| Paragonimiasis is a foodborne zoonosis caused by Paragonimus.Over 23 million people have suffered from paragonimiasis in the past few decades,and over 294 million people are at risk of infection.Humans become infected via consumption of crustaceans,such as raw or undercooked crabs or crayfish.Paragonimiasis causes significant harm to humans,seriously affects their quality of life and can even cause death,yet it is easily misdiagnosed as tuberculosis or a tumor.Therefore,it is considered as a Neglected Tropical Disease by the World Health Organization and is one of the most important public health issues in the world.More than 50 species of Paragonimus have been reported,and 49 species were registered with the National Center for Biotechnology Information(NCBI)Taxonomy Database.P.proliferus infections cause pulmonary injury in the host.However,its mechanisms of parasitism and pathogenicity are still unclear up to now.Through dual transcriptome sequencing of both parasite and host,bioinformatics analysis and a series of molecular biology techniques,this in-depth study assessed how P.proliferus successfully parasitizes rats and causes paragonimiasis as well as the mechanisms by which this occurs.This work provides a theoretical and experimental basis for the prevention and control of paragonimiasis.Part 1:Dynamic expression patterns of Paragonimus proliferus transcriptomes reveal mechanisms of its successful parasitismObjective:To explore the mechanisms by which P.proliferus successfully parasitizes rats,we analyzed the dynamic expression patterns of the transcriptomes of different developmental stages.Methods:Worms of P.proliferus at different developmental stages were collected.The transcriptomes of P.proliferus at four developmental stages,namely 0,14,28 and 70 d post-infection(PI),worms of 0 d were metacercariae,were sequenced using RNA-seq,and NOIseq was used to screen differentially expressed genes(DEGs).After Gene Ontology(GO)annotation,characteristics related to growth or development,metabolism and immunity were identified using hierarchical clustering(HCL),principal component analysis(PCA)and k-means clustering.These results were used to comprehensively examine the parasitic mechanisms of P.proliferus.Results1.Worms of P.proliferus reached to rat lungs at 7 d PI penetrated into the lung tissues at 14 d PI.Changes of expressions mainly occurred before 28 d PI,especially before 14 d PI.2.Gene expression patterns relating to growth or development were assigned to two categories.One category included genes that were highly expressed from 0 to 28 d but declined to their lowest levels on 70 d and included terms such as skin development.Genes in the second category were expressed at the highest levels on 0 and 14 d,maintained expression levels on 28 d and declined to their lowest levels on 70 d and included terms such as reproductive system and respiratory system development.The expression of catabolism-related and reduction-oxidation(REDOX)genes was highest on 0 and 14 d,maintained expression levels on 28 d and decreased until reaching their lowest levels on 70 d.Catabolism-related activities of P.proliferus cover an extremely wide range.At least 46 stimulus responses differed significantly among the different developmental stages.Responses in the first category were highly expressed only in 0 d and were related mainly to chemical stimulation.Responses in the second category were highly expressed on 0 and 14 d and were mainly involved in stress stimulation,such as the heat response,the cellular response to glucose starvation and removal of superoxide radicals.Responses in the third category were highly expressed on 0 and 28 d,and were mainly involved the cellular response to lipopolysaccharide and the response to hypoxia.At 14 d PI,accompanied by an increased immune response(such as antibacterial humoral response,antigen presentation interferon signaling、chemotaxis and migration of immune cells),genes related to bacterial detection were highly expressed on 14 and 70 d.Genes highly expressed at 70 d were also related to immune response.Conclusions1.Comprehensive adaptive responses rapidly occur at the transcriptome level PI of P.proliferus,and process of growth and development was initiated;P.proliferus has a strong ability of catabolism,REDOX which met the need of energy during its biological behavior,such as growth and development,migration and ovulation.Meanwhile,this species has a strong ability of response to stimulus to make sure that it can survive safely.2.Oxygen retrieval may be one of the mechanisms by which P.proliferus targets the lungs.A state of symbiosis between P.proliferus and bacteria may be essential for P.proliferus to successfully parasitize the host.Part 2:Study of possible molecular mechanisms of paragonimiasis caused by Paragonimus proliferus based on dynamic expression patterns of the transcriptomes of rat lung tissueObjective:To explore possible mechanisms of paragonimiasis caused by P.proliferus infection by examining the dynamic expression patterns of the transcriptomes of rat lung tissue at different stages of infection.Methods:Using Sprague-Dawley(SD)rats,an animal model of P.proliferus infection was established.Transcriptome sequencing was performed on the lung tissue of rats either uninfected(0 d)or infected with P.proliferus(3,7,14,28 and 70 d)using RNA-seq.NOIseq was used to screen DEGs.HCL and k-means clustering were performed after GO and Kyoto Encyclopedia of Genes and Genomes(KEGG)annotation.The functions and dynamic expression patterns of DEGs at different stages of infection were comprehensively analyzed to explore the possible mechanisms of paragonimiasis caused by P.proliferus infection.Then,for the screened key genes or signaling pathway,a series of experimental techniques of molecular biology,such as quantitative real time polymerase chain reaction(q-PCR),western blotting and immunohistochemistry(IHC),were used to deeply explore the molecular mechanism.Results1.There is mild inflammation in the lung tissue on 7 d,and 14～28 d reached to the most serious stage when some rats died,however,it was partially improved after 28 d PI.DEGs of rat lungs The process of P.proliferus infected was divided into three categories:the first one included those of 3 d and 7 d,14 d belonged to a separate category,and 28 d and 70 d were assigned to the third category.2.A number of DEGs related to energy metabolism were highly expressed before P.proliferus infection(0 d)as comprehensively lower-regulated during the early stage of infection(3 d).Highly expressed genes on 7 d were associated mainly with the viral defense response whereas highly expressed genes on 14～28 d were related to the innate immune response and the cytokine response.Highly expressed genes on 70 d were involved in the inflammatory response related to T and B cells.3.In the early stage of P.proliferus infection,expressions of mRNA and corresponding proteins of retinoic-acid-inducible gene I(RIG-I),interferon beta promoter stimulator 1(IPS-1),tumor necrosis factor receptor-associated factor 6(TRAF6),interferon regulatory factor 3(IRF3)and chemokine C-X-C motif ligand 10(CXCL10)were upregulated rapidly thereafter downregulated,expression patterns of which were in line with the levels of type helper T cells(Th)1 cytokines,such as interleukin(IL)-1,interferon y(IFN-y)and tumor necrosis factor α(TNF-α).Conclusions1.The process of P.proliferus infection can be divided into three stages:early-(before 14 d),mid-(14～28 d)and late-stage(after 28 d)infection.P.proliferus limites host metabolism which is conducive to intake energy and nutrients from host,during the early-stage of infection.A wide range of innate immune response and the cytokine response are initiated in host and cause severe immune or inflammatory injuries in the mid-stage when the severeness of paragonimiasis reaches to a peak.And in the late-stage which is also a chronization stage,immune response related to T and B cells causes a persistence of immune or inflammatory damage.2.Activation of host RIG-I/IPS-1 signaling,which is related to Pattern Recognition Receptor(PRR),induces the initiation of immune or inflammatory response after P.proliferus infection. |
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