Intracellular interferons in fish : a unique means to combat viral infection

Peer reviewedPublisher PD

The immunome and embryo quality in sea bream and sea bass

Gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) are teleosts belonging to Eupercaria and are the most important aquaculture fish species in the Mediterranean region. These two species are ranked second after the Atlantic salmon (Salmo salar) in production volume and value in the European Union (EU) aquaculture sector. Unpredictable fertilized egg/embryo quality and performance remain a bottleneck that threatens sustainability of sea bream and sea bass aquaculture, impeding the increased productivity of aquaculture that entirely depends on hatchery production. To address this issue, criteria and molecular markers linked to embryo quality that could be used to monitor and manage hatchery production were procured. Comparative molecular approaches using molecular biology, proteomics and transcriptomics were performed to analyze embryo performance and immunity in samples from several European commercial hatcheries. The core achievements were the: a) identification and characterization of lysozyme and complement 5 (C5) gene families and embryo and larval gene expression and enzyme activity from a diversity of hatcheries, b) characterization of the embryo proteome from three Mediterranean fish species [white sea bream (Diplodus sargus), meagre (Argyrosomus regius) and sea bream] 24h before hatch and at hatch and identification of common and species specific molecular patterns linked to biological function and putative quality-related proteins, c) comparative transcriptomics of good and poor quality sea bream embryos from several Mediterranean hatcheries. Qualitylinked transcripts and some elements of the regulatory epitranscriptome (non-coding RNA) were identified as well as the contribution of maternal proteins to embryos. Taken together, the results provide a comprehensive description of the molecular basis of sea bream and sea bass embryo development and reveal that immune-related molecules in fertilized eggs are low abundance. The development (quality)-related candidate markers identified will be of value for management of fish embryos in aquaculture hatcheries.O mar Mediterrâneo cobre apenas 0,7% da área oceânica mundial, mas é um dos principais reservatórios de biodiversidade marinha e costeira com cerca de 28% de espécies endémicas. A dourada (Sparus aurata) e o robalo (Dicentrarchus labrax), são duas espécies de peixe que pertencem à série Eupercaria e das mais comercializadas pela indústria de aquicultura nesta região. No sector da aquicultura da União Europeia (EU), estas duas espécies ocupam o segundo lugar no “ranking” da cadeia de valor, depois do salmão do Atlântico. Contudo, as suas características morfológicas e de crescimento são os parâmetros de qualidade relevantes considerados pela indústria. Esta abordagem deve-se à sua comercialização em formato de peixe inteiro, o que reduz o valor de mercado na cadeia de valor e também à ausência de critérios de qualidade para seleção de ovos e embriões, bem como, de marcadores moleculares de qualidade com maior grau de sensibilidade. Todos estes obstáculos, limitam substancialmente o desenvolvimento das indústrias associadas á comercialização destas duas espécies de peixe, impedindo a expansão da aquicultura e das “hatcheries” (maternidades incubadoras de ovos/embriões controlados artificialmente para fins comerciais). Para mitigar este problema, utilizou-se técnicas de biologia molecular e tecnologias ómicas e estabeleceu-se uma abordagem comparativa direcionada á descoberta de moléculas e vias metabólicas funcionais de importância crítica para o sistema imune dos peixes associada á “performance” de desenvolvimento de ovos e embriões. Esta tese está organizada em seis capítulos. Inicia-se com uma visão geral dos critérios morfológicos, físico-químicos e moleculares existentes para avaliar a qualidade de ovos e embriões para melhorar a gestão da aquicultura de peixes (Capítulo 1). Subsequentemente, caracterizou-se a família de genes do sistema imune: a) a das lisozimas em peixes teleósteos com enfase na sua caracterização molecular e funcional em dourada (Capítulo 2) e análises moleculares estruturais e evolutivas e b) a do complemento C5 (C5) em peixes especialmente em espécies da faamilia Cyprinidae (Capítulo 3). Os capítulos 4 e 5, integram abordagens de proteómica e transcriptómica em espécies de peixes mediterrânicos [pargo (Diplodus sargus), corvina (Argyrosomus regius) e dourada], focando os processos de desenvolvimento e de eclosão e na função da enzima “hatching enzyme” em dourada e robalo. Foi feita uma associação entre os capítulos e a análise integrada dos dados do transcritoma do embrião (Capítulo 5) revelou um padrão de expressão significativamente diferente (p-valor < 0,05) para o C5 (Capítulo 3) em diferentes lotes de embriões de dourada nas comparações entre graus de qualidade (Boa vrs Má) e entre estágios de desenvolvimento (Pré- eclosão vrs Eclosão). A variação do C5 em relação ao lote de embriões não foi afetada pela origem da “hatchery”, indicando que as prática de manejo ou os próprios reprodutores não influenciam a sua expressão. Os resultados sugerem que este gene e o seu produto proteico, são provavelmente importantes na proteção imunológica precoce e também em outras funções ainda não descritas na dourada ou em outras espécies de peixes. Também a integração dos resultados do proteoma (Capítulo 4) e do transcritoma (Capítulo 5) do embrião de dourada nos mesmos estágios de desenvolvimento, identificou um grupo de proteínas que se especula serem de origem materna. No último capítulo, sumarizou-se os resultados e são apresentadas perspetivas baseadas nos avanços e desafios atuais e propostas para o desenvolvimento de uma ferramenta integrada de monitorização da qualidade dos embriões e uma base biológica do desenvolvimento de ovos e embriões de peixes (Capítulo 6). Neste projeto foram: 1) identificadas duas importantes famílias de genes associadas à imunidade inata em peixes, a das lisozimas e a do C5. Caracterizou-se pela primeira vez a função das lisozimas através da sua expressão e atividade enzimática em embriões e em diferentes estágios larvares de uma diversidade de reprodutores de dourada. Estudou-se a função do C5 em peixes, através da construção de redes génicas, modelação por homologia e “docking” molecular entre o C5 e o seu receptor (C5R/CD88); 2) mapeou-se e caracterizou-se o proteoma do embrião de três espécies de peixes mediterrânicos (sargo, corvina e dourada) em duas fases do seu desenvolvimento (24h antes da eclosão e na eclosão) e identificou-se um grupo de proteínas potencialmente relacionadas com a imunidade e a qualidade dos embriões. Avaliou-se a função do gene para enzima “hatching enzyme”, com base na sua expressão em embriões de dourada e robalo nos estágios acima referidos; 3) mapeou-se e caracterizou-se o transcritoma de embriões de dourada com origem em diferentes “hatcheries” na região mediterrânica através de uma abordagem comparativa entre qualidade e estágios de desenvolvimento (qualidade- Boa vrs Má; estágios- Pré- eclosão vrs Eclosão) e identificou-se uma diversidade de transcritos, vias metabólicas e elementos do epitranscriptoma regulatório do RNA-não codificante. Foram identificados em comum 42 candidatos a marcadores de qualidade e enriquecidas duas vias metabólicas relacionadas com o sistema imunológico e associadas às “hatcheries”: a via de infeção por Salmonella (constituída por 7 genes relacionados com o sistema imune) e a via de sinalização MAPK (mitogen-activated protein kinase). Foram identificadas 543 proteínas que são expressas apenas no proteoma, sugerindo que podem ter origem materna e destas, 7 (diferencialmente expressas) estão potencialmente relacionadas com o sistema imune. Globalmente, os resultados forneceram um grupo de marcadores relacionados à imunidade e ao desenvolvimento (qualidade), com potencial de se traduzirem em critérios de qualidade de ovos e embriões para a indústria da aquicultura. Estes resultados, foram amplamente estudados para descrever a base molecular biológica entre os diferentes estágios de desenvolvimento de ovos e embriões de peixes e também, entre diferentes lotes de embriões de diferente qualidade. As ferramentas biológicas e critérios desenvolvidos neste trabalho, oferecem uma orientação para as “hatcheries” de peixes e a sua aplicação contribuirá para melhorar no futuro o sector da aquicultura.O apoio financeiro ao trabalho relatado na presente tese de doutoramento é reconhecido com gratidão, pois foi crucial para o progresso positivo e sucesso do trabalho científico. Uma diversidade de fontes de financiamento apoiou o trabalho desenvolvido nesta tese, quer diretamente através da compra de consumíveis/materiais (PerformFISH a European Union’s Horizon 2020 research and innovation grant, agreement Nº 727610), quer indiretamente através do financiamento do CCMAR de apoio a serviços e equipamentos utilizados durante a execução do trabalho (projetos da Fundação para a Ciência e Tecnologia (FCT) - UIDB/04326/2020, UIDP/04326/2020 e dos programas operacionais CRESC Algarve 2020 e COMPETE 2020 através do projeto EMBRC.PT ALG-01-0145-FEDER-022121)

On the molecular biology and evolution of plant parasitism by nematodes

Plant-parasitic nematodes (PPN) are among the most devastating plant pathogens. However, our understanding of how nematodes adapted to plant parasitism, and the molecular mechanisms that PPN use during infection is limited. Among the most important genomic changes that occurred in the free-living nematode ancestors of PPN were multiple horizontal gene transfer (HGT) events from bacteria. Though it is clear that HGT helped shape the genomes of many PPN, how this process occurred is unknown. Also, it is evident that successful parasitism occurs from the delivery of proteinaceous effectors into plant roots to hijack and modify host cellular processes. The research included in this dissertation aims at addressing several important questions regarding HGT in PPN, and investigates important molecular, cellular and developmental processes that are determined critical for successful parasitism. Of particular emphasis throughout this dissertation is the soybean cyst nematode, Heterodera glycines, due to a highly specialized and agronomically important interaction with its soybean host. Major findings for HGT in PPN include the identification of eighteen new H. glycines effectors, three of which are determined to have been part of more ancient HGT events from rhizosphere bacteria. Additionally, homologs of two of the three HGT genes are shown to have been transferred numerous different times from bacteria to diverse eukaryotes and archaea. The latter findings indicate the likely evolutionary advantages that these genes provided not just to PPN, but many different taxa. Intriguingly, we reveal that a group of retroviruses specific to distal nematode clades is genomically associated with HGT genes in PPN genomes. These retroviruses potentially have all of the elements that would be necessary for HGT to occur in PPN. Thus, we propose the tempting hypothesis that this specific group of retroviruses might have contributed to HGT in these nematodes. We also reveal several novelties for plant-nematode interactions. Major findings include the discovery of a strongly expressed H. glycines effector that is essential for virulence and efficiently targets plant cell nucleoli for suppression of innate immune responses. Also, this H. glycines effector contains marginal, but significant sequence similarity with an immunosuppressive effector found only in Plasmodium spp., the malaria parasites. Extensive database searches, phylogenetic analyses, and functional complementation experiments conclude that the similarities are best explained by sequence convergence due to similar immunosuppressive functions. Furthermore, we determine that a specific microRNA network in soybean that is essential for plant development delineates the formation of the H. glycines feeding site, and interfering with this network renders soybean roots much less susceptible to infection. In conclusion, the major findings included in this dissertation reveal novel insights into how nematodes adapted to plant parasitism, and for how PPN manipulate their host plants during infection to establish compatible interactions. Moreover, these findings will undoubtedly provide foundations for developing novel control measures against these important plant pathogens

A-to-I RNA Editing in Human Cells

RNA editing is a means of diversifying the transcriptome and regulating innate immunity. Among the different classes of enzymes that modify RNA, adenosine deaminase acting on RNA (ADAR) is a type that catalyzes adenosine-to-inosine editing on double-stranded RNA molecules to regulate cellular responses to endogenous and exogenous RNA. Of the three ADAR homologs in humans, dysregulation of ADAR1 editing due to inherited mutations leads to disorders such as Aicardi-Goutieres syndrome, an inflammatory disease that manifests in the brain and skin, and dyschromatosis symmetrica hereditaria, a skin pigmentation disorder. ADAR1 is the primary A-to-I editor of RNA in humans, and the majority of edit sites are found in a class of repetitive elements called Alu, many of which are located in introns and 3’ untranslated regions of RNA. The functional consequences of A-to-I editing are varied, although a complete lack of functional ADAR1 is usually not tolerated, as revealed by the MDA5-mediated embryonic lethality in mice lacking functional ADAR1. In human neural progenitor cells, loss of ADAR1 causes spontaneous upregulation of interferon and cell death, although the RNA triggers remain unknown. Given the importance of ADAR1-editing in maintaining homeostasis in various contexts, there is a need to understand in more detail how ADAR1 isoforms are regulated and how they individually contribute to the A-to-I RNA editome. Two ADAR1 protein isoforms, p110 (110 kDa) and p150 (150 kDa), are expressed constitutively and in response to interferon, respectively, but the contribution of each isoform to the editing landscape remains incompletely characterized, largely because of the challenges in expressing p150 without p110. We revealed that the p110 isoform can be expressed from the canonical p150-encoding mRNA due to leaky ribosome scanning downstream of the p150 start codon. Synonymous mutations introduced in the region between the p150 and p110 start codons reduce leaky scanning and usage of the p110 start codon, and cells expressing p150 constructs with these mutations produce significantly reduced levels of p110. With the ability to express p150 with significantly reduced levels of p110, the A-to-I editome can be classified in terms of p150-selective and p110-selective sites, allowing evaluation of the relative contributions of either isoform to global editing levels. Our editing analysis revealed that the majority of ADAR1-edit sites are p150-selective, although a significant proportion of ADAR1-edit sites are also shared between p150 and p110, being not dependent on presence of either isoform for editing to occur. Of the sites that are putatively p110- selective, the majority are located in introns. Finally, the ability of p150 mRNA to give rise to p110 means that p110 is also an interferon-inducible protein alongside the canonical interferon-stimulated ADAR1 isoform: p150. During the interferon response, the transcriptome changes, and many new mRNA structures, perhaps some immunogenic ones, will enter the nucleus and cytoplasm. The distribution of ADAR1 isoforms is such that p110 is mostly present in the nucleus, and p150 mostly in the cytoplasm. We propose that optimal editing in the nucleus and cytoplasm during the interferon response is achieved by the inducibility of p110 and p150, both of which share a large number of target sites

Identification and analysis of patterns in DNA sequences, the genetic code and transcriptional gene regulation

The present cumulative work consists of six articles linked by the topic ”Identification and Analysis of Patterns in DNA sequences, the Genetic Code and Transcriptional Gene Regulation”. We have applied a binary coding, to efficiently findpatterns within nucleotide sequences. In the first and second part of my work one single bit to encode all four nucleotides is used. The three possibilities of a one - bit coding are: keto (G,U) - amino (A,C) bases, strong (G,C) - weak (A,U) bases, and purines (G,A) - pyrimidines (C,U). We found out that the best pattern could be observed using the purine - pyrimidine coding. Applying this coding we have succeeded in finding a new representation of the genetic code which has been published under the title ”A New Classification Scheme of the Genetic Code” in ”Journal of Molecular Biology” and ”A Purine-Pyrimidine Classification Scheme of the Genetic Code” in ”BIOForum Europe”. This new representation enables to reduce the common table of the genetic code from 64 to 32 fields maintaining the same information content. It turned out that all known and even new patterns of the genetic code can easily be recognized in this new scheme. Furthermore, our new representation allows us for speculations about the origin and evolution of the translation machinery and the genetic code. Thus, we found a possible explanation for the contemporary codon - amino acid assignment and wide support for an early doublet code. Those explanations have been published in ”Journal of Bioinformatics and Computational Biology” under the title ”The New Classification Scheme of the Genetic Code, its Early Evolution, and tRNA Usage”. Assuming to find these purine - pyrimidine patterns at the DNA level itself, we examined DNA binding sites for the occurrence of binary patterns. A comprehensive statistic about the largest class of restriction enzymes (type II) has shown a very distinctive purine - pyrimidine pattern. Moreover, we have observed a higher G+C content for the protein binding sequences. For both observations we have provided and discussed several explanations published under the title ”Common Patterns in Type II Restriction Enzyme Binding Sites” in ”Nucleic Acid Research”. The identified patterns may help to understand how a protein finds its binding site. In the last part of my work two submitted articles about the analysis of Boolean functions are presented. Boolean functions are used for the description and analysis of complex dynamic processes and make it easier to find binary patterns within biochemical interaction networks. It is well known that not all functions are necessary to describe biologically relevant gene interaction networks. In the article entitled ”Boolean Networks with Biologically Relevant Rules Show Ordered Behavior”, submitted to ”BioSystems”, we have shown, that the class of required Boolean functions can strongly be restricted. Furthermore, we calculated the exact number of hierarchically canalizing functions which are known to be biologically relevant. In our work ”The Decomposition Tree for Analysis of Boolean Functions” submitted to ”Journal of Complexity”, we introduced an efficient data structure for the classification and analysis of Boolean functions. This permits the recognition of biologically relevant Boolean functions in polynomial time

Tetrahymena Metallothioneins Fall into Two Discrete Subfamilies

BACKGROUND: Metallothioneins are ubiquitous small, cysteine-rich, multifunctional proteins which can bind heavy metals. METHODOLOGY/PRINCIPAL FINDINGS: We report the results of phylogenetic and gene expression analyses that include two new Tetrahymena thermophila metallothionein genes (MTT3 and MTT5). Sequence alignments of all known Tetrahymena metallothioneins have allowed us to rationalize the structure of these proteins. We now formally subdivide the known metallothioneins from the ciliate genus Tetrahymena into two well defined subfamilies, 7a and 7b, based on phylogenetic analysis, on the pattern of clustering of Cys residues, and on the pattern of inducibility by the heavy metals Cd and Cu. Sequence alignment also reveals a remarkably regular, conserved and hierarchical modular structure of all five subfamily 7a MTs, which include MTT3 and MTT5. The former has three modules, while the latter has only two. Induction levels of the three T. thermophila genes were determined using quantitative real time RT-PCR. Various stressors (including heavy metals) brought about dramatically different fold-inductions for each gene; MTT5 showed the highest fold-induction. Conserved DNA motifs with potential regulatory significance were identified, in an unbiased way, upstream of the start codons of subfamily 7a MTs. EST evidence for alternative splicing in the 3′ UTR of the MTT5 mRNA with potential regulatory activity is reported. CONCLUSION/SIGNIFICANCE: The small number and remarkably regular structure of Tetrahymena MTs, coupled with the experimental tractability of this model organism for studies of in vivo function, make it an attractive system for the experimental dissection of the roles, structure/function relationships, regulation of gene expression, and adaptive evolution of these proteins, as well as for the development of biotechnological applications for the environmental monitoring of toxic substances

Molecular genetic tools for manipulation of the oleaginous yeast Rhodotorula toruloides

Rhodotorula (Rhodosporidium) toruloides is an oleaginous basidiomycete yeast with great biotechnological potential. Capable of accumulating lipid up to 76 % of its dry biomass and well suited to the metabolism of lignocellulosic hydrolysate, it is a good candidate for production of advanced biofuels as well as a host of other potential roles in industry. However, molecular genetic tools for manipulation of this yeast are lacking and its high genomic GC content can make routine cloning difficult. Agrobacterium tumefaciens-mediated transformation of R. toruloides CBS 14 was demonstrated, and plasmid vectors were developed for transformation of R. toruloides, including elements for Saccharomyces cerevisiae in-yeast assembly. In-yeast assembly is robust to the manipulation of GC-rich DNA and of large plasmids. Using these vectors and an EGFP reporter, a screen to identify inducible promoters was performed, and promoters from the genes NAR1, ICL1, CTR3, and MET16 identified. These promoters have independent induction/repression conditions and different levels and rates of induction. Minimal inducible promoters were determined, which are as small as 200 bp. As well as showing tight regulation of the EGFP marker, the NAR1 promoter was able to drive conditional rescue of a leu2 mutant strain. In parallel, as a proof of principle for production of advanced biofuels, hydrocarbon biosynthesis pathways were expressed in R. toruloides and analysed by GC-MS. After co-expression of Synechococcus elongatus fatty acyl-ACP reductase and fatty aldehyde decarbonylase, and E. coli ferredoxin and ferredoxin reductase, production of the alkane heptadecane was observed. To increase the availability of free fatty acids (FFA) for production of hydrocarbons by other pathways, Thermomyces lanuginosus lipase 2 was expressed, resulting in a 1.3-fold increase in the concentration of FFAs.BBSR