Transcriptomic responses of water buffalo liver to infection with the digenetic fluke Fasciola gigantica.
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ABSTRACT: Fasciola gigantica, the tropical liver fluke, infects buffaloes in Asian and African countries and causes significant economic losses and poses public health threat in these countries. However, little is known of the transcriptional response of buffaloes to infection with F. gigantica. The objective of the present study was to perform the first transcriptomic analysis of buffalo liver infected by F. gigantica. Understanding the mechanisms that underpin F. gigantica infection in buffaloes will contribute to our ability to control this parasite.We challenged buffaloes with 500 viable F. gigantica metacercariae and collected liver samples through a time course at 3, 42 and 70 days post-infection (dpi). Then, we performed gene expression analysis on liver samples using RNA sequencing (RNA-Seq) Illumina technology and confirmed the RNA-Seq data by quantitative RT-PCR analysis.Totals of 496, 880 and 441 differentially expressed transcripts were identified in the infected livers at 3, 42 and 70 dpi, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that transcriptional changes in the liver of infected buffaloes evolve over the course of infection. The predominant response of buffaloes to infection was mediated by certain pathways, such as MHC antigen processing and presentation, Toll-like receptor 4 (TLR4), transforming growth factor beta (TGF-?), and the cytochrome P450. Hepatic drug metabolizing enzymes and bile secretion were also affected.Fasciola gigantica can induce statistically significant and biologically plausible differences in the hepatic gene expression of infected buffaloes. These findings provide new insights into the response of buffaloes to F. gigantica over the course of infection, which may be useful in determining pathways that can modulate host-parasite interaction and thus potentially important for clearance of the parasite.
<h4>Background</h4>Fasciola gigantica, the tropical liver fluke, infects buffaloes in Asian and African countries and causes significant economic losses and poses public health threat in these countries. However, little is known of the transcriptional response of buffaloes to infection with F. gigantica. The objective of the present study was to perform the first transcriptomic analysis of buffalo liver infected by F. gigantica. Understanding the mechanisms that underpin F. gigantica infection i ...[more]
Project description:Fascioliasis, a neglected foodborne disease caused by liver flukes (genus Fasciola), affects more than 200 million people worldwide. Despite technological advances, little is known about the molecular biology and biochemistry of these flukes. We present the draft genome of Fasciola gigantica for the first time. The assembled draft genome has a size of ∼1.04 Gb with an N50 and N90 of 129 and 149 kb, respectively. A total of 20 858 genes were predicted. The de novo repeats identified in the draft genome were 46.85%. The pathway included all of the genes of glycolysis, Krebs cycle, and fatty acid metabolism but lacked the key genes of the fatty acid biosynthesis pathway. This indicates that the fatty acid required for survival of the fluke may be acquired from the host bile. It may be hypothesized that the relatively larger F. gigantica genome did not evolve through genome duplications but rather is interspersed with many repetitive elements. The genomic information will provide a comprehensive resource to facilitate the development of novel interventions for fascioliasis control.
Project description:Fasciola gigantica, responsible for the zoonotic disease fasciolosis, pose a great threat to the livestock and human health worldwide. The triclabendazole (TCBZ) has been used for decades as a broad spectrum anthelmintic to control this perilous disease but the emergence of resistance in flukes against TCBZ has prompted researchers across the world to explore for new drugs and antigenic targets. World Health Organization has strongly recommended the utilization of neurobiologically significant biomolecules as new drug/antigenic targets because of their significant role in the physiology of parasites. Monoamine Oxidase (MAO) is an important neurobiological enzyme which catabolizes aminergic neurotransmitters thus preventing prolonged excitation of neurons and in non-neuronal cells it prevents cellular toxicity due to accumulation of toxic monoamines. Owing to the important role of MAO in the survival and perpetuation of parasites, multipronged approaches were undertaken for the characterization of MAO-A in F. gigantica. The activity of MAO was found to be 1.5 times higher in the mitochondrial samples than the whole homogenate samples. The adult worms of the F. gigantica appeared to possess both the isoforms of MAO i.e., MAO-A and MAO-B. The zymographic studies revealed strong enzyme activity in its native state as assessed through prominent dark bands at 250KDa in the zymogram. The enzyme was also found to be highly immunogenic as revealed by high antibody titer at 1:6400 dilution. The immunogenicity of MAO-A enzyme was further established in the Western Blots in which a strong band of 50KDa was distinctly evident. Despite ubiquitous presence of MAO in F. gigantica some regions like tegumental surface and intestinal caecae displayed strong immunofluorescence as compared to other regions. The detection of MAO-A in the F. gigantica samples in Dot-Blot assay indicate a great potential of this molecule for the immunodiagnostics of fasciolosis, particularly in the field conditions. The enzyme activity was sensitive to the specific inhibitor clorgyline in a concentration dependant manner, particularly in the late incubation period. The zymographic results also exhibited similar trend. The strong intensity of spots in Dot-blots indicate high immunogenicity of the MAO protein. The intensity of bands/spots in the samples of worms treated with clorgyline also declined, clearly indicating that the tropical liver fluke possesses prominent MAO-A activity.
Project description:The liver fluke zoonoses, Fasciola spp. are parasitic helminths infecting humans and animals globally. Recent sequencing of the genome of Fasciola gigantica has provided a basis to understand the biochemistry of this parasite. Here, we identified the cytosolic malate dehydrogenase in F. gigantica (FgMDH) and characterized the enzyme biochemically and structurally. F. gigantica encodes a single cytosolic MDH, a key enzyme of the citric acid cycle. It catalyzes the reversible oxidation of malate to oxaloacetate using NAD+. The Fgmdh gene was amplified and cloned for expression of the recombinant protein. The purified protein showed a molecular weight of ~ 36 kDa that existed in a dimeric form in solution. The recombinant enzyme was catalytically active as it catalyzed both forward and reverse reactions efficiently. The kinetic parameters were determined for both directions. The structure of FgMDH and human MDH were modeled and validated. The superimposition of both the model structures showed overall structural similarity in the active site loop region, however, the conformation of the residues was different. Molecular docking elucidated the binding sites and affinities of the substrates and cofactors to the enzyme. Simulation of molecular dynamics and principal component analysis indicated the stability of the systems and collective motions, respectively. Understanding the structural and functional properties of MDH is important to better understand the roles of this enzyme in the biochemistry of the parasite.
Project description:The tropical liver fluke Fasciola gigantica is a parasitic helminth that has been frequently reported to infect mammals, typically involving water buffaloes. In this study, we characterized the tissue transcriptional landscape of buffaloes following infection by F. gigantica. RNAs were isolated from hepatic lymph nodes (hLNs), peripheral blood lymphocytes (pBLs), and spleen at 3-, 42- and 70-days post-infection (dpi), and all samples were subjected to RNA sequencing analyses. At 3 dpi, 2603, 460, and 162 differentially expressed transcripts (DETs) were detected in hLNs, pBLs, and spleen, respectively. At 42 dpi, 322, 937, and 196 DETs were detected in hLNs, pBLs, and spleen, respectively. At 70 dpi, 376, 334, and 165 DETs were detected in hLNs, pBLs, and spleen, respectively. Functional enrichment analysis identified upregulated immune-related pathways in the infected tissues involved in innate and adaptive immune responses, especially in hLNs at 42 and 70 dpi, and pBLs at 3 and 42 dpi. The upregulated transcripts in spleen were not enriched in any immune-related pathway. Co-expression network analysis further identified transcriptional changes associated with immune response to F. gigantica infection. Receiver operating characteristic (ROC) curve analysis showed that 107 genes in hLNs, 32 genes in pBLs, and 36 genes in spleen correlated with F. gigantica load. These findings provide new insight into molecular mechanisms and signaling pathways associated with F. gigantica infection in buffaloes.
Project description:BackgroundThe liver fluke Fasciola gigantica modulates several signaling pathways in infected buffaloes to facilitate its survival and establishment of persistent infection. In response to the parasite invasion, buffaloes activate innate and adaptive immune responses to counter the parasite infection. To detect new proteins that might be involved in the interaction between F. gigantica and the buffaloes, and that also might serve as biomarkers for fasciolosis, we used proteomic techniques to study the serum proteome of buffaloes during F. gigantica infection. Here, we used an isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic approach to identify serum proteins that are differentially expressed in infected buffaloes compared to uninfected control buffaloes. Additionally, we applied a parallel reaction monitoring (PRM) assay to validate specific proteins identified by the iTRAQ method.ResultsA total of 313, 459 and 399 proteins were identified at 3, 42 and 70 days post-infection, respectively; of these 92, 93 and 138 were differentially abundant proteins. Some of the identified differentially abundant proteins, including complement factor H related 5, complement component C6, complement component C7, amine oxidase, plasma serine protease inhibitor and lysozyme, are known to be involved in complement system activation, blood coagulation, platelet activation, lymphocyte's adhesion and lysozyme hydrolysis. Analysis of data for all three time points after infection identified six significantly upregulated proteins in infected serum that separated infected and uninfected buffaloes into distinct clusters. Further PRM analysis confirmed the expression of five proteins, namely MHC class I antigen, Beta-2-microglobulin, NID2 protein, Fetuin-B and Fibrinogen gamma-B chain.ConclusionsThese findings provide novel insights into the serum proteomics signature of buffaloes during F. gigantica infection.
Project description:BackgroundFasciola hepatica is not only responsible for major economic losses in livestock farming, but is also a major food-borne zoonotic agent, with 180 million people being at risk of infection worldwide. This parasite is sophisticated in manipulating the hosts' immune system to benefit its own survival. A better understanding of the mechanisms underpinning this immunomodulation is crucial for the development of control strategies such as vaccines.Methodology/principal findingsThis in vivo study investigated the global gene expression changes of ovine peripheral blood mononuclear cells (PBMC) response to both acute & chronic infection of F. hepatica, and revealed 6490 and 2364 differential expressed genes (DEGS), respectively. Several transcriptional regulators were predicted to be significantly inhibited (e.g. IL12 and IL18) or activated (e.g. miR155-5p) in PBMC during infection. Ingenuity Pathway Analysis highlighted a series of immune-associated pathways involved in the response to infection, including 'Transforming Growth Factor Beta (TGFβ) signaling', 'Production of Nitric Oxide in Macrophages', 'Toll-like Receptor (TLRs) Signaling', 'Death Receptor Signaling' and 'IL17 Signaling'. We hypothesize that activation of pathways relevant to fibrosis in ovine chronic infection, may differ from those seen in cattle. Potential mechanisms behind immunomodulation in F. hepatica infection are a discussed.SignificanceIn conclusion, the present study performed global transcriptomic analysis of ovine PBMC, the primary innate/adaptive immune cells, in response to infection with F. hepatica, using deep-sequencing (RNAseq). This dataset provides novel information pertinent to understanding of the pathological processes in fasciolosis, as well as a base from which to further refine development of vaccines.
