Molecular Mechanism of Duox 2 Regulating Innate Immunity against Bacteria in Procambarus clarkii Intestine

doi:10.13560/j.cnki.biotech.bull.1985.2024-0540

Abstract

Abstract:

【Objective】To reveal the regulatory function of Dual Oxidase 2（DUOX2）in the intestine of the Procambarus clarkii on the levels of reactive oxygen species（ROS）, as well as its impact on the expression of antimicrobial peptide genes.【Method】Initially, the expressions of the Duox 2 gene in hemocytes, hepatopancreas, intestine, and gill tissues following Staphylococcus aureus infection were detected using quantitative real-time PCR（RT-qPCR）. After RNA interference and subsequent stimulation with S. aureus, the survival rate of the P. clarkii was statistically analyzed, along with the bacterial clearance capability in the hemolymph observed on culture plates. Furthermore, the ROS levels in the P. clarkii intestine were measured using the fluorescent probe 2', 7'-dichlorodihydrofluorescein diacetate（DCFH-DA）. Finally, the expression changes of antimicrobial peptide-related genes（Pc-toll 1, Pc-toll 3, Pc-ALF 1, and Pc-lysozyme）in the intestine were detected through RT-qPCR.【Result】Under the stimulation of S. aureus, the expression of the Duox 2 gene in P. clarkii significantly increased in hemocytes, hepatopancreas, intestine, and gills. Furthermore, after the expression of the Duox 2 gene was suppressed using RNA interference technology, the resistance of P. clarkii to S. aureus decreased, the survival rate dropped, and the number of bacteria in the hemolymph increased. Moreover, the reduction in Duox 2 gene expression led to the suppression of ROS levels in the intestine, and the expressions of antimicrobial peptide-related genes were also suppressed, further confirming the important role of Duox 2 in regulating the immune defense of P. clarkii.【Conclusion】Duox 2 gene regulates the expression of antimicrobial peptide-related genes by modulating the levels of ROS in the P. clarkii intestine, thereby controlling the body's ability to clear S. aureus. Therefore, Duox 2 plays an important role in the innate immune response of P. clarkii against S. aureus infection.

Key words: Procambarus clarkii, dual oxidase 2, reactive oxygen species（ROS）, RNA interference, Staphylococcus aureus

WEN Jing, LI Qian-qian, ZHANG Ming-da, TAN Ming-yue, JIN Bo-yang, SHEN XIU-li, DU Zhi-qiang. Molecular Mechanism of Duox 2 Regulating Innate Immunity against Bacteria in Procambarus clarkii Intestine[J]. Biotechnology Bulletin, 2025, 41(1): 324-332.

Figures/Tables 6

Table 1 Primer list

引物Primer	引物序列Primer sequence（5'-3'）
Pc-Duox 2-iF	GCGTAATACGACTCACTATAGGGTGATGAAAAATGCGTTT
Pc-Duox 2-iR	GCGTAATACGACTCACTATAGGAAAGATGTCTCCCTGAAG
Pc-GFP-iF	GCGTAATACGACTCACTATAGGCGAGCTGGACGGCGACGTAAAC
Pc-GFP-iR	GCGTAATACGACTCACTATAGGCTTGAAGTTCACCTTGATGCC
Pc-Duox 2-RT-F	GCTGCCCAAGTTTCACTA
Pc-Duox 2-RT-R	AAAGGAGGCTGCACCAGC
18 S rRNA-RT-F	TCTTCTTAGAGGGATTAGCGG
18 S rRNA-RT-R	AAGGGGATTGAACGGGTTA
Pc-toll 1-RT-F	GACTTGTCCAAAAACGATATACG
Pc-toll 1-RT-R	TGCGTTACAGTAGTGAGCGAA
Pc-toll 3-RT-F	TCATTTGGCATCTGGCTCAC
Pc-toll 3-RT-R	GCAGGTGGTGGCGTTGA
Pc-ALF 1-RT-F	CGAGAGGCTGTAGAGGATGC
Pc-ALF 1-RT-R	CCCAGTTTGTTGATGATGAG
Pc-lysozyme-RT-F	GTCAACCCACCCTCAATAAC
Pc-lysozyme-RT-R	CTTGTGAATCAGGGCGTA

Fig. 1 Expression patterns of Duox 2 gene in P. clarkii stimulated by S. aureus Different letters indicate significant differences（P < 0.05）. The error bar indicates three repeated standard deviations（SD）, the same below

Fig. 2 Statistical results of P. clarkii survival after RNAi and S. aureus injection ** P < 0.01，**** P < 0.0001

Fig. 3 Results of the bacterial clearance assay in P. clarkii haemolymph after RNAi and injection of S. aureus

Fig. 4 Detection results of reactive oxygen species in P. clarkii intestinal tissue after RNAi and S. aureus injection

Fig. 5 Relative expressions of innate immune-related antimicrobial peptide genes in P. clarkii intestinal tissues after RNAi and S. aureus injection

References 37

[1]	邓灵, 赵康, 夏开, 等. 小龙虾(Procambarus clarkia)加工前后优势腐败菌的分离与鉴定[J]. 食品工业科技, 2020, 41(18): 100-104.
	Deng L, Zhao K, Xia K, et al. Isolation and identification of specific spoilage organisms in crayfish(Procambarus clarkii)before and after processing[J]. Sci Technol Food Ind, 2020, 41(18): 100-104.
[2]	Arfatahery N, Davoodabadi A, Abedimohtasab T. Characterization of toxin genes and antimicrobial susceptibility of Staphylococcus aureus isolates in fishery products in Iran[J]. Sci Rep, 2016, 6: 34216. doi: 10.1038/srep34216 pmid: 27694813
[3]	Shandilya S, Kumar S, Kumar JN, et al. Interplay of gut microbiota and oxidative stress: perspective on neurodegeneration and neuroprotection[J]. J Adv Res, 2021, 38: 223-244.
[4]	Poljsak B, Šuput D, Milisav I. Achieving the balance between ROS and antioxidants: when to use the synthetic antioxidants[J]. Oxid Med Cell Longev, 2013, 2013: 956792.
[5]	Yang HT, Yang MC, Sun JJ, et al. Catalase eliminates reactive oxygen species and influences the intestinal microbiota of shrimp[J]. Fish Shellfish Immunol, 2015, 47(1): 63-73.
[6]	De Deken X, Corvilain B, Dumont JE, et al. Roles of DUOX-mediated hydrogen peroxide in metabolism, host defense, and signaling[J]. Antioxid Redox Signal, 2014, 20(17): 2776-2793.
[7]	Hazime H, Ducasa GM, Santander AM, et al. Intestinal epithelial inactivity of dual oxidase 2 results in microbiome-mediated metabolic syndrome[J]. Cell Mol Gastroenterol Hepatol, 2023, 16(4): 557-572. doi: 10.1016/j.jcmgh.2023.06.009 pmid: 37369278
[8]	Sommer F, Bäckhed F. The gut microbiota engages different signaling pathways to induce Duox2 expression in the ileum and colon epithelium[J]. Mucosal Immunol, 2015, 8(2): 372-379. doi: 10.1038/mi.2014.74 pmid: 25160818
[9]	Lee KA, Kim B, You H, et al. Uracil-induced signaling pathways for DUOX-dependent gut immunity[J]. Fly, 2015, 9(3): 115-120.
[10]	Yang QH, Sun ZQ, Zhou YL, et al. SpATF2 participates in maintaining the homeostasis of hemolymph microbiota by regulating dual oxidase expression in mud crab[J]. Fish Shellfish Immunol, 2020, 104: 252-261.
[11]	Liu ZG, Zhang HY, Lemaitre B, et al. Duox activation in Drosophila Malpighian tubules stimulates intestinal epithelial renewal through a countercurrent flow[J]. Cell Rep, 2024, 43(4): 114109.
[12]	Dias RO, Cardoso C, Pimentel AC, et al. The roles of mucus-forming mucins, peritrophins and peritrophins with mucin domains in the insect midgut[J]. Insect Mol Biol, 2018, 27(1): 46-60. doi: 10.1111/imb.12340 pmid: 28833767
[13]	Zeng T, Jaffar S, Xu YJ, et al. The intestinal immune defense system in insects[J]. Int J Mol Sci, 2022, 23(23): 15132.
[14]	Zhou YL, Wang LZ, Gu WB, et al. Identification and functional analysis of immune deficiency(IMD)from Scylla paramamosain: the first evidence of IMD signaling pathway involved in immune defense against bacterial infection in crab species[J]. Fish Shellfish Immunol, 2018, 81: 150-160.
[15]	Li HY, Li QY, Wang S, et al. Stimulator of interferon genes defends against bacterial infection via IKKβ-mediated Relish activation in shrimp[J]. Front Immunol, 2022, 13: 977327.
[16]	Ha EM, Lee KA, Seo YY, et al. Coordination of multiple dual oxidase-regulatory pathways in responses to commensal and infectious microbes in drosophila gut[J]. Nat Immunol, 2009, 10(9): 949-957.
[17]	Donkó Á, Péterfi Z, Sum A, et al. Dual oxidases[J]. Phil Trans R Soc B, 2005, 360(1464): 2301-2308.
[18]	Sun ZQ, Hao SF, Gong Y, et al. Dual oxidases participate in the regulation of hemolymph microbiota homeostasis in mud crab Scylla paramamosain[J]. Dev Comp Immunol, 2018, 89: 111-121.
[19]	Inada M, Kihara K, Kono T, et al. Deciphering of the Dual oxidase(Nox family)gene from kuruma shrimp, Marsupenaeus japonicus: full-length cDNA cloning and characterization[J]. Fish Shellfish Immunol, 2013, 34(2): 471-485.
[20]	Schweikl H, Godula M, Petzel C, et al. Critical role of superoxide anions and hydroxyl radicals in HEMA-induced apoptosis[J]. Dent Mater, 2017, 33(1): 110-118. doi: S0109-5641(16)30620-0 pmid: 27887776
[21]	He HH, Chi YM, Yuan K, et al. Functional characterization of a reactive oxygen species modulator 1 gene in Litopenaeus vannamei[J]. Fish Shellfish Immunol, 2017, 70: 270-279.
[22]	Zhang L, Wang K, Lei YL, et al. Redox signaling: potential arbitrator of autophagy and apoptosis in therapeutic response[J]. Free Radic Biol Med, 2015, 89: 452-465.
[23]	de Almeida AJPO, de Oliveira JCPL, da Silva Pontes LV, et al. ROS: basic concepts, sources, cellular signaling, and its implications in aging pathways[J]. Oxid Med Cell Longev, 2022, 2022: 1225578.
[24]	Ray PD, Huang BW, Tsuji Y. Reactive oxygen species(ROS)homeostasis and redox regulation in cellular signaling[J]. Cell Signal, 2012, 24(5): 981-990.
[25]	Dryden M. Reactive oxygen species: a novel antimicrobial[J]. Int J Antimicrob Agents, 2018, 51(3): 299-303.
[26]	Wu XF, Yang MY, Kim JS, et al. Reactivity differences enable ROS for selective ablation of bacteria[J]. Angew Chem Int Ed Engl, 2022, 61(17): e202200808.
[27]	Nie JJ, Yu ZX, Yao DF, et al. Litopenaeus vannamei sirtuin 6 homolog(LvSIRT6)is involved in immune response by modulating hemocytes ROS production and apoptosis[J]. Fish Shellfish Immunol, 2020, 98: 271-284.
[28]	Benguettat O, Jneid R, Soltys J, et al. The DH31/CGRP enteroendocrine peptide triggers intestinal contractions favoring the elimination of opportunistic bacteria[J]. PLoS Pathog, 2018, 14(9): e1007279.
[29]	Mukherjee S, Hooper LV. Antimicrobial defense of the intestine[J]. Immunity, 2015, 42(1): 28-39. doi: 10.1016/j.immuni.2014.12.028 pmid: 25607457
[30]	Tassanakajon A, Somboonwiwat K, Supungul P, et al. Discovery of immune molecules and their crucial functions in shrimp immunity[J]. Fish Shellfish Immunol, 2013, 34(4): 954-967.
[31]	Yang L, Qiu LM, Fang Q, et al. Cellular and humoral immune interactions between Drosophila and its parasitoids[J]. Insect Sci, 2021, 28(5): 1208-1227.
[32]	Yu SC, Luo FZ, Xu YY, et al. Drosophila innate immunity involves multiple signaling pathways and coordinated communication between different tissues[J]. Front Immunol, 2022, 13: 905370.
[33]	Ding D, Sun XJ, Yan M, et al. The ECSIT mediated Toll3-dorsal-ALFs pathway inhibits bacterial amplification in kuruma shrimp[J]. Front Immunol, 2022, 13: 807326.
[34]	Li HY, Yin B, Wang S, et al. RNAi screening identifies a new Toll from shrimp Litopenaeus vannamei that restricts WSSV infection through activating Dorsal to induce antimicrobial peptides[J]. PLoS Pathog, 2018, 14(9): e1007109.
[35]	Wang Z, Chen YH, Dai YJ, et al. A novel vertebrates Toll-like receptor counterpart regulating the anti-microbial peptides expression in the freshwater crayfish, Procambarus clarkii[J]. Fish Shellfish Immunol, 2015, 43(1): 219-229.
[36]	Zhang HQ, Cheng WZ, Zheng LB, et al. Identification of a group D anti-lipopolysaccharide factor(ALF)from kuruma prawn(Marsupenaeus japonicus)with antibacterial activity against Vibrio parahaemolyticus[J]. Fish Shellfish Immunol, 2020, 102: 368-380.
[37]	Marra A, Hanson MA, Kondo S, et al. Drosophila antimicrobial peptides and lysozymes regulate gut microbiota composition and abundance[J]. mBio, 2021, 12(4): e0082421.