Intracellular Eukaryotic Pathogens Research Articles

Microsporidian EnP1 alters host cell H2B monoubiquitination and prevents ferroptosis facilitating microsporidia survival

Microsporidia are intracellular eukaryotic pathogens that pose a substantial threat to immunocompromised hosts. The way these pathogens manipulate host cells during infection remains poorly understood. Using a proximity biotinylation strategy we established that microsporidian EnP1 is a nucleus-targeted effector that modifies the host cell environment. EnP1's translocation to the host nucleus is meditated by nuclear localization signals (NLSs). In the nucleus, EnP1 interacts with host histone H2B. This interaction disrupts H2B monoubiquitination (H2Bub), subsequently impacting p53 expression. Crucially, this inhibition of p53 weakens its control over the downstream target gene SLC7A11, enhancing the host cell's resilience against ferroptosis during microsporidian infection. This favorable condition promotes the proliferation of microsporidia within the host cell. These findings shed light on the molecular mechanisms by which microsporidia modify their host cells to facilitate their survival.

Incomplete Recruitment of Protective T Cells Is Associated with Trypanosoma cruzi Persistence in the Mouse Colon.

ABSTRACTTrypanosoma cruzi is the etiological agent of Chagas disease. Following T cell-mediated suppression of acute-phase infection, this intracellular eukaryotic pathogen persists long-term in a limited subset of tissues at extremely low levels. The reasons for this tissue-specific chronicity are not understood. Using a dual bioluminescent-fluorescent reporter strain and highly sensitive tissue imaging that allows experimental infections to be monitored at single-cell resolution, we undertook a systematic analysis of the immunological microenvironments of rare parasitized cells in the mouse colon, a key site of persistence. We demonstrate that incomplete recruitment of T cells to a subset of colonic infection foci permits the occurrence of repeated cycles of intracellular parasite replication and differentiation to motile trypomastigotes at a frequency sufficient to perpetuate chronic infections. The lifelong persistence of parasites in this tissue site continues despite the presence, at a systemic level, of a highly effective T cell response. Overcoming this low-level dynamic host-parasite equilibrium represents a major challenge for vaccine development.

Characterisation of infection associated microRNA and protein cargo in extracellular vesicles of Theileria annulata infected leukocytes.

The protozoan parasites Theileria annulata and Theileria parva are unique amongst intracellular eukaryotic pathogens as they induce a transformation‐like phenotype in their bovine host cell. T. annulata causes tropical theileriosis, which is frequently fatal, with infected leukocytes becoming metastatic and forming foci in multiple organs resulting in destruction of the lymphoid system. Exosomes, a subset of extracellular vesicles (EV), are critical in metastatic progression in many cancers. Here, we characterised the cargo of EV from a control bovine lymphosarcoma cell line (BL20) and BL20 infected with T. annulata (TBL20) by comparative mass spectrometry and microRNA (miRNA) profiling (data available via ProteomeXchange, identifier PXD010713 and NCBI GEO, accession number GSE118456, respectively). Ingenuity pathway analysis that many infection‐associated proteins essential to migration and extracellular matrix digestion were upregulated in EV from TBL20 cells compared with BL20 controls. An altered repertoire of host miRNA, many with known roles in tumour and/or infection biology, was also observed. Focusing on the tumour suppressor miRNA, bta‐miR‐181a and bta‐miR‐181b, we identified putative messenger RNA targets and confirmed the interaction of bta‐miR181a with ICAM‐1. We propose that EV and their miRNA cargo play an important role in the manipulation of the host cell phenotype and the pathobiology of Theileria infection.

Subtelomere organization in the genome of the microsporidian Encephalitozoon cuniculi: patterns of repeated sequences and physicochemical signatures.

BackgroundThe microsporidian Encephalitozoon cuniculi is an obligate intracellular eukaryotic pathogen with a small nuclear genome (2.9 Mbp) consisting of 11 chromosomes. Although each chromosome end is known to contain a single rDNA unit, the incomplete assembly of subtelomeric regions following sequencing of the genome identified only 3 of the 22 expected rDNA units. While chromosome end assembly remains a difficult process in most eukaryotic genomes, it is of significant importance for pathogens because these regions encode factors important for virulence and host evasion.ResultsHere we report the first complete assembly of E. cuniculi chromosome ends, and describe a novel mosaic structure of segmental duplications (EXT repeats) in these regions. EXT repeats range in size between 3.5 and 23.8 kbp and contain four multigene families encoding membrane associated proteins. Twenty-one recombination sites were identified in the sub-terminal region of E. cuniculi chromosomes. Our analysis suggests that these sites contribute to the diversity of chromosome ends organization through Double Strand Break repair mechanisms. The region containing EXT repeats at chromosome extremities can be differentiated based on gene composition, GC content, recombination sites density and chromosome landscape.ConclusionTogether this study provides the complete structure of the chromosome ends of E. cuniculi GB-M1, and identifies important factors, which could play a major role in parasite diversity and host-parasite interactions. Comparison with other eukaryotic genomes suggests that terminal regions could be distinguished precisely based on gene content, genetic instability and base composition biais. The diversity of processes assciated with chromosome extremities and their biological consequences, as they are presented in the present study, emphasize the fact that great effort will be necessary in the future to characterize more carefully these regions during whole genome sequencing efforts.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1920-7) contains supplementary material, which is available to authorized users.

Intracellular eukaryotic pathogens in brown macroalgae in the Eastern Mediterranean, including LSU rRNA data for the oomycete Eurychasma dicksonii.

For the Mediterranean Sea, and indeed most of the world's oceans, the biodiversity and biogeography of eukaryotic pathogens infecting marine macroalgae remains poorly known, yet their ecological impact is probably significant. Based on 2 sampling campaigns on the Greek island of Lesvos in 2009 and 1 in northern Greece in 2012, this study provides first records of 3 intracellular eukaryotic pathogens infecting filamentous brown algae at these locations: Eurychasma dicksonii, Anisolpidium sphacellarum, and A. ectocarpii. Field and microscopic observations of the 3 pathogens are complemented by the first E. dicksonii large subunit ribosomal RNA (LSU rRNA) gene sequence analyses of isolates from Lesvos and other parts of the world. The latter highlights the monophyly of E. dicksonii worldwide and confirms the basal position of this pathogen within the oomycete lineage (Peronosporomycotina). The results of this study strongly support the notion that the geographic distribution of the relatively few eukaryotic seaweed pathogens is probably much larger than previously thought and that many of the world's marine bioregions remain seriously undersampled and understudied in this respect.

Treatment of Microsporidia Keratitis With Topical Voriconazole Monotherapy

Microsporidia are obligate intracellular eukaryotic pathogens known to cause superficial punctate keratitis and stromal keratitis in both immunocompromised and immuno-competent individuals. Traditionally, microsporidia were classified as primitive eukaryotes; however, recent genomic evidence supports their reclassification as fungi.1 Definitive treatment for micro-sporidial keratitis has not been established. Based on the classification of microsporidia as fungi, use of topical antifungal agents may be beneficial as monotherapy. Voriconazole is available through compounding pharmacies in topical form and is effective against several fungal pathogens.2 Herein, we report 2 cases of microsporidial superficial punctate keratitis that were responsive to treatment with topical voriconazole. To our knowledge, this is the first case series of the successful treatment of microsporidial superficial punctate keratitis with topical voriconazole.

The Transforming Parasite Theileria Co-opts Host Cell Mitotic and Central Spindles to Persist in Continuously Dividing Cells

Author SummaryAs part of their survival tactics, intracellular parasites often resort to cunning mechanisms to manipulate the cells they inhabit. Theileria, an important and particularly artful parasite of cattle in the tropics, transforms parasitized cells (that is, it induces continuous proliferation and protection from apoptosis—a state reminiscent of tumor cells). As a large, strictly intracellular syncytium, the transforming Theileria schizont cannot exit from the infected cell to invade other target cells. How then does the parasite ensure that each daughter cell, generated upon host cell division, remains infected and transformed? Our data show that the parasite co-opts the mitotic apparatus of the host cell and Plk1, a host protein kinase with a central regulatory role in mitosis and cytokinesis. As the host cell enters mitosis, the schizont binds to the microtubules that emanate symmetrically from the two spindle poles. This microtubule binding positions the schizont so that it spans the equatorial region of the mitotic cell where host cell chromosomes assemble. Then, as sister chromatids start to separate, the schizont associates with Plk1 and the central spindle that assembles between the separating chromosomes, with the activity of Plk1 presumably coordinating progression through mitosis with proper schizont positioning. This alignment with the central spindle positions the schizont to be included in the plane of cell division at the onset of cytokinesis, thus ensuring faithful passage of a Theileria schizont on to each daughter cell.

The malaria parasite Plasmodium falciparum: cell biological peculiarities and nutritional consequences

Apicomplexan parasites obligatorily invade and multiply within eukaryotic cells. Phylogenetically, they are related to a group of algae which, during their evolution, have acquired a secondary endosymbiont. This organelle, which in the parasite is called the apicoplast, is highly reduced compared to the endosymbionts of algae, but still contains many plant-specific biosynthetic pathways. The malaria parasite Plasmodium falciparum infects mammalian erythrocytes which are devoid of intracellular compartments and which largely lack biosynthetic pathways. Despite the limited resources of nutrition, the parasite grows and generates up to 32 merozoites which are the infectious stages of the complex life cycle. A large part of the intra-erythrocytic development takes place in the so-called parasitophorous vacuole, a compartment which forms an interface between the parasite and the cytoplasm of the host cell. In the course of parasite growth, the host cell undergoes dramatic alterations which on one hand contribute directly to the symptoms of severe malaria and which, on the other hand, are also required for parasite survival. Some of these alterations facilitate the acquisition of nutrients from the extracellular environment which are not provided by the host cell. Here, we describe the cell biologically unique interactions between an intracellular eukaryotic pathogen and its metabolically highly reduced host cell. We further discuss current models to explain the appearance of pathogen-induced novel physiological properties in a host cell which has lost its genetic programme.

Recognition of Leishmania Parasites by Innate Immunity

The host innate immune system represents the first line of defense against invasive pathogens. During the crucial early stages of infection, the host innate immune system must be able to rapidly detect and respond to foreign pathogens, enabling an efficient and successful adaptative immune response. Leishmania parasites are obligate intracellular eukaryotic pathogens living inside cells of the mononuclear phagocytic system. Recent data has proven distinct roles of various phagocytic cells, such as neutrophils, macrophages and dendritic cells during Leishmania infection. There is growing evidence that Leishmania modifies antigen presentation, apoptosis and immunoregulatory functions on these cells, leading to persistent and chronic infection. At the molecular level, the Toll-like receptors (TLR) family is a major player in the early host-pathogen interaction. The TLRs expressed intracellularly or at the surface of the cells involved in the innate immune response recognize conserved structures on foreign pathogens, such as Leishmania, playing a pivotal role in triggering innate and adaptative immune responses. Nonetheless, the same TLRs can be considered as a potential strategic target used by these organisms for their own advantage. In this review, we discussed the findings on the cellular processes involved in the innate host defense against intracellular pathogens, focusing on the Leishmania infection, from the initial host-parasite interactions involved in the parasite recognition to the mechanisms employed to eliminate the pathogen, presenting new data on the role of TLR2 in visceral leishmaniasis. Keywords: Leishmania, innate immunity, Toll-like receptors, TLR2, antigen presenting cells, host-parasite interaction

Evolutionary Biology: Microsporidia Sex — A Missing Link to Fungi

The evolutionary origins of the microsporidia, a group of intracellular eukaryotic pathogens, have been unclear. Genome analysis of a sex locus and other gene clusters has now revealed conserved synteny with zygomycete fungi, indicating that microsporidia are true fungi descended from a zygomycete ancestor.

Microsporidia Evolved from Ancestral Sexual Fungi

Microsporidia are obligate, intracellular eukaryotic pathogens that infect animal cells, including humans [1]. Previous studies suggested microsporidia share a common ancestor with fungi [2-7]. However, the exact nature of this phylogenetic relationship is unclear because of unusual features of microsporidial genomes, which are compact with fewer and highly divergent genes [8]. As a consequence, it is unclear whether microsporidia evolved from a specific fungal lineage, or whether microsporidia are a sister group to all fungi. Here, we present evidence addressing this controversial question that is independent of sequence-based phylogenetic reconstruction, but rather based on genome structure. In the zygomycete basal fungal lineage, the sex locus is a syntenic gene cluster governing sexual reproduction in which a high mobility group (HMG) transcription-factor gene is flanked by triose-phosphate transporter (TPT) and RNA helicase genes [9]. Strikingly, microsporidian genomes harbor a sex-related locus with the same genes in the same order. Genome-wide synteny analysis reveals multiple other loci conserved between microsporidia and zygomycetes to the exclusion of all other fungal lineages with sequenced genomes. These findings support the hypothesis that microsporidia are true fungi that descended from a zygomycete ancestor and suggest microsporidia may have an extant sexual cycle.

Toxoplasma co-opts host gene expression by injection of a polymorphic kinase homologue

Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa, can cause severe disease in humans with an immature or suppressed immune system. The outcome of Toxoplasma infection is highly dependent on the strain type, as are many of its in vitro growth properties. Here we use genetic crosses between type II and III lines to show that strain-specific differences in the modulation of host cell transcription are mediated by a putative protein kinase, ROP16. Upon invasion by the parasite, this polymorphic protein is released from the apical organelles known as rhoptries and injected into the host cell, where it ultimately affects the activation of signal transducer and activator of transcription (STAT) signalling pathways and consequent downstream effects on a key host cytokine, interleukin (IL)-12. Our findings provide a new mechanism for how an intracellular eukaryotic pathogen can interact with its host and reveal important differences in how different Toxoplasma lineages have evolved to exploit this interaction.

Lipid biology of Apicomplexa: perspectives for new drug targets, particularly for Toxoplasma gondii

Development of effective therapies for intracellular eukaryotic pathogens is a serious challenge, given the protected location of these pathogens and the similarity of their biology to that of the host. Identifying cellular processes that are unique to the parasite is therefore a crucial step towards defining appropriate drug targets. In the case of the apicomplexan parasite Toxoplasma gondii, the need to find alternative treatments is imperative because of the poor tolerability and frequent side-effects associated with existing therapeutic strategies. The discovery that the parasite uses lipid synthetic pathways which are different from, or absent in, the mammalian host is now driving a renewed interest in T. gondii lipid biology. Recent achievements in this field are promising and suggest that the elucidation of lipid pathways will provide new opportunities for designing potent antiparasitic strategies.

MyD88 is essential for clearance of Leishmania major: possible role for lipophosphoglycan and Toll-like receptor 2 signaling.

Leishmania major is an obligate intracellular eukaryotic pathogen of mononuclear phagocytes. Invasive promastigotes gain entry into target cells by receptor-mediated phagocytosis, transform into non-motile amastigotes and establish in the phagolysosome. Glycosylphosphatidylinositol-anchored lipophosphoglycan (LPG) is a virulence factor and a major parasite molecule involved in this process. We observed that mice lacking the Toll-like receptor (TLR) pathway adaptor protein MyD88 were more susceptible to infection with L. major than wild-type C57BL/6 mice, demonstrating a central role for this innate immune recognition pathway in control of infection, and suggesting that L. major possesses a ligand for TLR. We sought to identify parasite molecules capable of activating the protective Toll pathway, and found that purified Leishmania LPG, but not other surface glycolipids, activate innate immune signaling pathways via TLR2. Activation of cytokine synthesis by LPG required the presence of the lipid anchor and a functional MyD88 adaptor protein. LPG also induced the expression of negative regulatory pathways mediated by members of thesuppressors of cytokine signaling family SOCS-1 and SOCS-3. Thus, the Toll pathway is required for resistance to L. major and LPG is a defined TLR agonist from this important human pathogen.

Isolation and in vitro characteristics of chinook salmon ( Oncorhynchus tshawytscha) rosette agent

Isolation and in vitro characteristics of a significant obligate intracellular eukaryotic pathogen (rosette agent) of chinook salmon ( Oncorhynchus tshawytscha) are described. The pathogen was isolated by primary culture of infected spleen tissue followed by inoculation onto the chinook salmon embryo CHSE-214 cell line. Chicken and rabbit antibody was produced to a soluble antigen derived from the rosette agent. Mortalities and the disease were experimentally reproduced by inoculating chinook salmon with the isolate. The pathogen was reisolated 27 days post-inoculation from moribund fish. Identity of the isolate and the disease-causing organism was further supported by documenting antigenic and morphologic identity. Tested in CHSE-214 cells, the organisms are sensitive to the antibiotics oxytetracycline, chloramphenicol, amphotericin-B and nystatin. A ratio of about one organism per tissue culture cell at inoculation was required to develop an easily detectable infection. Adsorption of organisms to tissue culture cells at inoculation did not enhance the pathogen's infectivity. The pathogen replicated at an increasing rate from 5°C to 20°C. It could not be stored by freezing but storage in tissue culture at 5°C for at least 5 months proved possible. Early passages of the organism showed a high degree of host specificity in tissue culture which decreased in later passages. The taxonomic affinities of the organism are discussed.

A significant new systemic disease of net-pen reared chinook salmon ( Oncorhynchus tshawytscha) brood stock

During an 8-month period in 1983 and 1984, over 80% mortality occurred in groups of 3-year-old chinook salmon ( Oncorhynchus tshawytscha) brood stock which were being cultured in net-pens in Puget Sound, Washington. Despite thorough pathological and microbiological examination, these losses could not be attributed to known pathogens or poor nutrition. All sick fish were anemic with a marked lymphocytosis. The kidneys and spleens were enlarged at necropsy. Both light- and electron-microscopic examination of spleen and kidney tissue revealed numerous intracellular 3–7-μm spherical organisms with histochemical and ultrastructural characteristics suggesting that they had affinities to marine algae or fungi. The laboratory and field observations indicate that the disease reported here is a previously unknown and significant infectious disease of chinook salmon. Furthermore, this is the first report of a systemic disease of fish caused by an apparently obligate intracellular eukaryotic pathogen. The presumptive causative micro-organism was descriptively termed the chinook salmon rosette agent.