Re-Discovery of Giardiavirus: Genomic and Functional Analysis of Viruses from Giardia duodenalis Isolates

Giardiasis, caused by the protozoan parasite Giardia duodenalis, is an intestinal diarrheal disease affecting almost one billion people worldwide. A small endosymbiotic dsRNA viruses, G. lamblia virus (GLV), genus Giardiavirus, family Totiviridae, might inhabit human and animal isolates of G. duodenalis. Three GLV genomes have been sequenced so far, and only one was intensively studied; moreover, a positive correlation between GLV and parasite virulence is yet to be proved. To understand the biological significance of GLV infection in Giardia, the characterization of several GLV strains from naturally infected G. duodenalis isolates is necessary. Here we report high-throughput sequencing of four GLVs strains, from Giardia isolates of human and animal origin. We also report on a new, unclassified viral sequence (designed GdRV-2), unrelated to Giardiavirus, encoding and expressing for a single large protein with an RdRp domain homologous to Totiviridae and Botybirnaviridae. The result of our sequencing and proteomic analyses challenge the current knowledge on GLV and strongly suggest that viral capsid protein translation unusually starts with a proline and that translation of the RNA-dependent RNA polymerase (RdRp) occurs via a +1/−2 ribosomal frameshift mechanism. Nucleotide polymorphism, confirmed by mass-spectrometry analysis, was also observed among and between GLV strains. Phylogenetic analysis indicated the occurrence of at least two GLV subtypes which display different phenotypes and transmissibility in experimental infections of a GLV naïve Giardia isolate.

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Eudiplozoon nipponicum (Monogenea, Diplozoidae) and its adaptation to haematophagy as revealed by transcriptome and secretome profiling

BMC Genomics ◽

10.1186/s12864-021-07589-z ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Jiří Vorel ◽

Krystyna Cwiklinski ◽

Pavel Roudnický ◽

Jana Ilgová ◽

Lucie Jedličková ◽

...

Keyword(s):

High Throughput Sequencing ◽

De Novo ◽

Proteolytic Enzymes ◽

Current Knowledge ◽

Mass Spectrometry Analysis ◽

Fatty Acid Binding Proteins ◽

Rna Seq ◽

Fatty Acid Binding ◽

Spectrometry Analysis ◽

Eudiplozoon Nipponicum

Abstract Background Ectoparasites from the family Diplozoidae (Platyhelminthes, Monogenea) belong to obligate haematophagous helminths of cyprinid fish. Current knowledge of these worms is for the most part limited to their morphological, phylogenetic, and population features. Information concerning the biochemical and molecular nature of physiological processes involved in host–parasite interaction, such as evasion of the immune system and its regulation, digestion of macromolecules, suppression of blood coagulation and inflammation, and effect on host tissue and physiology, is lacking. In this study, we report for the first time a comprehensive transcriptomic/secretome description of expressed genes and proteins secreted by the adult stage of Eudiplozoon nipponicum (Goto, 1891) Khotenovsky, 1985, an obligate sanguivorous monogenean which parasitises the gills of the common carp (Cyprinus carpio). Results RNA-seq raw reads (324,941 Roche 454 and 149,697,864 Illumina) were generated, de novo assembled, and filtered into 37,062 protein-coding transcripts. For 19,644 (53.0%) of them, we determined their sequential homologues. In silico functional analysis of E. nipponicum RNA-seq data revealed numerous transcripts, pathways, and GO terms responsible for immunomodulation (inhibitors of proteolytic enzymes, CD59-like proteins, fatty acid binding proteins), feeding (proteolytic enzymes cathepsins B, D, L1, and L3), and development (fructose 1,6-bisphosphatase, ferritin, and annexin). LC-MS/MS spectrometry analysis identified 721 proteins secreted by E. nipponicum with predominantly immunomodulatory and anti-inflammatory functions (peptidyl-prolyl cis-trans isomerase, homolog to SmKK7, tetraspanin) and ability to digest host macromolecules (cathepsins B, D, L1). Conclusions In this study, we integrated two high-throughput sequencing techniques, mass spectrometry analysis, and comprehensive bioinformatics approach in order to arrive at the first comprehensive description of monogenean transcriptome and secretome. Exploration of E. nipponicum transcriptome-related nucleotide sequences and translated and secreted proteins offer a better understanding of molecular biology and biochemistry of these, often neglected, organisms. It enabled us to report the essential physiological pathways and protein molecules involved in their interactions with the fish hosts.

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Subgenomic flavivirus RNA binds the mosquito DEAD/H-box helicase ME31B and determines Zika virus transmission by Aedes aegypti

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1905617116 ◽

2019 ◽

Vol 116 (38) ◽

pp. 19136-19144 ◽

Cited By ~ 17

Author(s):

Giel P. Göertz ◽

Joyce W. M. van Bree ◽

Anwar Hiralal ◽

Bas M. Fernhout ◽

Carmen Steffens ◽

...

Keyword(s):

Aedes Aegypti ◽

Zika Virus ◽

Affinity Purification ◽

Mechanistic Model ◽

Virus Transmission ◽

Protein Translation ◽

Mass Spectrometry Analysis ◽

Small Interfering Rnas ◽

Spectrometry Analysis ◽

Mosquito Infection

Zika virus (ZIKV) is an arthropod-borne flavivirus predominantly transmitted by Aedes aegypti mosquitoes and poses a global human health threat. All flaviviruses, including those that exclusively replicate in mosquitoes, produce a highly abundant, noncoding subgenomic flavivirus RNA (sfRNA) in infected cells, which implies an important function of sfRNA during mosquito infection. Currently, the role of sfRNA in flavivirus transmission by mosquitoes is not well understood. Here, we demonstrate that an sfRNA-deficient ZIKV (ZIKVΔSF1) replicates similar to wild-type ZIKV in mosquito cell culture but is severely attenuated in transmission by Ae. aegypti after an infectious blood meal, with 5% saliva-positive mosquitoes for ZIKVΔSF1 vs. 31% for ZIKV. Furthermore, viral titers in the mosquito saliva were lower for ZIKVΔSF1 as compared to ZIKV. Comparison of mosquito infection via infectious blood meals and intrathoracic injections showed that sfRNA is important for ZIKV to overcome the mosquito midgut barrier and to promote virus accumulation in the saliva. Next-generation sequencing of infected mosquitoes showed that viral small-interfering RNAs were elevated upon ZIKVΔSF1 as compared to ZIKV infection. RNA-affinity purification followed by mass spectrometry analysis uncovered that sfRNA specifically interacts with a specific set of Ae. aegypti proteins that are normally associated with RNA turnover and protein translation. The DEAD/H-box helicase ME31B showed the highest affinity for sfRNA and displayed antiviral activity against ZIKV in Ae. aegypti cells. Based on these results, we present a mechanistic model in which sfRNA sequesters ME31B to promote flavivirus replication and virion production to facilitate transmission by mosquitoes.

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Selenoprotein P Regulates Synaptic Zinc and Reduces Tau Phosphorylation

Frontiers in Nutrition ◽

10.3389/fnut.2021.683154 ◽

2021 ◽

Vol 8 ◽

Author(s):

Arlene C. P. Kiyohara ◽

Daniel J. Torres ◽

Ayaka Hagiwara ◽

Jenna Pak ◽

Rachel H. L. H. Rueli ◽

...

Keyword(s):

Metal Binding ◽

Cultured Cells ◽

Hippocampal Slices ◽

Biological Significance ◽

Tau Phosphorylation ◽

Mass Spectrometry Analysis ◽

Selenoprotein P ◽

Normal Brain ◽

Spectrometry Analysis ◽

Extracellular Transport

Selenoprotein P (SELENOP1) is a selenium-rich antioxidant protein involved in extracellular transport of selenium (Se). SELENOP1 also has metal binding properties. The trace element Zinc (Zn2+) is a neuromodulator that can be released from synaptic terminals in the brain, primarily from a subset of glutamatergic terminals. Both Zn2+ and Se are necessary for normal brain function. Although these ions can bind together with high affinity, the biological significance of an interaction of SELENOP1 with Zn2+ has not been investigated. We examined changes in brain Zn2+ in SELENOP1 knockout (KO) animals. Timm-Danscher and N-(6-methoxy-8-quinolyl)-p-toluenesulphonamide (TSQ) staining revealed increased levels of intracellular Zn2+ in the SELENOP1−/− hippocampus compared to wildtype (WT) mice. Mass spectrometry analysis of frozen whole brain samples demonstrated that total Zn2+ was not increased in the SELENOP1−/− mice, suggesting only local changes in Zn2+ distribution. Unexpectedly, live Zn2+ imaging of hippocampal slices with a selective extracellular fluorescent Zn2+ indicator (FluoZin-3) showed that SELENOP1−/− mice have impaired Zn2+ release in response to KCl-induced neuron depolarization. The zinc/metal storage protein metallothionein 3 (MT-3) was increased in SELENOP1−/− hippocampus relative to wildtype, possibly in response to an elevated Zn2+ content. We found that depriving cultured cells of selenium resulted in increased intracellular Zn2+, as did inhibition of selenoprotein GPX4 but not GPX1, suggesting the increased Zn2+ in SELENOP1−/− mice is due to a downregulation of antioxidant selenoproteins and subsequent release of Zn2+ from intracellular stores. Surprisingly, we found increased tau phosphorylation in the hippocampus of SELENOP1−/− mice, possibly resulting from intracellular zinc changes. Our findings reveal important roles for SELENOP1 in the maintenance of synaptic Zn2+ physiology and preventing tau hyperphosphorylation.

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Giardia duodenalisin the UK: current knowledge of risk factors and public health implications

Parasitology ◽

10.1017/s0031182018001683 ◽

2018 ◽

Vol 146 (4) ◽

pp. 413-424 ◽

Cited By ~ 7

Author(s):

B. Horton ◽

H. Bridle ◽

C. L. Alexander ◽

F. Katzer

Keyword(s):

Public Health ◽

Current Knowledge ◽

Companion Animals ◽

Giardia Duodenalis ◽

Protozoan Parasite ◽

Epidemiological Data ◽

Food Sources ◽

Local Public Health ◽

Health Implications ◽

The Uk

AbstractGiardia duodenalisis a ubiquitous flagellated protozoan parasite known to cause giardiasis throughout the world. Potential transmission vehicles for this zoonotic parasite are both water and food sources. As such consumption of water contaminated by feces, or food sources washed in contaminated water containing parasite cysts, may result in outbreaks. This creates local public health risks which can potentially cause widespread infection and long-term post-infection sequelae. This paper provides an up-to-date overview ofG. duodenalisassemblages, sub-assemblages, hosts and locations identified. It also summarizes knowledge of potential infection/transmission routes covering water, food, person-to-person infection and zoonotic transmission from livestock and companion animals. Public health implications focused within the UK, based on epidemiological data, are discussed and recommendations for essentialGiardiadevelopments are highlighted.

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Super-Resolution Localisation of Nuclear PI(4)P and Identification of Its Interacting Proteome

Cells ◽

10.3390/cells9051191 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1191 ◽

Cited By ~ 1

Author(s):

Veronika Fáberová ◽

Ilona Kalasová ◽

Alžběta Krausová ◽

Pavel Hozák

Keyword(s):

Mass Spectrometry ◽

Cell Nucleus ◽

Current Knowledge ◽

Light And Electron Microscopy ◽

Super Resolution ◽

Mass Spectrometry Analysis ◽

Nuclear Speckles ◽

Spectrometry Analysis ◽

Interaction Partners ◽

Nuclear Processes

Phosphoinositides are glycerol-based phospholipids, and they play essential roles in cellular signalling, membrane and cytoskeletal dynamics, cell movement, and the modulation of ion channels and transporters. Phosphoinositides are also associated with fundamental nuclear processes through their nuclear protein-binding partners, even though membranes do not exist inside of the nucleus. Phosphatidylinositol 4-phosphate (PI(4)P) is one of the most abundant cellular phosphoinositides; however, its functions in the nucleus are still poorly understood. In this study, we describe PI(4)P localisation in the cell nucleus by super-resolution light and electron microscopy, and employ immunoprecipitation with a specific anti-PI(4)P antibody and subsequent mass spectrometry analysis to determine PI(4)P’s interaction partners. We show that PI(4)P is present at the nuclear envelope, in nuclear lamina, in nuclear speckles and in nucleoli and also forms multiple small foci in the nucleoplasm. Nuclear PI(4)P undergoes re-localisation to the cytoplasm during cell division; it does not localise to chromosomes, nucleolar organising regions or mitotic interchromatin granules. When PI(4)P and PI(4,5)P2 are compared, they have different nuclear localisations during interphase and mitosis, pointing to their functional differences in the cell nucleus. Mass spectrometry identified hundreds of proteins, including 12 potentially novel PI(4)P interactors, most of them functioning in vital nuclear processes such as pre-mRNA splicing, transcription or nuclear transport, thus extending the current knowledge of PI(4)P’s interaction partners. Based on these data, we propose that PI(4)P also plays a role in essential nuclear processes as a part of protein–lipid complexes. Altogether, these observations provide a novel insight into the role of PI(4)P in nuclear functions and provide a direction for further investigation.

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What about Dinner? Chemical and Microresidue Analysis Reveals the Function of Late Neolithic Ceramic Pans

Molecules ◽

10.3390/molecules26113391 ◽

2021 ◽

Vol 26 (11) ◽

pp. 3391

Author(s):

Jaromír Beneš ◽

Valentina Todoroska ◽

Kristýna Budilová ◽

Jaromír Kovárník ◽

Jaroslav Pavelka ◽

...

Keyword(s):

Mass Spectrometry Analysis ◽

Wild Plants ◽

Animal Origin ◽

Organic Residues ◽

Lake Ohrid ◽

Late Neolithic ◽

Spectrometry Analysis ◽

Relative Chronology ◽

Animal Remains ◽

Khapra Beetle

The Late Neolithic palafitte site, Ustie na Drim, in the northern part of Lake Ohrid (North Macedonia), excavated in 1962, offered ceramic fragments of large, flat, elongated pans. These artifacts could be dated by relative chronology to roughly around 5200–5000 BC. According to their shape and technological traits, the ceramic pans were probably used for baking. The attached materials on the surface of studied pan fragments were sampled for consequent chemical and microscopical analyses (i.e., analyses of starch, phytoliths, and microscopic animal remains). An immunological method revealed the presence of pork proteins in samples. The presence of organic residues of animal origin was, moreover, confirmed by the detection of cholesterol using gas chromatography coupled to mass spectrometry. Analysis of detected microscopic botanical objects revealed starch grains of several plants (i.e., oak, cattail, and grasses). An interesting find was the hair of a beetle larva, which could be interpreted contextually as the khapra beetle, a pest of grain and flour. Based on our data, we suppose that the ceramic pans from Ustie na Drim were used for the preparation of meals containing meat from common livestock in combination with cereals and wild plants.

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Translational GTPase BipA Is Involved in the Maturation of a Large Subunit of Bacterial Ribosome at Suboptimal Temperature

Frontiers in Microbiology ◽

10.3389/fmicb.2021.686049 ◽

2021 ◽

Vol 12 ◽

Author(s):

Kwok Jian Goh ◽

Rya Ero ◽

Xin-Fu Yan ◽

Jung-Eun Park ◽

Binu Kundukad ◽

...

Keyword(s):

Ribosome Biogenesis ◽

Large Subunit ◽

Protein A ◽

Protein Translation ◽

Rna Metabolism ◽

Soft Agar ◽

Mass Spectrometry Analysis ◽

Ribosome Assembly ◽

Spectrometry Analysis ◽

Bacterial Ribosome

BPI-inducible protein A (BipA), a highly conserved paralog of the well-known translational GTPases LepA and EF-G, has been implicated in bacterial motility, cold shock, stress response, biofilm formation, and virulence. BipA binds to the aminoacyl-(A) site of the bacterial ribosome and establishes contacts with the functionally important regions of both subunits, implying a specific role relevant to the ribosome, such as functioning in ribosome biogenesis and/or conditional protein translation. When cultured at suboptimal temperatures, the Escherichia coli bipA genomic deletion strain (ΔbipA) exhibits defects in growth, swimming motility, and ribosome assembly, which can be complemented by a plasmid-borne bipA supplementation or suppressed by the genomic rluC deletion. Based on the growth curve, soft agar swimming assay, and sucrose gradient sedimentation analysis, mutation of the catalytic residue His78 rendered plasmid-borne bipA unable to complement its deletion phenotypes. Interestingly, truncation of the C-terminal loop of BipA exacerbates the aforementioned phenotypes, demonstrating the involvement of BipA in ribosome assembly or its function. Furthermore, tandem mass tag-mass spectrometry analysis of the ΔbipA strain proteome revealed upregulations of a number of proteins (e.g., DeaD, RNase R, CspA, RpoS, and ObgE) implicated in ribosome biogenesis and RNA metabolism, and these proteins were restored to wild-type levels by plasmid-borne bipA supplementation or the genomic rluC deletion, implying BipA involvement in RNA metabolism and ribosome biogenesis. We have also determined that BipA interacts with ribosome 50S precursor (pre-50S), suggesting its role in 50S maturation and ribosome biogenesis. Taken together, BipA demonstrates the characteristics of a bona fide 50S assembly factor in ribosome biogenesis.

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