Systemic Immune Modulation by Gastrointestinal Nematodes.

Gastrointestinal nematode (GIN) infection has applied significant evolutionary pressure to the mammalian immune system and remains a global economic and human health burden. Upon infection, type 2 immune sentinels activate a common antihelminth response that mobilizes and remodels the intestinal tissue for effector function; however, there is growing appreciation of the impact GIN infection also has on the distal tissue immune state. Indeed, this effect is observed even in tissues through which GINs never transit. This review highlights how GIN infection modulates systemic immunity through (a) induction of host resistance and tolerance responses, (b) secretion of immunomodulatory products, and (c) interaction with the intestinal microbiome. It also discusses the direct consequences that changes to distal tissue immunity can have for concurrent and subsequent infection, chronic noncommunicable diseases, and vaccination efficacy.

Duplication and neofunctionalization of a horizontally transferred xyloglucanase as a facet of the Red Queen coevolutionary dynamic.

Oomycete protists share phenotypic similarities with fungi, including the ability to cause plant diseases, but branch in a distant region of the tree of life. It has been suggested that multiple horizontal gene transfers (HGTs) from fungi-to-oomycetes contributed to the evolution of plant-pathogenic traits. These HGTs are predicted to include secreted proteins that degrade plant cell walls, a barrier to pathogen invasion and a rich source of carbohydrates. Using a combination of phylogenomics and functional assays, we investigate the diversification of a horizontally transferred xyloglucanase gene family in the model oomycete species Phytophthora sojae. Our analyses detect 11 xyloglucanase paralogs retained in P. sojae. Using heterologous expression in yeast, we show consistent evidence that eight of these paralogs have xyloglucanase function, including variants with distinct protein characteristics, such as a long-disordered C-terminal extension that can increase xyloglucanase activity. The functional variants analyzed subtend a phylogenetic node close to the fungi-to-oomycete transfer, suggesting the horizontally transferred gene was a bona fide xyloglucanase. Expression of three xyloglucanase paralogs in Nicotiana benthamiana triggers high-reactive oxygen species (ROS) generation, while others inhibit ROS responses to bacterial immunogens, demonstrating that the paralogs differentially stimulate pattern-triggered immunity. Mass spectrometry of detectable enzymatic products demonstrates that some paralogs catalyze the production of variant breakdown profiles, suggesting that secretion of variant xyloglucanases increases efficiency of xyloglucan breakdown as well as diversifying the damage-associated molecular patterns released. We suggest that this pattern of neofunctionalization and the variant host responses represent an aspect of the Red Queen host-pathogen coevolutionary dynamic.

The parasite intraerythrocytic cycle and human circadian cycle are coupled during malaria infection.

During infections with the malaria parasites Plasmodium vivax, patients exhibit rhythmic fevers every 48 h. These fever cycles correspond with the time the parasites take to traverse the intraerythrocytic cycle (IEC). In other Plasmodium species that infect either humans or mice, the IEC is likely guided by a parasite-intrinsic clock [Rijo-Ferreiraet al., Science 368, 746-753 (2020); Smith et al., Science 368, 754-759 (2020)], suggesting that intrinsic clock mechanisms may be a fundamental feature of malaria parasites. Moreover, because Plasmodium cycle times are multiples of 24 h, the IECs may be coordinated with the host circadian clock(s). Such coordination could explain the synchronization of the parasite population in the host and enable alignment of IEC and circadian cycle phases. We utilized an ex vivo culture of whole blood from patients infected with P. vivax to examine the dynamics of the host circadian transcriptome and the parasite IEC transcriptome. Transcriptome dynamics revealed that the phases of the host circadian cycle and the parasite IEC are correlated across multiple patients, showing that the cycles are phase coupled. In mouse model systems, host-parasite cycle coupling appears to provide a selective advantage for the parasite. Thus, understanding how host and parasite cycles are coupled in humans could enable antimalarial therapies that disrupt this coupling.

LC3B labeling of the parasitophorous vacuole membrane of Plasmodium berghei liver stage parasites depends on the V-ATPase and ATG16L1.

The protozoan parasite Plasmodium, the causative agent of malaria, undergoes an obligatory stage of intra-hepatic development before initiating a blood-stage infection. Productive invasion of hepatocytes involves the formation of a parasitophorous vacuole (PV) generated by the invagination of the host cell plasma membrane. Surrounded by the PV membrane (PVM), the parasite undergoes extensive replication. During intracellular development in the hepatocyte, the parasites provoke the Plasmodium-associated autophagy-related (PAAR) response. This is characterized by a long-lasting association of the autophagy marker protein, and ATG8 family member, LC3B with the PVM. LC3B localization at the PVM does not follow the canonical autophagy pathway since upstream events specific to canonical autophagy are dispensable. Here, we describe that LC3B localization at the PVM of Plasmodium parasites requires the V-ATPase and its interaction with ATG16L1. The WD40 domain of ATG16L1 is crucial for its recruitment to the PVM. Thus, we provide new mechanistic insight into the previously described PAAR response targeting Plasmodium liver stage parasites.

Embracing nature's complexity: Immunoparasitology in the wild.

A wealth of research is dedicated to understanding how resistance against parasites is conferred and how parasite-driven pathology is regulated. This research is in part driven by the hope to better treatments for parasitic diseases of humans and livestock, and in part by immunologists who use parasitic infections as biomedical tools to evoke physiological immune responses. Much of the current mechanistic knowledge has been discovered in laboratory studies using model organisms, especially the laboratory mouse. However, wildlife are also hosts to a range of parasites. Through the study of host-parasite interactions in these non-laboratory systems we can gain a deeper understanding of parasite immunology in a more natural, complex environment. With a focus on helminth parasites, we here explore the insights gained into parasite-induced immune responses through (for immunologists) non-conventional experimental systems, and how current core findings from laboratory studies are reflected in these more natural conditions. The quality of the immune response is undoubtedly a central player in susceptibility versus resistance, as many laboratory studies have shown. Yet, in the wild, parasite infections tend to be chronic diseases. Whilst reading our review, we encourage the reader to consider the following questions which may (only) be answered by studying naturally occurring parasites in the wild: a) what type of immune responses are mounted against parasites in different hosts in the wild, and how do they vary within an individual over time, between individuals of the same species and between species? b) can we use wild or semi-wild study systems to understand the evolutionary drivers for tolerance versus resistance towards a parasite? c) what determines the ability of the host to cope with an infection and is there a link with the type of immune response mounted? d) can we modulate environmental factors to manipulate a wild animal's immune response to parasitic infections, with translation potential for humans, wildlife, and livestock? and e) in context of this special issue, what lessons for Type 2 immunity can we glean from studying animals in their natural environments? Further, we aim to integrate some of the knowledge gained in semi-wild and wild settings with knowledge gained from traditional laboratory-based research, and to raise awareness for the opportunities (and challenges) that come with integrating a multitude of naturally-occurring variables into immunoparasitological research.

Immune system modulation & virus transmission during parasitism identified by multi-species transcriptomics of a declining insect biocontrol system.

BACKGROUND: The Argentine stem weevil (ASW, Listronotus bonariensis) is a significant pasture pest in Aotearoa New Zealand, primarily controlled by the parasitoid biocontrol agent Microctonus hyperodae. Despite providing effective control of ASW soon after release, M. hyperodae parasitism rates have since declined significantly, with ASW hypothesised to have evolved resistance to its biocontrol agent. While the parasitism arsenal of M. hyperodae has previously been investigated, revealing many venom components and an exogenous novel DNA virus Microctonus hyperodae filamentous virus (MhFV), the effects of said arsenal on gene expression in ASW during parasitism have not been examined. In this study, we performed a multi-species transcriptomic analysis to investigate the biology of ASW parasitism by M. hyperodae, as well as the decline in efficacy of this biocontrol system. RESULTS: The transcriptomic response of ASW to parasitism by M. hyperodae involves modulation of the weevil's innate immune system, flight muscle components, and lipid and glucose metabolism. The multispecies approach also revealed continued expression of venom components in parasitised ASW, as well as the transmission of MhFV to weevils during parasitism and some interrupted parasitism attempts. Transcriptomics did not detect a clear indication of parasitoid avoidance or other mechanisms to explain biocontrol decline. CONCLUSIONS: This study has expanded our understanding of interactions between M. hyperodae and ASW in a biocontrol system of critical importance to Aotearoa-New Zealand's agricultural economy. Transmission of MhFV to ASW during successful and interrupted parasitism attempts may link to a premature mortality phenomenon in ASW, hypothesised to be a result of a toxin-antitoxin system. Further research into MhFV and its potential role in ASW premature mortality is required to explore whether manipulation of this viral infection has the potential to increase biocontrol efficacy in future.

Into the danger zone: How the within-host distribution of parasites controls virulence.

Despite the importance of virulence in epidemiological theory, the relative contributions of host and parasite to virulence outcomes remain poorly understood. Here, we use reciprocal cross experiments to disentangle the influence of host and parasite on core virulence components-infection and pathology-and understand dramatic differences in parasite-induced malformations in California amphibians. Surveys across 319 populations revealed that amphibians' malformation risk was 2.7× greater in low-elevation ponds, even while controlling for trematode infection load. Factorial experiments revealed that parasites from low-elevation sites induced higher per-parasite pathology (reduced host survival and growth), whereas there were no effects of host source on resistance or tolerance. Parasite populations also exhibited marked differences in within-host distribution: ~90% of low-elevation cysts aggregated around the hind limbs, relative to <60% from high-elevation. This offers a novel, mechanistic basis for regional variation in parasite-induced malformations while promoting a framework for partitioning host and parasite contributions to virulence.

Comparative secretome analysis of Striga and Cuscuta species identifies candidate virulence factors for two evolutionarily independent parasitic plant lineages.

BACKGROUND: Many parasitic plants of the genera Striga and Cuscuta inflict huge agricultural damage worldwide. To form and maintain a connection with a host plant, parasitic plants deploy virulence factors (VFs) that interact with host biology. They possess a secretome that represents the complement of proteins secreted from cells and like other plant parasites such as fungi, bacteria or nematodes, some secreted proteins represent VFs crucial to successful host colonisation. Understanding the genome-wide complement of putative secreted proteins from parasitic plants, and their expression during host invasion, will advance understanding of virulence mechanisms used by parasitic plants to suppress/evade host immune responses and to establish and maintain a parasite-host interaction. RESULTS: We conducted a comparative analysis of the secretomes of root (Striga spp.) and shoot (Cuscuta spp.) parasitic plants, to enable prediction of candidate VFs. Using orthogroup clustering and protein domain analyses we identified gene families/functional annotations common to both Striga and Cuscuta species that were not present in their closest non-parasitic relatives (e.g. strictosidine synthase like enzymes), or specific to either the Striga or Cuscuta secretomes. For example, Striga secretomes were strongly associated with 'PAR1' protein domains. These were rare in the Cuscuta secretomes but an abundance of 'GMC oxidoreductase' domains were found, that were not present in the Striga secretomes. We then conducted transcriptional profiling of genes encoding putatively secreted proteins for the most agriculturally damaging root parasitic weed of cereals, S. hermonthica. A significant portion of the Striga-specific secretome set was differentially expressed during parasitism, which we probed further to identify genes following a 'wave-like' expression pattern peaking in the early penetration stage of infection. We identified 39 genes encoding putative VFs with functions such as cell wall modification, immune suppression, protease, kinase, or peroxidase activities, that are excellent candidates for future functional studies. CONCLUSIONS: Our study represents a comprehensive secretome analysis among parasitic plants and revealed both similarities and differences in candidate VFs between Striga and Cuscuta species. This knowledge is crucial for the development of new management strategies and delaying the evolution of virulence in parasitic weeds.

Mitochondrial background can explain variable costs of immune deployment.

Organismal health and survival depend on the ability to mount an effective immune response against infection. Yet immune defence may be energy-demanding, resulting in fitness costs if investment in immune function deprives other physiological processes of resources. While evidence of costly immunity resulting in reduced longevity and reproduction is common, the role of energy-producing mitochondria on the magnitude of these costs is unknown. Here we employed Drosophila melanogaster cybrid lines, where several mitochondrial genotypes (mitotypes) were introgressed onto a single nuclear genetic background, to explicitly test the role of mitochondrial variation on the costs of immune stimulation. We exposed female flies carrying one of nine distinct mitotypes to either a benign, heat-killed bacterial pathogen (stimulating immune deployment while avoiding pathology) or a sterile control and measured lifespan, fecundity, and locomotor activity. We observed mitotype-specific costs of immune stimulation and identified a positive genetic correlation between life span and the proportion of time cybrids spent moving while alive. Our results suggest that costs of immunity are highly variable depending on the mitochondrial genome, adding to a growing body of work highlighting the important role of mitochondrial variation in host-pathogen interactions.

Eimeria falciformis extracellular vesicles differentially express host cell lncRNAs.

Long noncoding RNAs (lncRNAs) are regulatory transcripts during protozoan infections in the host intestinal epithelial cells (IECs). Apicomplexan Eimeria falciformis sporozoite extracellular vesicles (EVs) contain virulence factors that modulate host IECs pro-inflammatory genes and immune responses. In this study, E. falciformis sporozoites were made to interact with inactivated host cells, and the parasite EVs were separated from total secretome by ultracentrifugation and purified on density gradient medium. Dose-dependent bio-activity of E. falciformis EVs was investigated by RNA sequencing, functional annotation and quantitative PCR. It was found that E. falciformis EVs induced mRNA, circRNA, and lncRNA expressions in mouse IECs. Of 38, 217 lncRNAs assembled, 157 and 152 were upwardly and downwardly expressed respectively. Differentially expressed lncRNAs were associated with cytokines, pyroptosis, and immune signaling pathways including FoxO, NF-κB, MAPK, and TGF-ß. In essence, E. falciformis EVs altered host cell RNA expressions during the interaction with host IECs. Also, differentially expressed lncRNAs are potential diagnostic transcripts during Eimeria infections.

Host-parasite interaction in severe and uncomplicated malaria infection in ghanaian children.

PURPOSE: During malarial infection, both parasites and host red blood cells (RBCs) come under severe oxidative stress due to the production of free radicals. The host system responds in protecting the RBCs against the oxidative damage caused by these free radicals by producing antioxidants. In this study, we investigated the antioxidant enzyme; superoxide dismutase (SOD) activity and cytokine interactions with parasitaemia in Ghanaian children with severe and uncomplicated malaria. METHODOLOGY: One hundred and fifty participants aged 0-12 years were administered with structured questionnaires. Active case finding approach was used in participating hospitals to identify and interview cases before treatment was applied. Blood samples were taken from each participant and used to quantify malaria parasitaemia, measure haematological parameters and SOD activity. Cytokine levels were measured by commercial ELISA kits. DNA comet assay was used to evaluate the extent of parasite DNA damage due to oxidative stress. RESULTS: Seventy - Nine (79) and Twenty- Six (26) participants who were positive with malaria parasites were categorized as severe (56.75 × 103 ± 57.69 parasites/µl) and uncomplicated malaria (5.87 × 103 ± 2.87 parasites/µl) respectively, showing significant difference in parasitaemia (p < 0.0001). Significant negative correlation was found between parasitaemia and SOD activity levels among severe malaria study participants (p = 0.0428). Difference in cytokine levels (IL-10) amongst the control, uncomplicated and severe malaria groups was significant (p < 0.0001). The IFN-γ/IL-10 /TNF-α/IL-10 ratio differed significantly between the malaria infected and non- malaria infected study participants. DNA comet assay revealed damage to Plasmodium parasite DNA. CONCLUSION: Critical roles played by SOD activity and cytokines as anti-parasitic defense during P. falciparum malaria infection in children were established.

Metabolism of FAD, FMN and riboflavin (vitamin B2) in the human parasitic blood fluke Schistosoma mansoni.

BACKGROUND: Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. The intravascular worms acquire the nutrients necessary for their survival from host blood. Since all animals are auxotrophic for riboflavin (vitamin B2), schistosomes too must import it to survive. Riboflavin is an essential component of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD); these support key functions of dozens of flavoenzymes. METHODS: Here, using a combination of metabolomics, enzyme kinetics and in silico molecular analysis, we focus on the biochemistry of riboflavin and its metabolites in Schistosoma mansoni (Sm). RESULTS: We show that when schistosomes are incubated in murine plasma, levels of FAD decrease over time while levels of FMN increase. We show that live schistosomes cleave exogenous FAD to generate FMN and this ability is significantly blocked when expression of the surface nucleotide pyrophosphatase/phosphodiesterase ectoenzyme SmNPP5 is suppressed using RNAi. Recombinant SmNPP5 cleaves FAD with a Km of 178 ± 5.9 µM and Kcat/Km of 324,734 ± 36,347 M- 1.S- 1. The FAD-dependent enzyme IL-4I1 drives the oxidative deamination of phenylalanine to produce phenylpyruvate and H2O2. Since schistosomes are damaged by H2O2, we determined if SmNPP5 could impede H2O2 production by blocking IL-4I1 action in vitro. We found that this was not the case; covalently bound FAD on IL-4I1 appears inaccessible to SmNPP5. We also report that live schistosomes can cleave exogenous FMN to generate riboflavin and this ability is significantly impeded when expression of a second surface ectoenzyme (alkaline phosphatase, SmAP) is suppressed. Recombinant SmAP cleaves FMN with a Km of 3.82 ± 0.58 mM and Kcat/Km of 1393 ± 347 M- 1.S- 1. CONCLUSIONS: The sequential hydrolysis of FAD by tegumental ecto-enzymes SmNPP5 and SmAP can generate free vitamin B2 around the worms from where it can be conveniently imported by the recently described schistosome riboflavin transporter SmaRT. Finally, we identified in silico schistosome homologs of enzymes that are involved in intracellular vitamin B2 metabolism. These are riboflavin kinase (SmRFK) as well as FAD synthase (SmFADS); cDNAs encoding these two enzymes were cloned and sequenced. SmRFK is predicted to convert riboflavin to FMN while SmFADS could further act on FMN to regenerate FAD in order to facilitate robust vitamin B2-dependent metabolism in schistosomes.

Effects of Leishmania major infection on the gut microbiome of resistant and susceptible mice.

Cutaneous leishmaniasis, a parasitic disease caused by Leishmania major, is a widely frequent form in humans. To explore the importance of the host gut microbiota and to investigate its changes during L. major infection, two different groups of mouse models were assessed. The microbiome of two parts of the host gut-ileum and colon-from infected and non-infected mice were characterised by sequencing of 16S rDNA using an Ion Torrent PGM platform. Microbiome analysis was performed to reveal changes related to the susceptibility and the genetics of mice strains in two different gut compartments and to compare the results between infected and non-infected mice. The results showed that Leishmania infection affects mainly the ileum microbiota, whereas the colon bacterial community was more stable. Different biomarkers were determined in the gut microbiota of infected resistant mice and infected susceptible mice using LEfSe analysis. Lactobacillaceae was associated with resistance in the colon microbiota of all resistant mice strains infected with L. major. Genes related to xenobiotic biodegradation and metabolism and amino acid metabolism were primarily enriched in the small intestine microbiome of resistant strains, while genes associated with carbohydrate metabolism and glycan biosynthesis and metabolism were most abundant in the gut microbiome of the infected susceptible mice. These results should improve our understanding of host-parasite interaction and provide important insights into the effect of leishmaniasis on the gut microbiota. Also, this study highlights the role of host genetic variation in shaping the diversity and composition of the gut microbiome. KEY POINTS: • Leishmaniasis may affect mainly the ileum microbiota while colon microbiota was more stable. • Biomarkers related with resistance or susceptibility were determined in the gut microbiota of mice. • Several pathways were predicted to be upregulated in the gut microbiota of resistant or susceptible mice.

Association of ectoparasite Lepeophtheirus salmonis counts on farmed Atlantic salmon and wild sea trout in Scotland.

Parasitic sea lice (Copepoda: Caligidae) colonising marine salmonid (Salmoniformes: Salmonidae) aquaculture production facilities have been implicated as a possible pressure on wild salmon and sea trout populations. This investigation uses monitoring data from the mainland west coast and Western Isles of Scotland to estimate the association of the abundance of adult female Lepeophtheirus salmonis (Krøyer) colonising farmed Atlantic salmon Salmo salar L. with the occurrence of juvenile and mobile L. salmonis on wild sea trout, anadromous S. trutta L. The associations were evaluated using generalised linear mixed models incorporating farmed adult female salmon louse abundances which are temporally lagged relative to dependent wild trout values. The pattern of lags, which is consistent with time for L. salmonis development between egg and infective stage, was evaluated using model deviances. A significant positive association is identified between adult female L. salmonis abundance on farms and juvenile L. salmonis on wild trout. This association is consistent with a causal relationship in which increases in the number of L. salmonis copepodids originating from lice colonising farmed Atlantic salmon cause an increase of L. salmonis abundance on wild sea trout.

Influence of CK2 protein kinase activity on the interaction between Trypanosoma cruzi and its vertebrate and invertebrate hosts.

Chagas disease, endemic from Latin America, is caused by Trypanosoma cruzi and is transmitted by triatomine feces. This parasite undergoes complex morphological changes through its life cycle, promoted by significant changes in signal transduction pathways. The activity of protein kinase CK2 has been described in trypanosomatids. Using a specific peptide and radioactive ATP, we identified CK2 activity on the cellular surface and the cytoplasmic content in Trypanosoma cruzi, apart from the secreted form. Dephosphorylated casein promoted an increase of 48% in the secreted CK2 activity. Total extract of peritoneal macrophages from BALB/c and inactivated human serum promoted an increase of 67% and 36%, respectively, in this activity. The protein secreted by parasites was purified by HPLC and had shown compatibility with the catalytic subunit of mammalian CK2. Incubation of the parasites with CK2 inhibitors, added to the culture medium, prevented their growth. The opposite was observed when CK2 activators were used. Results of interaction between Trypanosoma cruzi and the gut of the vector have revealed that, in the presence of CK2 inhibitors, there is a reduction in the association rate. A similar inhibition profile was seen in the Trypanosoma cruzi-macrophages interaction, confirming the importance of this enzyme in the life cycle of this protozoan.

Toxoplasma gondii infection in ticks infesting migratory birds: the blackbird (Turdus merula) and the song thrush (Turdus philomelos).

Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is the zoonosis widespread all over the world. Birds constitute an important group of T. gondii intermediate hosts often attacked by definitive hosts, e.g. domestic cats. Due to confirmation of an additional way of T. gondii transmission via tick bite, the aim of our study was to state and evaluate the infection prevalence of ticks feeding on blackbirds (Turdus merula) and song thrushes (Turdus philomelos). The real-time PCR amplification of the B1 gene fragment was used for detection of T. gondii infection in 157 Ixodes ricinus ticks removed from captured birds. The results showed the thrushes as hosts intensively attacked by ticks (prevalence 88.5% and 70% for blackbirds and song thrushes, respectively), and T. gondii infected individuals were detected. Among all ticks infected, 7 (5.8%; n = 120) were collected from blackbirds, and 2 (5.4%; n = 37) from song thrushes. The thrushes small body sizes and their tendency to urban ecosystems colonization, suggest that they relatively often become a pray of domestic cats, and combined with our findings, are potentially involved in maintenance the T. gondii population, especially in anthropogenic habitats, where the presence of toxoplasmosis is likely to constitute a serious danger to public health.

An insight into the functional genomics and species classification of Eudiplozoon nipponicum (Monogenea, Diplozoidae), a haematophagous parasite of the common carp Cyprinus carpio.

BACKGROUND: Monogenea (Platyhelminthes, Neodermata) are the most species-rich class within the Neodermata superclass of primarily fish parasites. Despite their economic and ecological importance, monogenean research tends to focus on their morphological, phylogenetic, and population characteristics, while comprehensive omics analyses aimed at describing functionally important molecules are few and far between. We present a molecular characterisation of monogenean representative Eudiplozoon nipponicum, an obligate haematophagous parasite infecting the gills of the common carp. We report its nuclear and mitochondrial genomes, present a functional annotation of protein molecules relevant to the molecular and biochemical aspect of physiological processes involved in interactions with the fish hosts, and re-examinate the taxonomic position of Eudiplozoon species within the Diplozoidae family. RESULTS: We have generated 50.81 Gbp of raw sequencing data (Illumina and Oxford Nanopore reads), bioinformatically processed, and de novo assembled them into a genome draft 0.94 Gbp long, consisting of 21,044 contigs (N50 = 87 kbp). The final assembly represents 57% of the estimated total genome size (~ 1.64 Gbp), whereby repetitive and low-complexity regions account for ~ 64% of the assembled length. In total, 36,626 predicted genes encode 33,031 proteins and homology-based annotation of protein-coding genes (PCGs) and proteins characterises 14,785 (44.76%) molecules. We have detected significant representation of functional proteins and known molecular functions. The numbers of peptidases and inhibitors (579 proteins), characterised GO terms (16,016 unique assigned GO terms), and identified KEGG Orthology (4,315 proteins) acting in 378 KEGG pathways demonstrate the variety of mechanisms by which the parasite interacts with hosts on a macromolecular level (immunomodulation, feeding, and development). Comparison between the newly assembled E. nipponicum mitochondrial genome (length of 17,038 bp) and other diplozoid monogeneans confirms the existence of two distinct Eudiplozoon species infecting different fish hosts: Cyprinus carpio and Carassius spp. CONCLUSIONS: Although the amount of sequencing data and characterised molecules of monogenean parasites has recently increased, a better insight into their molecular biology is needed. The E. nipponicum nuclear genome presented here, currently the largest described genome of any monogenean parasite, represents a milestone in the study of monogeneans and their molecules but further omics research is needed to understand these parasites' biological nature.

Dissecting the Biology of Rafflesia Species: Current Progress and Future Directions Made Possible with High-Throughput Sequencing Data.

The angiosperm Rafflesia exhibits a unique biology, including a growth strategy that involves endophytic parasitism of a specific host, with only the gigantic flower externally visible. The Rafflesia possesses many unique evolutionary, developmental and morphological features that are rooted in yet-to-be-explained physiological processes. Although studies on the molecular biology of Rafflesia are limited by sampling difficulties due to its rarity in the wild and the short life span of its flower, current advances in high-throughput sequencing technology have allowed for the genome- and transcriptome-level dissection of the molecular mechanisms behind the unique characteristics of this parasitic plant. In this review, we summarize major findings on the cryptic biology of Rafflesia and provide insights into future research directions. The wealth of data obtained can improve our understanding of Rafflesia species and contribute toward the conservation strategy of this endangered plant.

Hijacking of the host cell Golgi by Plasmodium berghei liver stage parasites.

The intracellular lifestyle represents a challenge for the rapidly proliferating liver stage Plasmodium parasite. In order to scavenge host resources, Plasmodium has evolved the ability to target and manipulate host cell organelles. Using dynamic fluorescence-based imaging, we here show an interplay between the pre-erythrocytic stages of Plasmodium berghei and the host cell Golgi during liver stage development. Liver stage schizonts fragment the host cell Golgi into miniaturized stacks, which increases surface interactions with the parasitophorous vacuolar membrane of the parasite. Expression of specific dominant-negative Arf1 and Rab GTPases, which interfere with the host cell Golgi-linked vesicular machinery, results in developmental delay and diminished survival of liver stage parasites. Moreover, functional Rab11a is critical for the ability of the parasites to induce Golgi fragmentation. Altogether, we demonstrate that the structural integrity of the host cell Golgi and Golgi-associated vesicular traffic is important for optimal pre-erythrocytic development of P. berghei. The parasite hijacks the Golgi structure of the hepatocyte to optimize its own intracellular development. This article has an associated First Person interview with the first author of the paper.

Dissection of Barrier Dysfunction in Organoid-Derived Human Intestinal Epithelia Induced by Giardia duodenalis.

BACKGROUND & AIMS: The protozoa Giardia duodenalis is a major cause of gastrointestinal illness worldwide, but underlying pathophysiological mechanisms remain obscure, partly due to the absence of adequate cellular models. We aimed at overcoming these limitations and recapitulating the authentic series of pathogenic events in the primary human duodenal tissue by using the human organoid system. METHODS: We established a compartmentalized cellular transwell system with electrophysiological and barrier properties akin to duodenal mucosa and dissected the events leading to G. duodenalis-induced barrier breakdown by functional analysis of transcriptional, electrophysiological, and tight junction components. RESULTS: Organoid-derived cell layers of different donors showed a time- and parasite load-dependent leak flux indicated by collapse of the epithelial barrier upon G. duodenalis infection. Gene set enrichment analysis suggested major expression changes, including gene sets contributing to ion transport and tight junction structure. Solute carrier family 12 member 2 and cystic fibrosis transmembrane conductance regulator-dependent chloride secretion was reduced early after infection, while changes in the tight junction composition, localization, and structural organization occurred later as revealed by immunofluorescence analysis and freeze fracture electron microscopy. Functionally, barrier loss was linked to the adenosine 3',5'-cyclic monophosphate (cAMP)/protein kinase A-cAMP response element-binding protein signaling pathway. CONCLUSIONS: Data suggest a previously unknown sequence of events culminating in intestinal barrier dysfunction upon G. duodenalis infection during which alterations of cellular ion transport were followed by breakdown of the tight junctional complex and loss of epithelial integrity, events involving a cAMP/protein kinase A-cAMP response element-binding protein mechanism. These findings and the newly established organoid-derived model to study G. duodenalis infection may help to explore new options for intervening with disease and infection, in particular relevant for chronic cases of giardiasis.