RESUMO
Hsp40-Hsp70 typically function in concert as molecular chaperones, and their roles in post-infection immune responses are increasingly recognized. However, in the economically important fish species Scophthalmus maximus (turbot), there is still a lack in the systematic identification, interaction models, and binding site analysis of these proteins. Herein, 62 Hsp40 genes and 16 Hsp70 genes were identified in the turbot at a genome-wide level and were unevenly distributed on 22 chromosomes through chromosomal distribution analysis. Phylogenetic and syntenic analysis provided strong evidence in supporting the orthologies and paralogies of these HSPs. Protein-protein interaction and expression analysis was conducted to predict the expression profile after challenging with Aeromonas salmonicida. dnajb1b and hspa1a were found to have a co-expression trend under infection stresses. Molecular docking was performed using Auto-Dock Tool and PyMOL for this pair of chaperone proteins. It was discovered that in addition to the interaction sites in the J domain, the carboxyl-terminal domain of Hsp40 also plays a crucial role in its interaction with Hsp70. This is important for the mechanistic understanding of the Hsp40-Hsp70 chaperone system, providing a theoretical basis for turbot disease resistance breeding, and effective value for the prevention of certain diseases in turbot.
Assuntos
Doenças dos Peixes , Linguados , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70 , Filogenia , Animais , Linguados/imunologia , Linguados/genética , Linguados/microbiologia , Linguados/metabolismo , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Doenças dos Peixes/genética , Doenças dos Peixes/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Proteínas de Peixes/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/genética , Simulação de Acoplamento Molecular , Aeromonas salmonicida/imunologia , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genéticaRESUMO
Grass Carp Reovirus (GCRV) and Aeromonas hydrophila (Ah) are the causative agents of haemorrhagic disease in grass carp. This study aimed to investigate the molecular mechanisms and immune responses at the miRNA, mRNA, and protein levels in grass carp kidney cells (CIK) infected by Grass Carp Reovirus (GCRV, NV) and Aeromonas hydrophilus (Bacteria, NB) to gain insight into their pathogenesis. Within 48 h of infection with Grass Carp Reovirus (GCRV), 99 differentially expressed microRNA (DEMs), 2132 differentially expressed genes (DEGs), and 627 differentially expressed proteins (DEPs) were identified by sequencing; a total of 92 DEMs, 3162 DEGs, and 712 DEPs were identified within 48 h of infection with Aeromonas hydrophila. It is worth noting that most of the DEGs in the NV group were primarily involved in cellular processes, while most of the DEGs in the NB group were associated with metabolic pathways based on KEGG enrichment analysis. This study revealed that the mechanism of a grass carp haemorrhage caused by GCRV infection differs from that caused by the Aeromonas hydrophila infection. An important miRNA-mRNA-protein regulatory network was established based on comprehensive transcriptome and proteome analysis. Furthermore, 14 DEGs and 6 DEMs were randomly selected for the verification of RNA/small RNA-seq data by RT-qPCR. Our study not only contributes to the understanding of the pathogenesis of grass carp CIK cells infected with GCRV and Aeromonas hydrophila, but also serves as a significant reference value for other aquatic animal haemorrhagic diseases.
Assuntos
Aeromonas hydrophila , Carpas , MicroRNAs , RNA Mensageiro , Reoviridae , Transcriptoma , Animais , Carpas/genética , Carpas/microbiologia , Carpas/virologia , Carpas/imunologia , MicroRNAs/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reoviridae/fisiologia , Proteômica/métodos , Doenças dos Peixes/microbiologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Doenças dos Peixes/genética , Perfilação da Expressão Gênica , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/genética , Linhagem Celular , Infecções por Reoviridae/veterinária , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/genética , Redes Reguladoras de GenesRESUMO
Aeromonas veronii is associated with food spoilage and some human diseases, such as diarrhea, gastroenteritis, hemorrhagic septicemia or asymptomatic and even death. This research investigated the mechanism of the growth, biofilm formation, virulence, stress resistance, and spoilage potential of Bacillus subtilis lipopeptide against Aeromonas veronii. Lipopeptides suppressed the transmembrane transport of Aeromonas veronii by changing the cell membrane's permeability, the structure of membrane proteins, and Na+/K+-ATPase. Lipopeptide significantly reduced the activities of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) by 86.03% and 56.12%, respectively, ultimately slowing Aeromonas veronii growth. Lipopeptides also restrained biofilm formation by inhibiting Aeromonas veronii motivation and extracellular polysaccharide secretion. Lipopeptides downregulated gene transcriptional levels related to the virulence and stress tolerance of Aeromonas veronii. Furthermore, lipopeptides treatment resulted in a considerable decrease in the extracellular protease activity of Aeromonas veronii, which restrained the decomposing of channel catfish flesh. This research provides new insights into lipopeptides for controlling Aeromonas veronii and improving food safety.
Assuntos
Aeromonas , Doenças dos Peixes , Infecções por Bactérias Gram-Negativas , Ictaluridae , Animais , Humanos , Aeromonas veronii/genética , Aeromonas veronii/metabolismo , Bacillus subtilis/genética , Biofilmes , Lipopeptídeos/farmacologia , Lipopeptídeos/metabolismo , Infecções por Bactérias Gram-Negativas/genética , Aeromonas/genéticaRESUMO
Interleukin-6 (IL-6), a pleiotropic cytokine, plays a crucial role in acute stress induced by bacterial infection and is strongly associated with reactive oxygen species (ROS) production. However, the role of IL-6 in the liver of fish after Aeromonas hydrophila infection remains unclear. Therefore, this study constructed a zebrafish (Danio rerio) il-6 knockout line by CRISPR/Cas9 to investigate the function of IL-6 in the liver post bacterial infection. After infection with A. hydrophila, pathological observation showed that il-6-/- zebrafish exhibited milder liver damage than wild-type (WT) zebrafish. Moreover, liver transcriptome sequencing revealed that 2432 genes were significantly up-regulated and 1706 genes were significantly down-regulated in il-6-/- fish compared with WT fish after A. hydrophila infection. Further, gene ontology (GO) analysis showed that differentially expressed genes (DEGs) were significantly enriched in redox-related terms, including oxidoreductase activity, copper ion transport, etc. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEGs were significantly enriched in pathways such as the PPAR signaling pathway, suggesting that il-6 mutation has a significant effect on redox processes in the liver after A. hydrophila infection. Additionally, il-6-/- zebrafish exhibited lower malondialdehyde (MDA) levels and higher superoxide dismutase (SOD) activities in the liver compared with WT zebrafish following A. hydrophila infection, indicating that IL-6 deficiency mitigates oxidative stress induced by A. hydrophila infection in the liver. These findings provide a basis for further studies on the role of IL-6 in regulating oxidative stress in response to bacterial infections.
Assuntos
Infecções Bacterianas , Infecções por Bactérias Gram-Negativas , Interleucina-6 , Proteínas de Peixe-Zebra , Animais , Aeromonas hydrophila/fisiologia , Infecções Bacterianas/patologia , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/microbiologia , Interleucina-6/genética , Fígado/patologia , Mutação , Oxirredução , Estresse Oxidativo/genética , Peixe-Zebra/genética , Peixe-Zebra/microbiologia , Proteínas de Peixe-Zebra/metabolismo , Modelos Animais de DoençasRESUMO
As an opportunistic pathogen, Aeromonas veronii can cause hemorrhagic septicemia of various aquatic animals. In our present study, a dominant strain SJ4, isolated from naturally infected mandarin fish (Siniperca chuatsi), was identified as A. veronii according to the morphological, physiological, and biochemical features, as well as molecular identification. Intraperitoneal injection of A. veronii SJ4 into S. chuatsi revealed clinical signs similar to the natural infection, and the median lethal dosage (LD50) of the SJ4 to S. chuatsi in a week was 3.8 × 105 CFU/mL. Histopathological analysis revealed that the isolate SJ4 could cause cell enlargement, obvious hemorrhage, and inflammatory responses in S. chuatsi. Detection of virulence genes showed the isolate SJ4 carried act, fim, flgM, ompA, lip, hly, aer, and eprCAL, and the isolate SJ4 also produce caseinase, dnase, gelatinase, and hemolysin. In addition, the complete genome of A. veronii SJ4 was sequenced, and the size of the genome of A. veronii SJ4 was 4,562,694 bp, within a G + C content of 58.95%, containing 4079 coding genes. Nine hundred ten genes encoding for several virulence factors, such as type III and VI secretion systems, flagella, motility, etc., were determined based on the VFDB database. Besides, 148 antibiotic resistance-related genes in 27 categories related to tetracyclines, fluoroquinolones, aminoglycosides, macrolides, chloramphenicol, and cephalosporins were also annotated. The present results suggested that A. veronii was etiological agent causing the bacterial septicemia of S. chuatsi in this time, as well as provided a valuable base for revealing pathogenesis and resistance mechanism of A. veronii.
Assuntos
Doenças dos Peixes , Infecções por Bactérias Gram-Negativas , Animais , Aeromonas veronii/genética , Peixes , Virulência/genética , Fatores de Virulência/genética , Antibacterianos , Infecções por Bactérias Gram-Negativas/genética , Doenças dos Peixes/genéticaRESUMO
Hepcidin is a small peptide of defensins with antibacterial activity, and plays an important role in innate immunity against pathogenic microorganisms, which can also participate in the regulation of iron metabolism. The hepcidin gene in Yellow River carp (Cyprinus carpio haematopterus) (CcHep) was cloned and identified. The total length of CcHep cDNA was 480 bp, containing an open reading frame (ORF) that encoded 91 amino acids (aa), which contained a 24-aa signal peptide, a 42-aa propeptide, and a 25-aa mature peptide. The mature peptide had a typical RX (K/R) R motif and eight conserved cysteine residues forming four pairs of disulfide bonds. Homology and phylogenetic tree analysis showed that CcHep had the closest relationship with that of crucian carp. The expression levels of hepcidin mRNA in healthy and Aeromonas hydrophila stimulated fish were measured by real-time fluorescence quantitative PCR. The results showed that CcHep mRNA was expressed in different tissues of healthy fish with the highest relative expression level in liver, followed by kidney and intestine, and the lowest expression level was observed in heart. The hepcidin gene was extremely significantly up-regulated in head kidney, intestine, liver, skin, spleen, and gill at 6 h and 12 h after A. hydrophila infection. Furthermore, the immunoregulation effect of dietary recombinant protein was evaluated. The recombinant hepcidin protein (rCcHep) was successfully expressed by Pichia pastoris X-33 and showed strong antibacterial activity against A. hydrophila, Escherichia coli, Vibrio anguillarum and Bacillus subtilis in vitro. In order to evaluate the preventive effect of rCcHep, fish were fed with basal diet or diet supplemented with different doses of rCcHep, and then challenged with A. hydrophila. The results showed that immune genes were up-regulated to varying degrees, and feed additive groups exhibited a significantly improved up-regulation expressions of Lysozyme, Toll-like receptor 5 (TLR 5), Major histocompatibility complex classâ ¡ (MHCâ ¡), while inhibited up-regulation expressions of Interleukin 1ß (IL-1ß), Interleukin 8 (IL-8), and Tumor necrosis factor α (TNF-α) in liver and spleen compared to the control. Meanwhile, the relative immune protection rate in 120 mg/kg feed additive group was 28%, and the bacterial clearance rate in tissues of this group was higher than that of the control. Collectively, these results indicated that rCcHep had antibacterial activity and showed an immune protection effect against A. hydrophila, and could be considered as a dietary supplement to apply in aquaculture.
Assuntos
Carpas , Doenças dos Peixes , Infecções por Bactérias Gram-Negativas , Animais , Carpas/metabolismo , Aeromonas hydrophila/fisiologia , Hepcidinas/metabolismo , Filogenia , Infecções por Bactérias Gram-Negativas/prevenção & controle , Infecções por Bactérias Gram-Negativas/veterinária , Infecções por Bactérias Gram-Negativas/genética , Suplementos Nutricionais/análise , Imunidade Inata/genética , RNA Mensageiro/metabolismo , Proteínas de Peixes/químicaRESUMO
CD68 is a highly glycosylated transmembrane glycoprotein that belongs to the lysosome-associated membrane glycoprotein family and is involved in various immune processes. In this study, Megalobrama amblycephala CD68 (MaCD68) was cloned and characterized, and its expression patterns and evolutionary characteristics were analyzed. The coding region of MaCD68 was 987 bp, encoding 328 amino acids, and the predicted protein molecular weight was 34.9 kDa. MaCD68 contained two transmembrane helical structures and 18 predicted N-glycosylation sites. Multiple sequence alignments showed that the MaCD68 protein had high homology with other fish, and their functional sites were also highly conserved. Phylogenetic analysis revealed that MaCD68 and other cypriniformes fish clustered into one branch. Adaptive evolution analysis identified several positively selected sites of teleost CD68 using site and branch-site models, indicating that it was under positive selection pressure during evolution. Quantitative real-time reverse transcription polymerase chain reaction analysis showed that MaCD68 was highly expressed in the head kidney, spleen, and heart. After Aeromonas hydrophila infection, MaCD68 was significantly upregulated in all tested tissues, peaking at 12 h post-infection (hpi) in the kidney and head kidney and at 120 hpi in the liver and spleen, suggesting that MaCD68 participated in the innate immune response of the host against bacterial infection. Immunohistochemical and immunofluorescence analyses also showed that positive signals derived from the MaCD68 protein were further enhanced after bacterial and lipopolysaccharide treatment, which suggested that MaCD68 is involved in the immune response and could be used as a macrophage marker. Biological activity analysis indicated that recombinant MaCD68 (rMaCD68) protein had no agglutination or bactericidal effects on A. hydrophila but did have these effects on Escherichia coli. In conclusion, these results suggest that MaCD68 plays a vital role in the immune response against pathogens, which is helpful in understanding the immune responses and mechanisms of M. amblycephala.
Assuntos
Cyprinidae , Cipriniformes , Doenças dos Peixes , Infecções por Bactérias Gram-Negativas , Animais , Cyprinidae/genética , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/veterinária , Filogenia , Sequência de Aminoácidos , Clonagem Molecular , Sequência de Bases , Aeromonas hydrophila/genética , Cipriniformes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Peixes/metabolismoRESUMO
A growing number of studies identified long non-coding RNAs (lncRNAs) to be closely associated with immune function through the regulation of immune cell differentiation and immune cell effector function. Here we tested whether lncRNAs are involved in immune function in black carp (Mylopharyngodon piceus) through the exposure to Aeromonas hydrophila and analysis of the spleen gene expression response using RNA-seq. A total of 9036 lncRNAs were identified with high confidence. Differential expression analysis identified a total of 3558 DElncRNAs (Differential expression lncRNA) involved in A. hydrophila infection and 4526 target genes corresponding to DElncRNAs. After screening 4526 target genes in the InnateDB database, a total of 150 immunity genes were identified. After GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis of the obtained immunity genes, the Toll-like receptor (TLR) signaling pathway, TLR2, TLR3, TLR5, and TLR8 were identified as particularly significant in A. hydrophyla-resistant black carp. At the same time, the Ras signaling pathway was particularly enriched in the spleen of susceptible black carp. Analysis of PPI (protein-protein interaction) networks of the obtained immune genes identified SRC (SRC Proto-Oncogene), MYD88 (Myeloid differentiation primary response 88), MAPK3 (Mitogen-Activated Protein Kinase 3), MYC (MYC Proto-Oncogene) as main hub genes regulated by lncRNA and possibly mediating a mechanism of susceptibility to bacteria. These results establish a functional role of lncRNAs and a mechanistic base for the immune response in black carp resistant to A. hydrophila.
Assuntos
Carpas , Doenças dos Peixes , Infecções por Bactérias Gram-Negativas , RNA Longo não Codificante , Aeromonas hydrophila/fisiologia , Animais , Carpas/genética , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/veterinária , RNA Longo não Codificante/genéticaRESUMO
BACKGROUND: Aeromonas veronii is a Gram-negative rod-shaped motile bacterium that inhabits mainly freshwater environments. A. veronii is a pathogen of aquatic animals, causing diseases in fish. A. veronii is also an emerging human enteric pathogen, causing mainly gastroenteritis with various severities and also often being detected in patients with inflammatory bowel disease. Currently, limited information is available on the genomic information of A. veronii strains that cause human gastrointestinal diseases. Here we sequenced, assembled and analysed 25 genomes (one complete genome and 24 draft genomes) of A. veronii strains isolated from patients with gastrointestinal diseases using combine sequencing technologies from Illumina and Oxford Nanopore. We also conducted comparative analysis of genomes of 168 global A. veronii strains isolated from different sources. RESULTS: We found that most of the A. veronii strains isolated from patients with gastrointestinal diseases were closely related to each other, and the remaining were closely related to strains from other sources. Nearly 300 putative virulence factors were identified. Aerolysin, microbial collagenase and multiple hemolysins were present in all strains isolated from patients with gastrointestinal diseases. Type III Secretory System (T3SS) in A. veronii was in AVI-1 genomic island identified in this study, most likely acquired via horizontal transfer from other Aeromonas species. T3SS was significantly less present in A. veronii strains isolated from patients with gastrointestinal diseases as compared to strains isolated from fish and domestic animals. CONCLUSIONS: This study provides novel information on source of infection and virulence of A. veronii in human gastrointestinal diseases.
Assuntos
Aeromonas veronii , Gastroenteropatias , Genoma Bacteriano , Infecções por Bactérias Gram-Negativas , Aeromonas veronii/genética , Aeromonas veronii/patogenicidade , Animais , Doenças dos Peixes/microbiologia , Gastroenteropatias/genética , Gastroenteropatias/microbiologia , Infecções por Bactérias Gram-Negativas/genética , Humanos , Virulência/genéticaRESUMO
BACKGROUND: The host response to bacterial sepsis is reported to be nonspecific regardless of the causative pathogen. However, newer paradigms indicated that the host response of Gram-negative sepsis may be different from Gram-positive sepsis, and the difference has not been clearly clarified. The current study aimed to explore the difference by identifying the differential gene sets using the genome-wide technique. METHODS: The training dataset GSE6535 and the validation dataset GSE13015 were used for bioinformatics analysis. The distinct gene sets of sepsis with different infections were screened using gene set variation analysis (GSVA) and gene set enrichment analysis (GSEA). The intersection gene sets based on the two algorithms were confirmed through Venn analysis. Finally, the common gene sets between GSE6535 and GSE13015 were determined by GSEA. RESULTS: Two immunological gene sets in GSE6535 were identified based on GSVA, which could be used to discriminate sepsis caused by Gram-positive, Gram-negative, or mixed infection. A total of 19 gene sets were obtained in GSE6535 through Venn analysis based on GSVA and GSEA, which revealed the heterogeneity of Gram-negative and Gram-positive sepsis at the molecular level. The result was also verified by analysis of the validation set GSE13015, and 40 common differential gene sets were identified between dataset GSE13015 and dataset GSE6535 by GSEA. CONCLUSIONS: The identified differential gene sets indicated that host response may differ dramatically depending on the inciting organism. The findings offer new insight to investigate the pathophysiology of bacterial sepsis.
Assuntos
Infecções por Bactérias Gram-Negativas , Infecções por Bactérias Gram-Positivas , Sepse , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Infecções por Bactérias Gram-Negativas/genética , Humanos , Sepse/genéticaRESUMO
BACKGROUND: Genome-wide expression profiles have been previously employed as clinical research diagnostic tools for newborn sepsis. We aimed to determine if transcriptomic profiles could discriminate between Gram-positive and Gram-negative bacterial sepsis in preterm infants. METHODS: Prospective, observational, double-cohort study was conducted in very low birth weight infants with clinical signs and culture-positive sepsis. Blood samples were collected when clinical signs became apparent. Total RNA was processed for transcriptomic analysis. Results were validated by both reverse-transcription polymerase chain reaction and a mathematical model. RESULTS: We included 25 septic preterm infants, 17 with Gram-positive and 8 with Gram-negative bacteria. The principal component analysis identified these two clusters of patients. We performed a predictive model based on 21 genes that showed an area under the receiver-operating characteristic curve of 1. Eight genes were overexpressed in Gram-positive septic infants: CD37, CSK, MAN2B2, MGAT1, MOB3A, MYO9B, SH2D3C, and TEP1. The most significantly overexpressed pathways were related to metabolic and immunomodulating responses that translated into an equilibrium between pro- and anti-inflammatory responses. CONCLUSIONS: The transcriptomic profile allowed identification of whether the causative agent was Gram-positive or Gram-negative bacteria. The overexpression of genes such as CD37 and CSK, which control cytokine production and cell survival, could explain the better clinical outcome in sepsis caused by Gram-positive bacteria. IMPACT: Transcriptomic profiles not only enable an early diagnosis of sepsis in very low birth weight infants but also discriminate between Gram-positive and Gram-negative bacteria as causative agents. The overexpression of some genes related to cytokine production and cell survival could explain the better clinical outcome in sepsis caused by Gram-positive bacteria, and could lead us to a future, targeted therapy.
Assuntos
Bacteriemia , Infecções por Bactérias Gram-Negativas , Sepse , Antibacterianos/uso terapêutico , Bacteriemia/microbiologia , Estudos de Coortes , Citocinas/genética , Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/diagnóstico , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/microbiologia , Bactérias Gram-Positivas/genética , Humanos , Lactente , Recém-Nascido , Recém-Nascido Prematuro , Estudos Prospectivos , Sepse/diagnóstico , Sepse/genética , TranscriptomaRESUMO
Linker histone H1 (LHH1) is an abundant nuclear protein that condenses chromatin to form higher-order structure. The present study reported cloning and sequencing of 942 bp of LHH1 from liver tissue of rohu, Labeo rohita, with a complete coding sequence of 792 bp of having 263 amino acids. The phylogenetic tree of L. rohita LHH1 (LrLHH1) shared maximum similarity with that of Carassius auratus. The three dimensional model and domain architecture of LrLHH1 protein was also predicted using Swiss-Prot and SMART domain software. The expression of LHH1 during ontogeny showed significantly higher transcript level in milt, unfertilized eggs and up to 3 h post-fertilization followed by a dramatic decrease thereafter. The tissue-specific expression showed constitutive expression of LrLHH1 in all examined tissues. The expression of LHH1 during different infection models, namely, bacteria (Aeromonas hydrophila); ectoparasite (Argulus siamensis) and poly I:C induction revealed modulation in the level of expression at varied time points post-exposure in the liver and anterior kidney tissues of rohu. However, a synthetic peptide derived from LHH1 sequence of rohu did not have any detectable antibacterial activity. The present study provided necessary information on the role of this protein during ontogeny and innate immunity in Indian major carp species.
Assuntos
Cyprinidae , Infecções por Bactérias Gram-Negativas , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cyprinidae/genética , Cyprinidae/metabolismo , Proteínas de Peixes/química , Proteínas de Peixes/genética , Perfilação da Expressão Gênica , Infecções por Bactérias Gram-Negativas/genética , Histonas/genética , FilogeniaRESUMO
Probiotic administration is a potential strategy against enteric pathogen infection in either clinical treatment or animal nutrition industry, but the administration duration of probiotics varied and the underlying mechanisms remain unclear. A strain (YC) affiliated to Pediococcus pentosaceus, a commonly used probiotic, was isolated from fish gut and the potential role of YC against Aeromonas hydrophila was detected in zebrafish. We found that 3- or 4-week YC administration (YC3W or YC4W) increased the resistance against A. hydrophila while 1- or 2-week treatment (YC1W or YC2W) did not. To determine the possible reason, intestinal microbiota analysis and RNAseq were conducted. The results showed that compared with CON and YC1W, YC4W significantly increased the abundance of short-chain fatty acids (SCFAs) producing bacteria and elevated the gene expression of nlrp3. Higher butyrate content and enhanced expression of IL1ß were subsequently found in YC4W. To identify the causal relationship between butyrate and the higher pathogen resistance, different concentrations of sodium butyrate (SB) were supplemented. The results suggested that 10 mmol/kg SB addition mirrored the protective effect of YC4W by increasing the production of IL-1ß. Furthermore, the increased IL-1ß raised the percentage of intestinal neutrophils, which endued the zebrafish with A. hydrophila resistance. In vivo knockdown of intestinal il1b eliminated the anti-infection effect. Collectively, our data suggested that the molecular mechanism of probiotics determined the administration duration, which is vital for the efficiency of probiotics. Promoting host inflammation by probiotic pretreatment is one potential way for probiotics to provide their protective effects against pathogens.
Assuntos
Aeromonas hydrophila , Resistência à Doença , Doenças dos Peixes , Infecções por Bactérias Gram-Negativas , Pediococcus pentosaceus , Probióticos , Animais , Ácidos Graxos Voláteis/metabolismo , Feminino , Doenças dos Peixes/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/metabolismo , Doenças dos Peixes/microbiologia , Microbioma Gastrointestinal , Expressão Gênica , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/metabolismo , Infecções por Bactérias Gram-Negativas/microbiologia , Interleucina-1beta/imunologia , Intestinos/imunologia , Intestinos/microbiologia , Masculino , Neutrófilos/imunologia , Peixe-ZebraRESUMO
Microbial challenges, such as widespread bacterial infection in sepsis, induce endotoxin tolerance, a state of hyporesponsiveness to subsequent infections. The participation of DNA methylation in this process is poorly known. In this study, we perform integrated analysis of DNA methylation and transcriptional changes following in vitro exposure to gram-negative bacterial lipopolysaccharide, together with analysis of ex vivo monocytes from septic patients. We identify TET2-mediated demethylation and transcriptional activation of inflammation-related genes that is specific to toll-like receptor stimulation. Changes also involve phosphorylation of STAT1, STAT3 and STAT5, elements of the JAK2 pathway. JAK2 pathway inhibition impairs the activation of tolerized genes on the first encounter with lipopolysaccharide. We then confirm the implication of the JAK2-STAT pathway in the aberrant DNA methylome of patients with sepsis caused by gram-negative bacteria. Finally, JAK2 inhibition in monocytes partially recapitulates the expression changes produced in the immunosuppressive cellular state acquired by monocytes from gram-negative sepsis, as described by single cell-RNA-sequencing. Our study evidences both the crucial role the JAK2-STAT pathway in epigenetic regulation and initial response of the tolerized genes to gram-negative bacterial endotoxins and provides a pharmacological target to prevent exacerbated responses.
Assuntos
Tolerância à Endotoxina/genética , Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/imunologia , Monócitos/imunologia , Monócitos/microbiologia , Sepse/genética , Sepse/imunologia , Estudos de Casos e Controles , Metilação de DNA/genética , Metilação de DNA/imunologia , Tolerância à Endotoxina/efeitos dos fármacos , Tolerância à Endotoxina/imunologia , Endotoxinas/toxicidade , Epigênese Genética , Feminino , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Técnicas In Vitro , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Janus Quinase 2/imunologia , Lipopolissacarídeos/toxicidade , Masculino , Monócitos/efeitos dos fármacos , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/imunologia , Sepse/microbiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologiaRESUMO
The current treatments applied in aquaculture to limit disease dissemination are mostly based on the use of antibiotics, either as prophylactic or therapeutic agents, with vaccines being available for a limited number of fish species and pathogens. Antimicrobial peptides are considered as promising novel substances to be used in aquaculture, due to their antimicrobial and immunomodulatory activities. Hepcidin, the major iron metabolism regulator, is found as a single gene in most mammals, but in certain fish species, including the European sea bass (Dicentrarchus labrax), two different hepcidin types are found, with specialized roles: the single type 1 hepcidin is involved in iron homeostasis trough the regulation of ferroportin, the only known iron exporter; and the various type 2 hepcidins present antimicrobial activity against a number of different pathogens. In this study, we tested the administration of sea bass derived hepcidins in models of infection and iron overload. Administration with hamp2 substantially reduced fish mortalities and bacterial loads, presenting itself as a viable alternative to the use of antibiotics. On the other hand, hamp1 seems to attenuate the effects of iron overload. Further studies are necessary to test the potential protective effects of hamp2 against other pathogens, as well as to understand how hamp2 stimulate the inflammatory responses, leading to an increased fish survival upon infection.
Assuntos
Peptídeos Antimicrobianos/uso terapêutico , Bass/imunologia , Doenças dos Peixes/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/veterinária , Hepcidinas/uso terapêutico , Sobrecarga de Ferro/veterinária , Photobacterium , Sequência de Aminoácidos , Animais , Apoferritinas/biossíntese , Apoferritinas/genética , Carga Bacteriana , Bass/microbiologia , Proteínas de Transporte de Cátions/biossíntese , Proteínas de Transporte de Cátions/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Perfilação da Expressão Gênica , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/imunologia , Hepcidinas/biossíntese , Hepcidinas/genética , Ferro/análise , Sobrecarga de Ferro/tratamento farmacológico , Sobrecarga de Ferro/genética , Sobrecarga de Ferro/imunologia , Fígado/química , Photobacterium/isolamento & purificaçãoRESUMO
Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis, has extensive negative effects on wild and farmed salmonids worldwide. Vaccination induces some protection under certain conditions but disease outbreaks occur even in vaccinated fish. Therefore, alternative disease control approaches are required to ensure the sustainable expansion of rainbow trout aquaculture. Selective breeding can be applied to enhance host resistance to pathogens. The present work used genome-wide association study (GWAS) to identify quantitative trait loci (QTL) associated with A. salmonicida resistance in rainbow trout. A total 798 rainbow trout exposed to A. salmonicida by bath challenge revealed 614 susceptible and 138 resistant fish. Genotyping was conducted using the 57 K single nucleotide polymorphism (SNP) array and the GWAS was performed for survival and time to death phenotypes. We identified a QTL on chromosome 16 and located positional candidate genes in the proximity of the most significant SNPs. In addition, samples from exposed fish were examined for expression of 24 immune-relevant genes indicating a systematic immune response to the infection. The present work demonstrated that resistance to A. salmonicida is moderately heritable with oligogenic architecture. These result will be useful for the future breeding programs for improving the natural resistance of rainbow trout against furunculosis.
Assuntos
Aeromonas salmonicida , Resistência à Doença/genética , Doenças dos Peixes/microbiologia , Furunculose/microbiologia , Infecções por Bactérias Gram-Negativas/genética , Oncorhynchus mykiss/microbiologia , Locos de Características Quantitativas , Animais , Doenças dos Peixes/genética , Furunculose/genética , Estudo de Associação Genômica Ampla , Oncorhynchus mykiss/genéticaRESUMO
Chemokines are crucial regulators of cell mobilization for development, homeostasis, and immunity. Chemokines signal through binding to chemokine receptors, a superfamily of seven-transmembrane domain G-coupled receptors. In the present study, seventeen CXC chemokine ligands (SsCXCLs) and nine CXC chemokine receptors (SsCXCRs) were systematically identified from Sebastes schlegelii genome. Phylogeny, synteny, and evolutionary analyses were performed to annotate these genes, indicating that the tandem duplications (CXCL8, CXCL11, CXCL32, CXCR2, and CXCR3), the whole genome duplications (CXCL8, CXCL12, CXCL18, and CXCR4), and the teleost-specific members (CXCL18, CXCL19, and CXCL32) led to the expansion of SsCXCLs and SsCXCRs. In addition, SsCXCLs and SsCXCRs were ubiquitously expressed in nine examined healthy tissues, with high expression levels observed in head kidney, liver, gill and spleen. Moreover, most SsCXCLs and SsCXCRs were significantly differentially expressed in head kidney, liver, and gill after Aeromonas salmonicida infection, and exhibited tissue-specific and time-dependent manner. Finally, protein-protein interaction network (PPI) analysis indicated that SsCXCLs and SsCXCRs interacted with a few immune-related genes such as interleukins, cathepsins, CD genes, and TLRs, etc. These results should be valuable for comparative immunological studies and provide insights for further functional characterization of chemokines and receptors in teleost.
Assuntos
Aeromonas salmonicida/patogenicidade , Quimiocinas CXC/genética , Proteínas de Peixes/genética , Furunculose/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Imunidade Inata/genética , Perciformes/microbiologia , Receptores de Quimiocinas/genética , Aeromonas salmonicida/imunologia , Animais , Bases de Dados Genéticas , Furunculose/genética , Furunculose/imunologia , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/microbiologia , Interações Hospedeiro-Patógeno , Perciformes/genética , Perciformes/imunologia , Filogenia , Mapas de Interação de Proteínas , Transdução de Sinais , Fatores de TempoRESUMO
Lipopolysaccharide (LPS) is a component of the cell wall of Gram-negative bacteria, and triggers an inflammatory response both in vitro and in vivo. Here, we used LPS from Escherichia coli serotype enteritidis to stimulate chicken macrophages (HD11) and conducted the transcriptome analysis using a bioinformatics approach to explore the functions of immune-related genes and miRNAs. In total, 1759 differentially expressed genes (DEGs) and 18 differentially expressed (DE)-miRNAs were detected during LPS infection. At 6 h post infection, 1025 DEGs and 10 miRNAs were up-regulated, and 734 DEGs and 8 DE-miRNAs were down-regulated. Based on both RNA hybrid and miRanda systems, 55 DEGs could be targeted by 14 DE-miRNAs. The target genes were related to the immune response, such as IRF8, STAT3, TRAF7, and other potential candidate genes. The DE-miRNAs miR146a-3p, miR6583-5p, and miR30c-2-3p were investigated further. They were predicted to target 34 genes that may also be candidates for immune-related miRNAs and genes. Our results enhanced our understanding of the pathogenic mechanisms of Gram-negative bacteria in chickens.
Assuntos
Infecções por Bactérias Gram-Negativas/metabolismo , Macrófagos/metabolismo , MicroRNAs/genética , RNA Mensageiro/genética , Transcriptoma , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Linhagem Celular , Galinhas , Infecções por Bactérias Gram-Negativas/genética , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Circular RNAs (circRNAs), a class of non-coding RNAs, play an important role in regulating various biological processes. In the present study, circRNAs from the Megalobrama amblycephala liver were identified at five different time points post Aeromonas hydrophila using RNA-seq technology. A total of 250 circRNAs were identified, of which 106 were differentially expressed (DE) in ten pairwise comparisons. GO and KEGG analyses showed that the parental genes of DE circRNAs were enriched in phagocytosis, complement and coagulation cascades, and Fc gamma R-mediated phagocytosis pathways. According to ceRNA hypothesis, the interaction network of circRNAs, miRNAs and mRNAs was constructed. Moreover, WGCNA was conducted, and five specific modules significantly related to bacterial infection were identified. All the above results reveal the important role of circRNAs in immune response, which enriches the information of circRNAs in teleost, and helps to understand the immune response mechanism of M. amblycephala to A. hydrophila.
Assuntos
Aeromonas hydrophila/fisiologia , Cipriniformes/imunologia , Doenças dos Peixes/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Fígado/imunologia , RNA Circular/imunologia , Animais , Cipriniformes/genética , Cipriniformes/microbiologia , Doenças dos Peixes/genética , Doenças dos Peixes/microbiologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Expressão Gênica , Redes Reguladoras de Genes/imunologia , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/microbiologia , MicroRNAs/genética , RNA Circular/genética , RNA Mensageiro/genéticaRESUMO
NK-lysin, an effector of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), not only exhibits cytotoxic effect in fish cells, but also participates in the immune defense against pathogenic infection. In this study, ORF sequences of RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin were 369 bp. Tissue-specific analysis revealed that the highest expressions of RCC-NK-lysin and WCC-NK-lysin were observed in gill, while the peaked level of WR-NK-lysin mRNA was observed in spleen. A. hydrophila infection sharply increased RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin mRNA expression in liver, trunk kidney and spleen. In addition, elevated levels of NK-lysin mRNA were observed in cultured fin cell lines of red crucian carp (RCC), white crucian carp (WCC) and their hybrid offspring (WR) after Lipopolysaccharide (LPS) challenge. RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin exerted regulatory roles in inducing ROS generation, modulating mitochondrial membrane potential, decreasing fish cell viability and antagonizing survival signalings, respectively. RCC/WCC/WR-NK-lysin-overexpressing fish could up-regulate expressions of inflammatory cytokines and decrease bacterial loads in spleen. These results indicated that NK-lysin in hybrid fish contained close sequence similarity to those of its parents, possessing the capacities of cytotoxicity and immune defense against bacterial infection.