RESUMO
Schistosomiasis is one of the most detrimental neglected tropical diseases. Controlling the spread of this parasitic illness requires effective sanitation, access to chemotherapeutic drugs, and control over populations of the freshwater snails, such as Biomphalaria glabrata, that are essential intermediate hosts for schistosomes. Effectively controlling this disease, while minimising ecological implications of such control, will require an extensive understanding of the immunological interactions between schistosomes and their molluscan intermediate hosts. Here we histologically characterise the clearance of schistosome larvae by snails that exhibit allelic variation at a single genomic region, the Guadeloupe resistance complex. We show that snails with a resistant Guadeloupe resistance complex genotype clear schistosomes within the first 24-48â¯h, and that this resistance can be transferred to susceptible snails via whole hemolymph but not cell-free plasma. These findings imply that Guadeloupe resistance complex-coded proteins help to coordinate hemocyte-mediated immune responses to schistosome infections in Guadeloupean snails.
Assuntos
Biomphalaria/genética , Biomphalaria/parasitologia , Genótipo , Schistosoma mansoni/fisiologia , Animais , Hemolinfa , Interações Hospedeiro-Parasita/genéticaRESUMO
This corrects the article DOI: 10.1038/ncomms15451.
RESUMO
Biomphalaria snails are instrumental in transmission of the human blood fluke Schistosoma mansoni. With the World Health Organization's goal to eliminate schistosomiasis as a global health problem by 2025, there is now renewed emphasis on snail control. Here, we characterize the genome of Biomphalaria glabrata, a lophotrochozoan protostome, and provide timely and important information on snail biology. We describe aspects of phero-perception, stress responses, immune function and regulation of gene expression that support the persistence of B. glabrata in the field and may define this species as a suitable snail host for S. mansoni. We identify several potential targets for developing novel control measures aimed at reducing snail-mediated transmission of schistosomiasis.
Assuntos
Biomphalaria/genética , Biomphalaria/parasitologia , Genoma , Esquistossomose mansoni/transmissão , Comunicação Animal , Animais , Biomphalaria/imunologia , Elementos de DNA Transponíveis , Evolução Molecular , Água Doce , Regulação da Expressão Gênica , Interações Hospedeiro-Parasita , Feromônios , Proteoma , Schistosoma mansoni , Análise de Sequência de DNA , Estresse FisiológicoRESUMO
We previously observed that exposure to a complex mixture of high molecular weight polycyclic aromatic hydrocarbons (PAHs) increased sensitivity of rainbow trout (Oncorhynchus mykiss) to subsequent challenge with Aeromonas salmonicida, the causative agent of furunculosis. In this study, we evaluate potential mechanisms associated with disease susceptibility from combined environmental factors of dietary PAH exposure and pathogen challenge. Rainbow trout were fed a mixture of ten high molecular weight PAHs at an environmentally relevant concentration (7.82µg PAH mixture/g fish/day) or control diet for 50 days. After 50 days of PAH exposure, fish were challenged with either Aeromonas salmonicida at a lethal concentration 30 (LC30) or growth media without the pathogen (mock challenge). Head kidneys were collected 2, 4, 10 and 20 days after challenge and gene expression (q<0.05) was evaluated among treatments. In animals fed the PAH contaminated diet, we observed down-regulation of expression for innate immune system genes in pathways (p<0.05) for the terminal steps of the complement cascade (complement component C6) and other bacteriolytic processes (lysozyme type II) potentially underlying increased disease susceptibility after pathogen challenge. Increased expression of genes associated with hemorrhage/tissue remodeling/inflammation pathways (p<0.05) was likely related to more severe head kidney damage due to infection in PAH-fed compared to control-fed fish. This study is the first to evaluate transcriptional signatures associated with the impact of chronic exposure to an environmentally relevant mixture of PAHs in disease susceptibility and immunity.
Assuntos
Aeromonas salmonicida/patogenicidade , Rim Cefálico/imunologia , Imunidade Inata/efeitos dos fármacos , Oncorhynchus mykiss/microbiologia , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Transcrição Gênica/efeitos dos fármacos , Animais , Regulação para Baixo , Imunidade Inata/genética , Muramidase/metabolismo , Oncorhynchus mykiss/imunologia , Oncorhynchus mykiss/metabolismoRESUMO
Schistosomiasis is one of the most important neglected tropical diseases. Despite effective chemotherapeutic treatments, this disease continues to afflict hundreds of millions of people. Understanding the natural intermediate snail hosts of schistosome parasites is vital to the suppression of this disease. A recently identified genomic region in Caribbean Biomphalaria glabrata snails strongly influences their resistance to infection by Schistosoma mansoni. This region contains novel genes having structural similarity to known pathogen recognition proteins. Here we elaborate on the probable structure and role of one of these genes, grctm6. We characterised the expression of Grctm6 in a population of Caribbean snails, and performed a siRNA knockdown of Grctm6. We show that this protein is not only expressed in B. glabrata hemolymph, but that it also has a role in modulating the number of S. mansoni cercariae released by infected snails, making it a possible target for the biological control of schistosomiasis.
Assuntos
Biomphalaria/imunologia , Biomphalaria/parasitologia , Vetores de Doenças , Interações Hospedeiro-Patógeno , Schistosoma mansoni/crescimento & desenvolvimento , Schistosoma mansoni/imunologia , Animais , Região do Caribe , Perfilação da Expressão Gênica , Inativação Gênica , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismoRESUMO
BACKGROUND: New strategies to combat the global scourge of schistosomiasis may be revealed by increased understanding of the mechanisms by which the obligate snail host can resist the schistosome parasite. However, few molecular markers linked to resistance have been identified and characterized in snails. METHODOLOGY/PRINCIPAL FINDINGS: Here we test six independent genetic loci for their influence on resistance to Schistosoma mansoni strain PR1 in the 13-16-R1 strain of the snail Biomphalaria glabrata. We first identify a genomic region, RADres, showing the highest differentiation between susceptible and resistant inbred lines among 1611 informative restriction-site associated DNA (RAD) markers, and show that it significantly influences resistance in an independent set of 439 outbred snails. The additive effect of each RADres resistance allele is 2-fold, similar to that of the previously identified resistance gene sod1. The data fit a model in which both loci contribute independently and additively to resistance, such that the odds of infection in homozygotes for the resistance alleles at both loci (13% infected) is 16-fold lower than the odds of infection in snails without any resistance alleles (70% infected). Genome-wide linkage disequilibrium is high, with both sod1 and RADres residing on haplotype blocks >2 Mb, and with other markers in each block also showing significant effects on resistance; thus the causal genes within these blocks remain to be demonstrated. Other candidate loci had no effect on resistance, including the Guadeloupe Resistance Complex and three genes (aif, infPhox, and prx1) with immunological roles and expression patterns tied to resistance, which must therefore be trans-regulated. CONCLUSIONS/SIGNIFICANCE: The loci RADres and sod1 both have strong effects on resistance to S. mansoni. Future approaches to control schistosomiasis may benefit from further efforts to characterize and harness this natural genetic variation.
Assuntos
Biomphalaria/parasitologia , Schistosoma mansoni/crescimento & desenvolvimento , Animais , Biomphalaria/imunologia , Loci Gênicos , Marcadores Genéticos , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Parasita , Humanos , Desequilíbrio de Ligação , Camundongos , Dados de Sequência Molecular , Schistosoma mansoni/imunologia , Análise de Sequência de DNARESUMO
Continuing transmission of human intestinal schistosomiasis depends on the parasite's access to susceptible snail intermediate hosts (often Biomphalaria glabrata). Transmission fails when parasite larvae enter resistant individuals in wild snail populations. The genetic basis for differences in snail susceptibility/resistance is being intensively investigated as a means to devise novel control strategies based on resistance genes. Reactive oxygen species produced by the snail's defence cells (haemocytes) are effectors of resistance. We hypothesised that genes relevant to production and consumption of reactive oxygen species would be expressed differentially in the haemocytes of snail hosts with different susceptibility/resistance phenotypes. By restricting the genetic diversity of snails, we sought to facilitate identification of resistance genes. By inbreeding, we procured from a 13-16-R1 snail population with both susceptible and resistant individuals 52 lines of B. glabrata (expected homozygosity ~87.5%), and determined the phenotype of each in regard to susceptibility/resistance to Schistosoma mansoni. The inbred lines were found to have line-specific differences in numbers of spreading haemocytes; these were enumerated in both juvenile and adult snails. Lines with high cell numbers were invariably resistant to S. mansoni, whereas lines with lower cell numbers could be resistant or susceptible. Transcript levels in haemocytes were quantified for 18 potentially defence-related genes. Among snails with low cell numbers, the different susceptibility/resistance phenotypes correlated with differences in transcript levels for two redox-relevant genes: an inferred phagocyte oxidase component and a peroxiredoxin. Allograft inflammatory factor (potentially a regulator of leucocyte activation) was expressed at higher levels in resistant snails regardless of spread cell number. Having abundant spreading haemocytes is inferred to enable a snail to kill parasite sporocysts. In contrast, snails with fewer spreading haemocytes seem to achieve resistance only if specific genes are expressed constitutively at levels that are high for the species.
Assuntos
Biomphalaria/parasitologia , Hemócitos/parasitologia , Interações Hospedeiro-Parasita , Schistosoma mansoni/crescimento & desenvolvimento , Animais , Biomphalaria/imunologia , Cruzamento , Contagem de Células , Perfilação da Expressão Gênica , Hemócitos/imunologia , Redes e Vias Metabólicas/genética , Dados de Sequência Molecular , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/toxicidade , Schistosoma mansoni/imunologia , Análise de Sequência de DNARESUMO
Development of protocols and media for culturing immune cells from marine invertebrates has not kept pace with advancements in mammalian immune cell culture, the latter having been driven by the need to understand the causes of and develop therapies for human and animal diseases. However, expansion of the aquaculture industry and the diseases that threaten these systems creates the need to develop cell and tissue culture methods for marine invertebrates. Such methods will enable us to better understand the causes of disease outbreaks and to develop means to avoid and remedy epidemics. We report a method for the short-term culture of phagocytes from the purple sea urchin, Strongylocentrotus purpuratus, by modifying an approach previously used to culture cells from another sea urchin species. The viability of cultured phagocytes from the purple sea urchin decreases from 91.6% to 57% over six days and phagocyte morphology changes from single cells to aggregates leading to the formation of syncytia-like structures. This process is accelerated in the presence of lipopolysaccharide suggesting that phagocytes are capable of detecting this molecular pattern in culture conditions. Sea urchin immune response proteins, called Sp185/333, are expressed on the surface of a subset of phagocytes and have been associated with syncytia-like structures. We evaluated their expression in cultured phagocytes to determine their possible role in cell aggregation and in the formation of syncytia-like structures. Between 0 and 3 hr, syncytia-like structures were observed in cultures when only ~10% of the cells were positive for Sp185/333 proteins. At 24 hr, ~90% of the nuclei were Sp185/333-positive when all of the phagocytes had aggregated into syncytia-like structures. Consequently, we conclude that the Sp185/333 proteins do not have a major role in initiating the aggregation of cultured phagocytes, however the Sp185/333 proteins are associated with the clustered nuclei within the syncytia-like structures.
Assuntos
Lipopolissacarídeos/farmacologia , Fagócitos/citologia , Strongylocentrotus purpuratus/citologia , Strongylocentrotus purpuratus/efeitos dos fármacos , Animais , Agregação Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Análise por Conglomerados , Meios de Cultura/farmacologia , Células Gigantes/citologia , Células Gigantes/efeitos dos fármacos , Humanos , Fagócitos/efeitos dos fármacos , Fagócitos/metabolismo , Fatores de TempoRESUMO
Allelic variation at the Cu-Zn superoxide dismutase (SOD1) locus has been shown to be associated with resistance of the snail, Biomphalaria glabrata, to infection by the trematode parasite, Schistosoma mansoni. SOD1 catalyses the production of hydrogen peroxide, a known cytotoxic component of the oxidative burst used in defence against pathogens. In our laboratory population of B. glabrata, the most resistant allele at SOD1 is over-expressed relative to the other two alleles. Because hydrogen peroxide also causes oxidative stress on host tissues, we hypothesised that over-expression of SOD1 might be compensated by epistatic interactions with other loci involved in oxidation-reduction (redox) pathways. Catalase, peroxiredoxins and glutathione peroxidases all degrade hydrogen peroxide. We tested whether alleles at each of these loci were in linkage disequilibrium with SOD1 in our population, as might be expected given strong epistatic selection. We found that SOD1, catalase (CAT) and a peroxiredoxin locus (PRX4) are in strong linkage disequilibrium in our population. We also found that these loci are tightly linked, within 1-2cM of each other, which explains the high linkage disequilibrium. This result raises the possibility that there is a linked cluster of redox genes, and perhaps other defence-relevant genes, in the B. glabrata genome. Whether epistatic interactions for fitness actually exist among these loci still needs to be tested. However the close physical linkage among SOD1, PRX4 and CAT, and subsequent high disequilibrium, makes such interactions a plausible hypothesis.
Assuntos
Biomphalaria/enzimologia , Catalase/genética , Redes e Vias Metabólicas/genética , Peroxirredoxinas/genética , Explosão Respiratória , Superóxido Dismutase/genética , Animais , Biomphalaria/genética , Catalase/metabolismo , Desequilíbrio de Ligação , Dados de Sequência Molecular , Família Multigênica , Peroxirredoxinas/metabolismo , Análise de Sequência de DNA , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1RESUMO
Resistance of the snail Biomphalaria glabrata to the trematode Schistosoma mansoni is correlated with allelic variation at copper-zinc superoxide dismutase (sod1). We tested whether there is a fitness cost associated with carrying the most resistant allele in three outbred laboratory populations of snails. These three populations were derived from the same base population, but differed in average resistance. Under controlled laboratory conditions we found no cost of carrying the most resistant allele in terms of fecundity, and a possible advantage in terms of growth and mortality. These results suggest that it might be possible to drive resistant alleles of sod1 into natural populations of the snail vector for the purpose of controlling transmission of S. mansoni. However, we did observe a strong effect of genetic background on the association between sod1 genotype and resistance. sod1 genotype explained substantial variance in resistance among individuals in the most resistant genetic background, but had little effect in the least resistant genetic background. Thus, epistatic interactions with other loci may be as important a consideration as costs of resistance in the use of sod1 for vector manipulation.
Assuntos
Biomphalaria/enzimologia , Biomphalaria/fisiologia , Superóxido Dismutase/genética , Alelos , Animais , Biomphalaria/genética , Biomphalaria/parasitologia , Fertilidade , Variação Genética , Genótipo , Reprodução , Schistosoma mansoni/patogenicidade , Análise de SobrevidaRESUMO
Elasmobranchs are the most commonly used experimental models among the jawed, cartilaginous fish (Chondrichthyes). Previously we developed cell lines from embryos of two elasmobranchs, Squalus acanthias the spiny dogfish shark (SAE line), and Leucoraja erinacea the little skate (LEE-1 line). From these lines cDNA libraries were derived and expressed sequence tags (ESTs) generated. From the SAE cell line 4303 unique transcripts were identified, with 1848 of these representing unknown sequences (showing no BLASTX identification). From the LEE-1 cell line, 3660 unique transcripts were identified, and unknown, unique sequences totaled 1333. Gene Ontology (GO) annotation showed that GO assignments for the two cell lines were in general similar. These results suggest that the procedures used to derive the cell lines led to isolation of cell types of the same general embryonic origin from both species. The LEE-1 transcripts included GO categories "envelope" and "oxidoreductase activity" but the SAE transcripts did not. GO analysis of SAE transcripts identified the category "anatomical structure formation" that was not present in LEE-1 cells. Increased organelle compartments may exist within LEE-1 cells compared to SAE cells, and the higher oxidoreductase activity in LEE-1 cells may indicate a role for these cells in responses associated with innate immunity or in steroidogenesis. These EST libraries from elasmobranch cell lines provide information for assembly of genomic sequences and are useful in revealing gene diversity, new genes and molecular markers, as well as in providing means for elucidation of full-length cDNAs and probes for gene array analyses. This is the first study of this type with members of the Chondrichthyes.
Assuntos
Etiquetas de Sequências Expressas/química , Rajidae/genética , Squalus acanthias/genética , Animais , Linhagem Celular , Rajidae/embriologia , Squalus acanthias/embriologiaRESUMO
To identify gene(s) that may be associated with resistance/susceptibility in the intermediate snail host Biomphalaria glabrata to Schistosoma mansoni infection, a snail albumen gland cDNA library was differentially screened and a partial cDNA encoding an antioxidant enzyme thioredoxin peroxidase (Tpx), or peroxiredoxin (Prx), was identified. The 753bp full-length, single-copy, constitutively expressed gene now referred to as BgPrx4 was later isolated. BgPrx4 is a 2-Cys peroxiredoxin containing the conserved peroxidatic cysteine (C(P)) in the N-terminus and the resolving cysteine (C(R)) in the C-terminus. Sequence analysis of BgPrx4 from both resistant and susceptible snails revealed the presence of several (at least 7) single nucleotide polymorphisms (SNPs). Phylogenetic analysis indicated BgPrx4 to resemble a homolog of human peroxiredoxin, PRDX4. Northern analysis of hepatopancreas RNA from both resistant and susceptible snails showed that upon parasite exposure there were qualitative changes in gene expression. Quantitative real-time RT-PCR analysis showed differences in the levels of BgPrx4 transcript induction following infection, with the transcript up-regulated in resistant snails during the early phase (5h) of infection compared to susceptible snails in which it was down-regulated within the early time period. While there was an increase in transcription in susceptible snails later (48h) post-infection, this never reached the levels detected in resistant snails. A similar trend - higher, earlier up-regulation in the resistant snails but lower, slower protein expression in susceptible snails - was observed by Western blot analysis. Enzymatic analysis of the purified, recombinant BgPrx4 revealed the snail sequence to function as Prx but with an unusual ability to use both thioredoxin and glutathione as substrates.
Assuntos
Biomphalaria/enzimologia , Biomphalaria/fisiologia , Regulação da Expressão Gênica , Peroxirredoxinas/biossíntese , Schistosoma mansoni/imunologia , Esquistossomose mansoni/imunologia , Sequência de Aminoácidos , Animais , Biomphalaria/genética , DNA/química , DNA/genética , Glutationa/metabolismo , Dados de Sequência Molecular , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Tiorredoxinas/metabolismoRESUMO
Schistosomiasis, caused by infections by human blood flukes (Trematoda), continues to disrupt the lives of over 200,000,000 people in over 70 countries, inflicting misery and precluding the individuals' otherwise reasonable expectations of productive lives. Infection requires contact with freshwater in which infected snails (the intermediate hosts of schistosomes) have released cercariae larvae. Habitats suitable for the host snails continue to expand as a consequence of water resource development. No vaccine is available, and resistance has emerged towards the single licensed schistosomicide drug. Since human infections would cease if parasite infections in snails were prevented, efforts are being made to discover requirements of intra-molluscan development of these parasites. Wherever blood flukes occur, naturally resistant conspecific snails are present. To understand the mechanisms used by parasites to ensure their survival in immunocompetent hosts, one must comprehend the interior defense mechanisms that are available to the host. For one intermediate host snail (Biomphalaria glabrata) and trematodes for which it serves as vector, molecular genetic and proteomic surveys for genes and proteins influencing the outcomes on infections are yielding lists of candidates. A comparative approach drawing on data from studies in divergent species provides a robust basis for hypothesis generation to drive decisions as to which candidates merit detailed further investigation. For example, reactive oxygen and nitrogen species are known mediators or effectors in battles between infectious agents and their hosts. An approach targeting genes involved in relevant pathways has been fruitful in the Schistosoma mansoni -- B. glabrata parasitism, leading to discovery of a functionally relevant gene set (encoding enzymes responsible for the leukocyte respiratory burst) that associates significantly with host resistance phenotype. This review summarizes advances in the understanding of strategies used by both this trematode parasite and its molluscan host to ensure their survival.
Assuntos
Biomphalaria/parasitologia , Interações Hospedeiro-Parasita/fisiologia , Schistosoma mansoni/fisiologia , Animais , Vetores de Doenças , Interações Hospedeiro-Parasita/genética , Humanos , Schistosoma mansoni/genética , Esquistossomose mansoni/transmissãoRESUMO
Sessile inhabitants of marine intertidal environments commonly face heat stress, an important component of summer mortality syndrome in the Pacific oyster Crassostrea gigas. Marker-aided selection programs would be useful for developing oyster strains that resist summer mortality; however, there is currently a need to identify candidate genes associated with stress tolerance and to develop molecular markers associated with those genes. To identify candidate genes for further study, we used cDNA microarrays to test the hypothesis that oyster families that had high (>64%) or low (<29%) survival of heat shock (43 degrees C, 1 h) differ in their transcriptional responses to stress. Based upon data generated by the microarray and by real-time quantitative PCR, we found that transcription after heat shock increased for genes putatively encoding heat shock proteins and genes for proteins that synthesize lipids, protect against bacterial infection, and regulate spawning, whereas transcription decreased for genes for proteins that mobilize lipids and detoxify reactive oxygen species. RNAs putatively identified as heat shock protein 27, collagen, peroxinectin, S-crystallin, and two genes with no match in Genbank had higher transcript concentrations in low-surviving families than in high-surviving families, whereas concentration of putative cystatin B mRNA was greater in high-surviving families. These ESTs should be studied further for use in marker-aided selection programs. Low survival of heat shock could result from a complex interaction of cell damage, opportunistic infection, and metabolic exhaustion.
Assuntos
Crassostrea/fisiologia , Perfilação da Expressão Gênica , Temperatura Alta , Adaptação Fisiológica , Animais , Cruzamento , Crassostrea/genética , Crassostrea/metabolismo , Etiquetas de Sequências ExpressasRESUMO
The snail Biomphalaria glabrata kills the blood fluke Schistosoma mansoni by a mechanism involving production of hydrogen peroxide, the enzymatic product of cytosolic Cu/Zn superoxide dismutase (SOD1). This enzyme exhibits higher activity in blood cells (hemocytes) from a predominantly resistant strain of B. glabrata than in hemocytes from a susceptible strain. Additionally, B. glabrata SOD1 polymorphisms have been associated with susceptibility/resistance to the parasite. To address the hypothesis that SOD1 transcription levels differ in accordance with variation at the SOD1 locus, quantitative PCR was performed using hemocyte-derived cDNA prepared from SOD1-genotyped snails. Here we report that individuals possessing the allele previously associated with resistance to S. mansoni express significantly higher levels of hemocyte SOD1 transcripts than individuals lacking this allele. A causal relationship between SOD1 expression and susceptibility/resistance to S. mansoni is supported by the correlation of transcript quantity with data (from a previous study) on the probability of infection.
Assuntos
Biomphalaria/enzimologia , Biomphalaria/parasitologia , Regulação Enzimológica da Expressão Gênica , Variação Genética/genética , Schistosoma mansoni/fisiologia , Esquistossomose mansoni/enzimologia , Superóxido Dismutase/metabolismo , Alelos , Animais , Biomphalaria/genética , Suscetibilidade a Doenças , Genótipo , RNA Mensageiro/genética , Esquistossomose mansoni/genética , Esquistossomose mansoni/parasitologia , Esquistossomose mansoni/patologia , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
We have established a cartilaginous fish cell line [Squalus acanthias embryo cell line (SAE)], a mesenchymal stem cell line derived from the embryo of an elasmobranch, the spiny dogfish shark S. acanthias. Elasmobranchs (sharks and rays) first appeared >400 million years ago, and existing species provide useful models for comparative vertebrate cell biology, physiology, and genomics. Comparative vertebrate genomics among evolutionarily distant organisms can provide sequence conservation information that facilitates identification of critical coding and noncoding regions. Although these genomic analyses are informative, experimental verification of functions of genomic sequences depends heavily on cell culture approaches. Using ESTs defining mRNAs derived from the SAE cell line, we identified lengthy and highly conserved gene-specific nucleotide sequences in the noncoding 3' UTRs of eight genes involved in the regulation of cell growth and proliferation. Conserved noncoding 3' mRNA regions detected by using the shark nucleotide sequences as a starting point were found in a range of other vertebrate orders, including bony fish, birds, amphibians, and mammals. Nucleotide identity of shark and human in these regions was remarkably well conserved. Our results indicate that highly conserved gene sequences dating from the appearance of jawed vertebrates and representing potential cis-regulatory elements can be identified through the use of cartilaginous fish as a baseline. Because the expression of genes in the SAE cell line was prerequisite for their identification, this cartilaginous fish culture system also provides a physiologically valid tool to test functional hypotheses on the role of these ancient conserved sequences in comparative cell biology.
Assuntos
Regiões 3' não Traduzidas , Peixes/genética , RNA/genética , Animais , Sequência de Bases , Linhagem Celular , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Humanos , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
Inflammatory stimuli elicit liver synthesis and subsequent release into the plasma of several proteins (positive acute phase proteins, APP) with functions in innate immunity, tissue repair and restoration of homeostasis. To expand the basis for evaluating the degree of conservation of the APR in vertebrates and to assess the extent to which genes encoding both cellular and plasma proteins are affected, we profiled transcriptional changes in livers of individual rainbow trout (Oncorhynchus mykiss) after intraperitoneal injection of Listonella (Vibrio) anguillarum bacterin in Freund's Incomplete Adjuvant. Twenty genes were down-regulated, some unexpectedly such as complement component 3 and alpha2-macroglobulin. Sixteen up-regulated genes included three encoding proteins involved in iron metabolism (hepcidin, haptoglobin, and intelectin), from which we infer that sequestration of iron is likely to be a major component of the trout APR. Activated genes encoding proteins of unknown functions included precerebellin-like plasma protein, and differentially regulated trout protein which is predicted to be cell surface associated. The only complement component that increased was C7. Genes encoding proteins that are probably not released into plasma included two fatty acid binding proteins, two transport proteins (SEC61 and a Na - Ca exchanger), GAPDH, an amino transferase, and a hydrolase. When microarray data and quantitative RT-PCR analyses were used to evaluate specific transcripts, variations were notable between individual fish, possibly a basis for natural variation in susceptibility to infectious diseases. This study suggests novel hypotheses relating to NFkappaB, albumin-related protein, pentraxin, hypoferremia and the complement cascade. While the capacity to mount an APR is conserved throughout vertebrate evolution, the responding genes vary from species to species, and considerable variation is observed from individual to individual within a species.
Assuntos
Proteínas de Fase Aguda/biossíntese , Reação de Fase Aguda/imunologia , Expressão Gênica/imunologia , Fígado/fisiopatologia , Oncorhynchus mykiss/imunologia , Proteínas de Fase Aguda/análise , Proteínas de Fase Aguda/genética , Reação de Fase Aguda/genética , Animais , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Primers do DNA/química , Regulação para Baixo , Perfilação da Expressão Gênica/veterinária , Ferro/metabolismo , Leucócitos/imunologia , Listonella/imunologia , Fígado/imunologia , Análise de Sequência com Séries de Oligonucleotídeos/veterinária , Oncorhynchus mykiss/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterináriaRESUMO
Variation in susceptibility of the snail Biomphalaria glabrata to infection by the parasite Schistosoma mansoni is, at least in part, genetically determined. Functional studies have demonstrated that hemocyte-mediated killing of the parasite involves hydrogen peroxide, the enzymatic product of superoxide dismutase (SOD). The present study identifies alleles of the gene coding for cytosolic copper/zinc SOD (SOD1). The resistance/susceptibility phenotypes and SOD1 genotypes were determined for 354 snails of the predominantly resistant 13-16-R1 strain of B. glabrata. Resistance to the parasite was found to be significantly associated with one allele of the SOD1 gene. Conversely, a separate SOD1 allele was significantly associated with susceptibility.
Assuntos
Biomphalaria/enzimologia , Biomphalaria/imunologia , Citosol/enzimologia , Schistosoma mansoni/patogenicidade , Superóxido Dismutase/genética , Alelos , Animais , Biomphalaria/parasitologia , Cobre/metabolismo , Imunidade Inata , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Superóxido Dismutase/metabolismo , Zinco/metabolismoRESUMO
Transcriptome profiling is a powerful means of simultaneously identifying large numbers of genes that respond transcriptionally to stimuli of any sort. Whereas individuality at the level of genomic sequence is readily revealed and can be expected to influence transcriptional responses, knowledge of the global transcriptomic consequences of genomic individuality is in its infancy. Appreciation of the inherent variability of biological systems gives us confidence in predicting that no two individuals in any outbred population will respond identically to a stimulus. More critical for comparative studies, even unstimulated transcriptomes will be distinctive for each individual. To assess the confidence with which inferences may be drawn from transcriptome profiling when genetically identical samples can be assured, we examined the unprovoked transcriptomes of hepatic and pronephric (head kidney) tissues in three clonal lines of Rainbow trout (Oncorhynchus mykiss). Clonal individuals derived from three allopatric populations presented transcriptional profiles for both liver and pronephros that were not statistically significantly different within each clonal line; however each clonal line was distinguished by a subset of genes with constitutively different transcript abundance. Among these, immunologically-relevant genes were over-represented, possibly reflecting evolutionarily recent, pathogen-driven genetic sweeps.