Volume 41 Issue 4
Jul.  2020
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Lu-Ying Wang, Zi-Xu Liu, Ling-Min Zhao, Li-Xing Huang, Ying-Xue Qin, Yong-Quan Su, Wei-Qiang Zheng, Fan Wang, Qing-Pi Yan. Dual RNA-seq provides novel insight into the roles of dksA from Pseudomonas plecoglossicida in pathogen-host interactions with large yellow croakers (Larimichthys crocea). Zoological Research, 2020, 41(4): 410-422. doi: 10.24272/j.issn.2095-8137.2020.048
Citation: Lu-Ying Wang, Zi-Xu Liu, Ling-Min Zhao, Li-Xing Huang, Ying-Xue Qin, Yong-Quan Su, Wei-Qiang Zheng, Fan Wang, Qing-Pi Yan. Dual RNA-seq provides novel insight into the roles of dksA from Pseudomonas plecoglossicida in pathogen-host interactions with large yellow croakers (Larimichthys crocea). Zoological Research, 2020, 41(4): 410-422. doi: 10.24272/j.issn.2095-8137.2020.048

Dual RNA-seq provides novel insight into the roles of dksA from Pseudomonas plecoglossicida in pathogen-host interactions with large yellow croakers (Larimichthys crocea)

doi: 10.24272/j.issn.2095-8137.2020.048
Funds:  This work was supported by the National Natural Science Foundation of China (31672694), Fujian Provincial Special Fund for Marine and Fishery Protection and Development (MCZ[2019]062), and Open Fund of Fujian Province Key Laboratory of Special Aquatic Formula Feed (2019-01)
More Information
  • Corresponding author: E-mail: yanqp@jmu.edu.cn
  • Received Date: 2020-02-20
  • Accepted Date: 2020-04-21
  • Available Online: 2020-05-13
  • Publish Date: 2020-07-18
  • Pseudomonas plecoglossicida is a rod-shaped, gram-negative bacterium with flagella. It causes visceral white spot disease and high mortality in Larimichthys crocea during culture, resulting in serious economic loss. Analysis of transcriptome and quantitative real-time polymerase chain reaction (PCR) data showed that dksA gene expression was significantly up-regulated after 48 h of infection with Epinephelus coioides (log2FC=3.12, P<0.001). RNAi of five shRNAs significantly reduced the expression of dksA in P. plecoglossicida, and the optimal silencing efficiency was 96.23%. Compared with wild-type strains, the symptoms of visceral white spot disease in L. crocea infected with RNAi strains were reduced, with time of death delayed by 48 h and mortality reduced by 25%. The dksA silencing led to a substantial down-regulation in cellular component-, flagellum-, and ribosome assembly-related genes in P. plecoglossicida, and the significant up-regulation of fliC may be a way in which virulence is maintained in P. plecoglossicida. The GO and KEGG results showed that RNAi strain infection in L. crocea led to the down-regulation of inflammatory factor genes in immune-related pathways, which were associated with multiple immune response processes. Results also showed that dksA was a virulence gene in P. plecoglossicida. Compared with the wild-type strains, RNAi strain infection induced a weaker immune response in L. crocea.

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  • [1]
    Alcamo E, Mizgerd JP, Horwitz BH, Bronson R, Beg AA, Scott M, Doerschuk CM, Hynes RO, Baltimore D. 2001. Targeted mutation of TNF receptor I rescues the RelA-deficient mouse and reveals a critical role for NF-κB in leukocyte recruitment. The Journal of Immunology, 167(3): 1592−1600. doi: 10.4049/jimmunol.167.3.1592
    Anders S, Huber W. 2010. Differential expression analysis for sequence count data. Genome Biology, 11(10): R106. doi: 10.1186/gb-2010-11-10-r106
    Chen JP, Pang W, Zhao ZW, Bi YH, Chen XW. 2019. Transcription profiles of skin and head kidney from goldfish suffering hemorrhagic septicemia with an emphasis on the TLR signaling pathway. Zoological Research, 40(4): 337−342. doi: 10.24272/j.issn.2095-8137.2019.028
    Collette Y, Gilles A, Pontarotti P, Olive D. 2003. A co-evolution perspective of the TNFSF and TNFRSF families in the immune system. Trends in Immunology, 24(7): 387−394. doi: 10.1016/S1471-4906(03)00166-2
    Crofts AA, Giovanetti SM, Rubin EJ, Poly FM, Gutierrez RL, Talaat KR, Porter CK, Riddle MS, DeNearing B, Brubaker J, Maciel M Jr, Alcala AN, Chakraborty S, Prouty MG, Savarino SJ, Davies BW, Trent MS. 2018. Enterotoxigenic E. coli virulence gene regulation in human infections. Proceedings of the National Academy of Sciences of the United States of America, 115(38): E8968−E8976. doi: 10.1073/pnas.1808982115
    Dalebroux ZD, Yagi BF, Sahr T, Buchrieser C, Swanson MS. 2010. Distinct roles of ppGpp and DksA in Legionella pneumophila differentiation. Molecular Microbiology, 76(1): 200−219. doi: 10.1111/j.1365-2958.2010.07094.x
    Delves PJ. 2020. Innate and adaptive systems of immunity. In: Rose NR, Mackay IR. The Autoimmune Diseases. 6th ed. San Diego: Academic Press, 45–61.
    Gardell SJ, Craik CS, Clauser E, Goldsmith EJ, Stewart CB, Graf M, Rutter WJ. 1988. A novel rat carboxypeptidase, CPA2: characterization, molecular cloning, and evolutionary implications on substrate specificity in the carboxypeptidase gene family. Journal of Biological Chemistry, 263(33): 17828−17836.
    Godahewa GI, Wickramaarachchi WDN, Whang I, Bathige SDNK, Lim BS, Choi CY, De Zoysa M, Noh JK, Lee J. 2014. Two carboxypeptidase counterparts from rock bream (Oplegnathus fasciatus): molecular characterization, genomic arrangement and immune responses upon pathogenic stresses. Veterinary Immunology and Immunopathology, 162(3-4): 180−191. doi: 10.1016/j.vetimm.2014.10.005
    Guanzon DAV, Maningas MBB. 2018. Functional elucidation of LvToll 3 receptor from P. vannamei through RNA interference and its potential role in the shrimp antiviral response. Developmental & Comparative Immunology, 84: 172−180.
    Guo LN, Huang LX, Su YQ, Qin YX, Zhao LM, Yan QP. 2018. secA, secD, secF, yajC, and yidC contribute to the adhesion regulation of Vibrio alginolyticus. Microbiologyopen, 7(2): e00551. doi: 10.1002/mbo3.551
    He Y, Xu TT, Fossheim LE, Zhang XH. 2012. FliC, a flagellin protein, is essential for the growth and virulence of fish pathogen Edwardsiella tarda. PLoS One, 7(9): e45070. doi: 10.1371/journal.pone.0045070
    Huang LX, Liu WJ, Jiang QL, Zuo YF, Su YQ, Zhao LM, Qin YX, Yan QP. 2018. Integration of Transcriptomic and Proteomic Approaches Reveals the Temperature-Dependent Virulence of Pseudomonas plecoglossicida. Frontiers in Cellular and Infection Microbiology, 8: 207. doi: 10.3389/fcimb.2018.00207
    Huang LX, Zhao LM, Qi WL, Xu XJ, Zhang JN, Zhang JL, Yan QP. 2020. Temperature-specific expression of cspA1 contributes to activation of sigX during pathogenesis and intracellular survival in Pseudomonas plecoglossicida. Aquaculture, 518: 734861. doi: 10.1016/j.aquaculture.2019.734861
    Huang LX, Zuo YF, Jiang QL, Su YQ, Qin YX, Xu XJ, Zhao LM, Yan QP. 2019. A metabolomic investigation into the temperature-dependent virulence of Pseudomonas plecoglossicida from large yellow croaker (Pseudosciaena crocea). Journal of Fish Diseases, 42(3): 431−446. doi: 10.1111/jfd.12957
    Joosten LAB, Abdollahi-Roodsaz S, Dinarello CA, O'Neill L, Netea MG. 2016. Toll-like receptors and chronic inflammation in rheumatic diseases: new developments. Nature Reviews Rheumatology, 12(6): 344−357. doi: 10.1038/nrrheum.2016.61
    Kamarthapu V, Epshtein V, Benjamin B, Proshkin S, Mironov A, Cashel M, Nudler E. 2016. ppGpp couples transcription to DNA repair in E. coli. Science, 352(6288): 993−996. doi: 10.1126/science.aad6945
    Kasai K, Ishikawa T, Komata T, Fukuchi K, Chiba M, Nozaka H, Nakamura T, Sato T, Miura T. 2010. Novel L-amino acid oxidase with antibacterial activity against methicillin-resistant Staphylococcus aureus isolated from epidermal mucus of the flounder Platichthys stellatus. The FEBS Journal, 277(2): 453−465. doi: 10.1111/j.1742-4658.2009.07497.x
    Kim D, Langmead B, Salzberg SL. 2015. HISAT: a fast spliced aligner with low memory requirements. Nature Methods, 12(4): 357−360. doi: 10.1038/nmeth.3317
    Langmead B, Salzberg SL. 2012. Fast gapped-read alignment with Bowtie 2. Nature Methods, 9(4): 357−359. doi: 10.1038/nmeth.1923
    Lemke JJ, Durfee T, Gourse RL. 2009. DksA and ppGpp directly regulate transcription of the Escherichia coli flagellar cascade. Molecular Microbiology, 74(6): 1368−1379. doi: 10.1111/j.1365-2958.2009.06939.x
    Li CH, Xiong JB, Ding FF, Chen J. 2020. Immune and gut bacterial successions of large yellow croaker (Larimichthys crocea) during Pseudomonas plecoglossicida infection. Fish & Shellfish Immunology, 99: 176−183.
    Liu WJ, Huang LX, Su YQ, QinYX, Zhao LM, Y an, Q P. 2017. Contributions of the oligopeptide permeases in multistep of vibrio alginolyticus pathogenesis. Microbiologyopen, 6(1): e00511.
    Lu XJ, Chen J. 2019. Specific function and modulation of teleost monocytes/macrophages: polarization and phagocytosis. Zoological Research, 40(3): 146−150. doi: 10.24272/j.issn.2095-8137.2019.035
    Luo G, Sun YJ, Huang LX, Su YQ, Zhao LM, Qin YX, Xu XJ, Yan QP. 2020. Time-resolved dual RNA-seq of tissue uncovers Pseudomonas plecoglossicida key virulence genes in host-pathogen interaction with Epinephelus coioides. Environmental Microbiology, 22(2): 677−693. doi: 10.1111/1462-2920.14884
    Luo G, Xu XJ, Zhao LM, Qin YX, Huang LX, Su YQ, Yan QP. 2019b. clpV is a key virulence gene during in vivo Pseudomonas plecoglossicida infection. Journal of Fish Diseases, 42(7): 991−1000.
    Luo G, Zhao LM, Xu XJ, Qin YX, Huang LX, Su YQ, Zheng WQ, Yan QP. 2019a. Integrated dual RNA-seq and dual iTRAQ of infected tissue reveals the functions of a diguanylate cyclase gene of Pseudomonas plecoglossicida in host-pathogen interactions with Epinephelus coioides. Fish & shellfish Immunology, 95: 481−490.
    Mallik P, Paul BJ, Rutherford ST, Gourse RL, Osuna R. 2006. DksA is required for growth phase-dependent regulation, growth rate-dependent control, and stringent control of fis expression in Escherichia coli. Journal of Bacteriology, 188(16): 5775−5782. doi: 10.1128/JB.00276-06
    Mandel M, Higa A. 1970. Calcium-dependent bacteriophage DNA infection. Journal of Molecular Biology, 53(1): 159−162. doi: 10.1016/0022-2836(70)90051-3
    Mantovani A, Dinarello CA, Molgora M, Garlanda C. 2019. Interleukin-1 and Related Cytokines in the Regulation of Inflammation and Immunity. Immunity, 50(4): 778−795. doi: 10.1016/j.immuni.2019.03.012
    Marshall JS, Warrington R, Watson W, Kim HL. 2018. An introduction to immunology and immunopathology. Allergy, Asthma & Clinical Immunology, 14(S2): 49.
    Nishimori E, Kita-Tsukamoto K, Wakabayashi H. 2000. Pseudomonas plecoglossicida sp. nov., the causative agent of bacterial haemorrhagic ascites of ayu, Plecoglossus altivelis. International Journal of Systematic and Evolutionary Microbiology, 50(1): 83−89. doi: 10.1099/00207713-50-1-83
    Nuss AM, Beckstette M, Pimenova M, Schmühl C, Opitz W, Pisano F, Heroven AK, Dersch P. 2017. Tissue dual RNA-seq allows fast discovery of infection-specific functions and riboregulators shaping host-pathogen transcriptomes. Proceedings of the National Academy of Sciences of the United States of America, 114(5): E791−E800. doi: 10.1073/pnas.1613405114
    Palti Y. 2011. Toll-like receptors in bony fish: from genomics to function. Developmental & Comparative Immunology, 35(12): 1263−1272.
    Paul BJ, Barker MM, Ross W, Schneider DA, Webb C, Foster JW, Gourse RL. 2004. DksA: a critical component of the transcription initiation machinery that potentiates the regulation of rRNA promoters by ppGpp and the initiating NTP. Cell, 118(3): 311−322. doi: 10.1016/j.cell.2004.07.009
    Robinson MD, McCarthy DJ, Smyth GK. 2010. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics, 26(1): 139−140. doi: 10.1093/bioinformatics/btp616
    Rong DL, Wu QP, Xu MF, Zhang JM, Yu SB. 2017. Prevalence, virulence genes, antimicrobial susceptibility, and genetic diversity of Staphylococcus aureus from retail aquatic products in China. Frontiers in Microbiology, 8: 714. doi: 10.3389/fmicb.2017.00714
    Saleh M, Kumar G, Abdel-Baki AA, Dkhil MA, El-Matbouli M, Al-Quraishy S. 2016. In vitro gene silencing of the fish microsporidian Heterosporis saurida by RNA interference. Nucleic Acid Therapeutics, 26(4): 250−256. doi: 10.1089/nat.2016.0613
    Sharma A, Steichen AL, Jondle CN, Mishra BB, Sharma J. 2014. Protective role of Mincle in bacterial pneumonia by regulation of neutrophil mediated phagocytosis and extracellular trap formation. The Journal of Infectious Disease, 209(11): 1837−1846. doi: 10.1093/infdis/jit820
    Sharma AK, Payne SM. 2006. Induction of expression of hfq by DksA is essential for Shigella flexneri virulence. Molecular Microbiology, 62(2): 469−479. doi: 10.1111/j.1365-2958.2006.05376.x
    Stokes BA, Yadav S, Shokal U, Smith LC, Eleftherianos I. 2015. Bacterial and fungal pattern recognition receptors in homologous innate signaling pathways of insects and mammals. Frontiers in Microbiology, 6: 19.
    Sun YJ, Luo G, Zhao LM, Huang LX, Qin YX, Su YQ, Yan QP. 2018. Integration of RNAi and RNA-seq Reveals the Immune Responses of Epinephelus coioides to sigX Gene of Pseudomonas plecoglossicida. Frontiers in Immunology, 9: 1624. doi: 10.3389/fimmu.2018.01624
    Sun YJ, Nie P, Zhao LM, Huang LX, Qin YX, Xu XJ, Zhang JN, Yan QP. 2019a. Dual RNA-Seq unveils the role of the Pseudomonas plecoglossicida fliA gene in pathogen-host interaction with Larimichthys crocea. Microorganisms, 7(10): 443. doi: 10.3390/microorganisms7100443
    Sun YJ, Zhuang ZX, Wang XR, Huang HB, Fu Q, Yan QP. 2019b. Dual RNA-seq reveals the effect of the flgM gene of Pseudomonas plecoglossicida on the immune response of Epinephelus coioides. Fish & Shellfish Immunology, 87: 515−523.
    Takeda K, Akira S. 2015. Toll-like receptors. Current Protocols in Immunology, 109(14.12.1): 14.12.10.
    Tan SY, Chowdhury S, Polak N, Gorrell MD, Weninger W. 2017. Fibroblast activation protein is dispensable in the anti-influenza immune response in mice. PLoS One, 12(2): e0171194. doi: 10.1371/journal.pone.0171194
    Tang RQ, Luo G, Zhao LM, Huang LX, Qin YX, Xu XJ, Su YQ, Yan QP. 2019b. The effect of a LysR-type transcriptional regulator gene of Pseudomonas plecoglossicida on the immune responses of Epinephelus coioides. Fish & Shellfish Immunology, 89: 420−427.
    Tang RQ, Zhao LM, Xu XJ, Huang LX, Qin YX, Su YQ, Yan QP. 2019a. Dual RNA-Seq uncovers the function of an ABC transporter gene in the host-pathogen interaction between Epinephelus coioides and Pseudomonas plecoglossicida. Fish & Shellfish Immunology, 92: 45−53.
    Tang Y, Sun YJ, Zhao LM, Xu XJ, Huang LX, Qin YX, Su YQ, Yi GF, Yan QP. 2019c. Mechanistic insight into the roles of Pseudomonas plecoglossicida clpV gene in host-pathogen interactions with Larimichthys crocea by dual RNA-seq. Fish & Shellfish Immunology, 93: 344−353.
    Tang Y, Xin G, Zhao LM, Huang LX, Qin YX, Su YQ, Zheng WQ, Wu B, Lin N, Yan QP. 2020. Novel insights into host-pathogen interactions of large yellow croakers (Larimichthys crocea) and pathogenic bacterium Pseudomonas plecoglossicida using time-resolved dual RNA-seq of infected spleens. Zoological Research, 41(3): 341−327.
    Tao Z, Xu Y, Zhou SM, Qian D, Liu MH, Li WY, Xu WJ, Yan XJ. 2020. Acquisition of a type VI secretion system is critical for Pseudomonas plecoglossicida induced granulomas in fish internal organs. Aquaculture, 516: 734629. doi: 10.1016/j.aquaculture.2019.734629
    Tao Z, Zhou T, Zhou SM, Wang GL. 2016. Temperature-regulated expression of type VI secretion systems in fish pathogen Pseudomonas plecoglossicida revealed by comparative secretome analysis. FEMS Microbiology Letters, 363(22): fnw261. doi: 10.1093/femsle/fnw261
    Valenzuela-Miranda D, Gallardo-Escarate C. 2018. Dual RNA-seq uncovers metabolic amino acids dependency of the intracellular bacterium Piscirickettsia salmonis infecting Atlantic salmon. Frontiers in Microbiology, 9: 2877. doi: 10.3389/fmicb.2018.02877
    Wang LY, Sun YJ, Zhao LM, Xu XJ, Huang LX, Qin YX, Su YQ, Zhang JN, Yan QP. 2019a. Dual RNA-seq uncovers the immune response of Larimichthys crocea to the secY gene of Pseudomonas plecoglossicida from the perspective of host-pathogen interactions. Fish & Shellfish Immunology, 93: 949−957.
    Wang YQ, Wang XY, Ali F, Li ZQ, Fu YY, Yang XJ, Lin WX, Lin XM. 2019b. Comparative extracellular proteomics of Aeromonas hydrophila reveals iron-regulated secreted proteins as potential vaccine candidates. Frontiers in Immunology, 10: 256. doi: 10.3389/fimmu.2019.00256
    Westermann AJ, Barquist L, Vogel J. 2017. Resolving host-pathogen interactions by dual RNA-seq. PLoS Pathogens, 13(2): e1006033. doi: 10.1371/journal.ppat.1006033
    Westermann AJ, Förstner KU, Amman F, Barquist L, Chao YJ, Schulte LN, Müller L, Reinhardt R, Stadler PF, Vogel J. 2016. Dual RNA-seq unveils noncoding RNA functions in host-pathogen interactions. Nature, 529(7587): 496−501. doi: 10.1038/nature16547
    Westermann AJ, Gorski SA, Vogel J. 2012. Dual RNA-seq of pathogen and host. Nature Reviews Microbiology, 10(9): 618−630. doi: 10.1038/nrmicro2852
    Xie C, Mao XZ, Huang JJ, Ding Y, Wu JM, Dong S, Kong L, Gao G, Li CY, Wei LP. 2011. KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Research, 39(S2): W316−W322.
    Yang J, Lu XJ, Chai FC, Chen J. 2016. Molecular characterization and functional analysis of a piscidin gene in large yellow croaker (Larimichthys crocea). Zoological Research, 37(6): 347−355.
    Yang Y, Adeola AC, Xie HB, Zhang YP. 2018. Genomic and transcriptomic analyses reveal selection of genes for puberty in Bama Xiang pigs. Zoological Research, 39(6): 424−430. doi: 10.24272/j.issn.2095-8137.2018.068
    Yao ZJ, Guo Z, Wang YQ, Li WX, Fu YY, Lin YX, Lin WX, Lin XM. 2019. Integrated succinylome and metabolome profiling reveals crucial role of S-ribosylhomocysteine lyase in quorum sensing and metabolism of Aeromonas hydrophila. Molecular & Cellular Proteomics, 18(2): 200−215.
    Ye C, An X, Jiang YD, Ding BY, Shang F, Christiaens O, Taning CNT, Smagghe G, Niu JZ, Wang JJ. 2018. Induction of RNAi core machinery's gene expression by exogenous dsRNA and the effects of pre-exposure to dsRNA on the gene silencing efficiency in the pea aphid (Acyrthosiphon pisum). Frontiers in Physiology, 9: 1906.
    Zhang BB, Luo G, Zhao LM, Huang LX, Qin YX, Su YQ, Yan QP. 2018a. Integration of RNAi and RNA-seq uncovers the immune responses of Epinephelus coioides to L321_RS19110 gene of Pseudomonas plecoglossicida. Fish & Shellfish Immunology, 81: 121−129.
    Zhang BB, Zhuang ZX, Wang XR, Huang HB, Fu Q, Yan QP. 2019b. Dual RNA-Seq reveals the role of a transcriptional regulator gene in pathogen-host interactions between Pseudomonas plecoglossicida and Epinephelus coioides. Fish & Shellfish Immunology, 87: 778−787.
    Zhang J, Wang Y, Guo H, Mao Z, Ge C. 2017. Identification and characterization of a phospholipase A1 activity type three secreted protein, PP_ExoU from Pseudomonas plecoglossicida NB2011, the causative agent of visceral granulomas disease in large yellow croaker (Larimichthys crocea). Journal of Fish Diseases, 40(6): 831−840. doi: 10.1111/jfd.12565
    Zhang JT, Zhou SM, An SW, Chen L, Wang GL. 2014. Visceral granulomas in farmed large yellow croaker, Larimichthys crocea (Richardson), caused by a bacterial pathogen, Pseudomonas plecoglossicida. Journal of Fish Diseases, 37(2): 113−121. doi: 10.1111/jfd.12075
    Zhang MM, Qin YX, Huang LX, Yan QP, Mao LL, Xu XJ, Wang SY, Zhang MM, Chen LW. 2019a. The role of sodA and sodB in Aeromonas hydrophila resisting oxidative damage to survive in fish macrophages and escape for further infection. Fish & Shellfish Immunology, 88: 489−495.
    Zhang MM, Yan QP, Mao LL, Wang SY, Huang LX, Xu XJ, Qin YX. 2018b. KatG plays an important role in Aeromonas hydrophila survival in fish macrophages and escape for further infection. Gene, 672: 156−164. doi: 10.1016/j.gene.2018.06.029
    Zuo YF, Zhao LM, Xu XJ, Zhang JN, Zhang JL, Yan QP, Huang LX. 2019. Mechanisms underlying the virulence regulation of new Vibrio alginolyticus ncRNA Vvrr1 with a comparative proteomic analysis. Emerging Microbes & Infections, 8(1): 1604−1618.
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