytokinesis, the symbionts are positioned close to each nucleus to ensure that each daughter cell will inherit a single copy of the bacterium. These observations indicated that the association of the bacterium with the protozoan nucleus coordinates the cell cycle in both organisms.

Trypanosomatids are common and diverse parasites of Drosophila.

Drosophila melanogaster is an important model system of immunity and parasite resistance, yet most studies use parasites that do not naturally infect this organism. We have studied trypanosomatids in natural populations to assess the prevalence and diversity of these gut parasites. We collected several species of Drosophila from Europe and surveyed them for trypanosomatids using conserved primers for two genes. We have used the conserved GAPDH sequence to construct a phylogenetic tree and the highly variable spliced leader RNA to assay genetic diversity. All 5 of the species that we examined were infected, and the average prevalence ranged from 1 to 6%. There are several different groups of trypanosomatids, related to other monoxenous Trypanosomatidae. These may represent new trypanosomatid species and were found in different species of European Drosophila from different geographical locations. The detection of a little studied natural pathogen in D. melanogaster and related species provides new opportunities for research into both the Drosophila immune response and the evolution of hosts and parasites.

Trypanosomatid Flagellar Pocket from Structure to Function.

The trypanosomatids Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. are flagellate eukaryotic parasites that cause serious diseases in humans and animals. These parasites have cell shapes defined by a subpellicular microtubule array and all share a number of important cellular features. One of these is the flagellar pocket, an invagination of the cell membrane around the proximal end of the flagellum, which is an important organelle for endo/exocytosis. The flagellar pocket plays a crucial role in parasite pathogenicity and persistence in the host and has a great influence on cell morphogenesis and cell division. Here, we compare the morphology and function of the flagellar pockets between different trypanosomatids, with their life cycles and ecological niches likely influencing these differences.

How monoxenous trypanosomatids revealed hidden feeding habits of their tsetse fly hosts.

Tsetse flies are well-known vectors of trypanosomes pathogenic for humans and livestock. For these strictly blood-feeding viviparous flies, the host blood should be the only source of nutrients and liquids, as well as any exogenous microorganisms colonising their intestine. Here we describe the unexpected finding of several monoxenous trypanosomatids in their gut. In a total of 564 individually examined Glossina (Austenia) tabaniformis (Westwood) (436 specimens) and Glossina (Nemorhina) fuscipes fuscipes (Newstead) (128 specimens) captured in the Dzanga-Sangha Protected Areas, Central African Republic, 24 (4.3%) individuals were infected with monoxenous trypanosomatids belonging to the genera Crithidia Léger, 1902; Kentomonas Votýpka, Yurchenko, Kostygov et Lukes, 2014; Novymonas Kostygov et Yurchenko, 2020; Obscuromonas Votýpka et Lukes, 2021; and Wallacemonas Kostygov et Yurchenko, 2014. Moreover, additional 20 (3.5%) inspected tsetse flies harboured free-living bodonids affiliated with the genera Dimastigella Sandon, 1928; Neobodo Vickerman, 2004; Parabodo Skuja, 1939; and Rhynchomonas Klebs, 1892. In the context of the recently described feeding behaviour of these dipterans, we propose that they become infected while taking sugar meals and water, providing indirect evidence that blood is not their only source of food and liquids.

The flagellum-mitogen-activated protein kinase connection in Trypanosomatids: a key sensory role in parasite signalling and development?

Trypanosomatid parasites are the causative agents of severe human diseases such as sleeping sickness, Chagas disease and leishmaniases. These microorganisms are transmitted via different insect vectors and hence are confronted to changing environments during their infectious cycle in which they activate specific and complex patterns of differentiation. Several studies in Trypanosoma brucei and in different subspecies of Leishmania have shed light on the role of mitogen-activated protein (MAP) kinases in these processes. Surprisingly, several MAP kinases turned out to be involved in the control of flagellum length in the promastigote stage of Leishmania. Recently, a sensory function has been recognized for cilia and flagella in unicellular and multicellular eukaryotes. This review aims to stimulate discussions on the possibility that the Trypanosomatid flagellum could act as a sensory organ through the MAP kinase pathway, with the objective to encourage investigation of this new hypothesis through a series of proposed experimental approaches.

Proteins involved in the biosynthesis of lipophosphoglycan in Leishmania: a comparative genomic and evolutionary analysis.

BACKGROUND: Leishmania spp. are digenetic parasites capable of infecting humans and causing a range of diseases collectively known as leishmaniasis. The main mechanisms involved in the development and permanence of this pathology are linked to evasion of the immune response. Crosstalk between the immune system and particularities of each pathogenic species is associated with diverse disease manifestations. Lipophosphoglycan (LPG), one of the most important molecules present on the surface of Leishmania parasites, is divided into four regions with high molecular variability. Although LPG plays an important role in host-pathogen and vector-parasite interactions, the distribution and phylogenetic relatedness of the genes responsible for its synthesis remain poorly explored. The recent availability of full genomes and transcriptomes of Leishmania parasites offers an opportunity to leverage insight on how LPG-related genes are distributed and expressed by these pathogens. RESULTS: Using a phylogenomics-based framework, we identified a catalog of genes involved in LPG biosynthesis across 22 species of Leishmania from the subgenera Viannia and Leishmania, as well as 5 non-Leishmania trypanosomatids. The evolutionary relationships of these genes across species were also evaluated. Nine genes related to the production of the glycosylphosphatidylinositol (GPI)-anchor were highly conserved among compared species, whereas 22 genes related to the synthesis of the repeat unit presented variable conservation. Extensive gain/loss events were verified, particularly in genes SCG1-4 and SCA1-2. These genes act, respectively, on the synthesis of the side chain attached to phosphoglycans and in the transfer of arabinose residues. Phylogenetic analyses disclosed evolutionary patterns reflective of differences in host specialization, geographic origin and disease manifestation. CONCLUSIONS: The multiple gene gain/loss events identified by genomic data mining help to explain some of the observed intra- and interspecies variation in LPG structure. Collectively, our results provide a comprehensive catalog that details how LPG-related genes evolved in the Leishmania parasite specialization process.

Lysophosphatidylcholine triggers cell differentiation in the protozoan parasite Herpetomonas samuelpessoai through the CK2 pathway.

BACKGROUND: Protozoa are distantly related to vertebrates but present some features of higher eukaryotes, making them good model systems for studying the evolution of basic processes such as the cell cycle. Herpetomonas samuelpessoai is a trypanosomatid parasite isolated from the hemipteran insect Zelus leucogrammus. Lysophosphatidylcholine (LPC) is implicated in the transmission and establishment of Chagas disease, whose etiological agent is Trypanosoma cruzi. LPC is synthesized by T. cruzi and its vectors, the hemipteran Rhodnius prolixus and Triatoma infestans. Platelet-activating factor (PAF), a phospholipid with potent and diverse physiological and pathophysiological actions, is a powerful inducer of cell differentiation in Herpetomonas muscarum muscarum and T. cruzi. The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the 2-ester bond of 3-sn-phosphoglyceride, transforming phosphatidylcholine (PC) into LPC. METHODS: In this study, we evaluated cellular differentiation, PLA2 activity and protein kinase CK2 activity of H. samuelpessoai in the absence and in the presence of LPC and PAF. RESULTS: We demonstrate that both PC and LPC promoted a twofold increase in the cellular differentiation of H. samuelpessoai, through CK2, with a concomitant inhibition of its cell growth. Intrinsic PLA2 most likely directs this process by converting PC into LPC. CONCLUSIONS: Our results suggest that the actions of LPC on H. samuelpessoai occur upon binding to a putative PAF receptor and that the protein kinase CK2 plays a major role in this process. Cartoon depicting a model for the synthesis and functions of LPC in Herpetomonas samuelpessoai, based upon our results regarding the role of LPC on the cell biology of Trypanosoma cruzi [28-32]. N nucleus, k kinetoplast, PC phosphatidylcholine, LPC lysophosphatidylcholine, PLA2 phospholipase A2, PAFR putative PAF receptor in trypanosomatids [65], CK2 protein kinase CK2 [16].

Cytokinesis in trypanosomatids.

The process of cytokinesis, where the cytoplasm of one cell is divided to produce two daughter cells, is intricate in trypanosomatids because of the requirement to replicate and segregate a number of single copy organelles, including the nucleus, kinetoplast, Golgi apparatus, and flagellum. Identifying regulators of the three stages of cytokinesis, initiation, furrow ingression, and abscission is complicated by the fact that cell division in trypanosomatids is easily perturbed and aberrant cells are readily produced during functional characterization of gene products. In this review, we discuss direct and indirect effects on cytokinesis, using Trypanosoma brucei as a model.

A postgenomic view of the heat shock proteins in kinetoplastids.

The kinetoplastids Leishmania major, Trypanosoma brucei and Trypanosoma cruzi are causative agents of a diverse spectrum of human diseases: leishmaniasis, sleeping sickness and Chagas' disease, respectively. These protozoa possess digenetic life cycles that involve development in mammalian and insect hosts. It is generally accepted that temperature is a triggering factor of the developmental programme allowing the adaptation of the parasite to the mammalian conditions. The heat shock response is a general homeostatic mechanism that protects cells from the deleterious effects of environmental stresses, such as heat. This response is universal and includes the synthesis of the heat-shock proteins (HSPs). In this review, we summarize the salient features of the different HSP families and describe their main cellular functions. In parallel, we analyse the composition of these families in kinetoplastids according to literature data and our understanding of genome sequence data. The genome sequences of these parasites have been recently completed. The HSP families described here are: HSP110, HSP104, group I chaperonins, HSP90, HSP70, HSP40 and small HSPs. All these families are widely represented in these parasites. In particular, kinetoplastids possess an unprecedented number of members of the HSP70, HSP60 and HSP40 families, suggesting key roles for these HSPs in their biology.

Unique Aspects of rRNA Biogenesis in Trypanosomatids.

Trypanosomatids are protozoan parasites that cycle between an insect and a mammalian host. The large-subunit rRNA of these organisms undergoes unique processing events absent in other eukaryotes. Recently, small nucleolar RNAs (snoRNAs) that mediate these specific cleavages were identified. Trypanosomatid rRNA is rich in RNA modifications such as 2'-O-methylation (Nm) and pseudouridylation (Ψ) that are also guided by these snoRNAs. A subset of these modifications is developmentally regulated and increased in the parasite form that propagates in the mammalian host. Such hypermodification contributes the temperature adaptation and hence infectivity during cycling of the parasite. rRNA processing and modification should be considered promising drug targets for fighting the diseases caused by these parasites.