斑马鱼新型肽聚糖结合蛋白KRT8的模式识别活性和多肽抑菌活性鉴定
Pattern Recognition and Antibacterial Activity Identification of Zebrafish Novel Peptidoglycan-Binding Protein KRT8
DOI: 10.12677/hjfns.2026.154041, PDF,    科研立项经费支持
作者: 刘龙霄, 何 静, 范传浩, 王 霞*：青岛科技大学生物工程学院，山东 青岛；殷丽娟：山东新华制药股份有限公司技术质量保证部，山东 淄博
关键词: 细菌耐药性；KRT8；抗菌肽；mP90-103；抑菌活性；Bacterial Resistance； KRT8； Antimicrobial Peptides； mP90-103； Antibacterial Activity
摘要: 目的：研究角蛋白KRT8在斑马鱼中的免疫相关功能，并在该蛋白的基础上筛选抗菌肽。方法：以斑马鱼KRT8为研究对象，通过蛋白免疫印记和酶联免疫吸附实验，研究该蛋白是否具有与肽聚糖、脂多糖以及革兰氏阴性与阳性菌结合的能力；通过脂多糖诱导斑马鱼发生炎症，探究krt8在炎症反应中的作用；通过斑马鱼体内细菌感染实验，进一步研究KRT8对炎症的作用；在该蛋白的基础上筛选抗菌肽，然后验证筛选出候选肽的抑菌能力。结果：本研究发现，KRT8可以与肽聚糖、脂多糖、革兰氏阴性和革兰氏阳性细菌结合。在脂多糖诱导的斑马鱼炎症模型中，krt8的表达水平发生变化，这说明krt8可能参与炎症反应。进一步研究发现，KRT8可缓解细菌诱导的炎症反应。从KRT8中筛选出的抗菌肽mP90-103具有直接杀菌作用，且无溶血活性。结论：KRT8可作为模式识别受体参与斑马鱼炎症免疫调控；其衍生肽mP90-103具有优良的抗菌活性和生物安全性。本研究为解释多种动物体内角蛋白的免疫功能提供了新见解。
Abstract: Objective: To investigate the immune-related functions of keratin KRT8 in zebrafish, and screen antimicrobial peptides derived from KRT8. Methods: Taking zebrafish KRT8 as the research object, Western blot and ELISA were performed to detect the binding capacity of KRT8 to peptidoglycan (PGN), lipopolysaccharide (LPS), Gram-negative bacteria and Gram-positive bacteria. An LPS-induced zebrafish inflammatory model was constructed to explore the role of krt8 in inflammatory responses. In vivo bacterial infection assays in zebrafish were conducted to further clarify the regulatory effect of KRT8 on inflammation. Moreover, candidate antimicrobial peptides were screened based on the KRT8 sequence, and their antibacterial activities were verified subsequently. Results: KRT8 was capable of binding to PGN, LPS, Gram-negative and Gram-positive bacteria. In the LPS-induced inflammatory model, the expression of krt8 was significantly altered, suggesting that krt8 is involved in the inflammatory response. Further in vivo experiments confirmed that KRT8 could alleviate bacteria-triggered inflammatory reaction. The derived peptide mP90-103 screened from KRT8 exerted direct bactericidal activity with no hemolytic toxicity. Conclusion: KRT8 acts as a pattern recognition receptor and participates in the regulation of inflammatory immunity in zebrafish. Its derivative peptide mP90-103 possesses favorable antibacterial activity and high biosafety. This study provides novel insights into the immunological functions of keratins among diverse animal species.
文章引用：刘龙霄, 何静, 范传浩, 殷丽娟, 王霞. 斑马鱼新型肽聚糖结合蛋白KRT8的模式识别活性和多肽抑菌活性鉴定[J]. 食品与营养科学, 2026, 15(4): 362-377. https://doi.org/10.12677/hjfns.2026.154041

参考文献

[1] Zhu, Y., Huang, W.E. and Yang, Q. (2022) Clinical Perspective of Antimicrobial Resistance in Bacteria. Infection and Drug Resistance, 15, 735-746.
[2] Di Somma, A., Moretta, A., Canè, C., Cirillo, A. and Duilio, A. (2020) Antimicrobial and Antibiofilm Peptides. Biomolecules, 10, Article 652. [Google Scholar] [CrossRef] [PubMed]
[3] Wu, R., Patocka, J., Nepovimova, E., Oleksak, P., Valis, M., Wu, W., et al. (2021) Marine Invertebrate Peptides: Antimicrobial Peptides. Frontiers in Microbiology, 12, Article 785085. [Google Scholar] [CrossRef] [PubMed]
[4] Bragulla, H.H. and Homberger, D.G. (2009) Structure and Functions of Keratin Proteins in Simple, Stratified, Keratinized and Cornified Epithelia. Journal of Anatomy, 214, 516-559.
[5] Pegoraro, A.F., Janmey, P. and Weitz, D.A. (2017) Mechanical Properties of the Cytoskeleton and Cells. Cold Spring Harbor Perspectives in Biology, 9, a022038. [Google Scholar] [CrossRef] [PubMed]
[6] Xiong, F., Guo, T., Wang, X., Wu, G., Liu, W., Wang, Q., et al. (2022) Keratin 8 Is an Inflammation-Induced and Prognosis-Related Marker for Pancreatic Adenocarcinoma. Disease Markers, 2022, 1-25. [Google Scholar] [CrossRef] [PubMed]
[7] Schaffeld, M., Knappe, M., Markl, J. and Hunzinger, C. (2003) cDNA Sequences of the Authentic Keratins 8 and 18 in Zebrafish. Differentiation, 71, 73-82. [Google Scholar] [CrossRef] [PubMed]
[8] Toivola, D.M., Boor, P., Alam, C. and Strnad, P. (2015) Keratins in Health and Disease. Current Opinion in Cell Biology, 32, 73-81. [Google Scholar] [CrossRef] [PubMed]
[9] Stenvall, C.A., Tayyab, M., Grönroos, T.J., Ilomäki, M.A., Viiri, K., Ridge, K.M., et al. (2021) Targeted Deletion of Keratin 8 in Intestinal Epithelial Cells Disrupts Tissue Integrity and Predisposes to Tumorigenesis in the Colon. Cellular and Molecular Life Sciences, 79, Article No. 10. [Google Scholar] [CrossRef] [PubMed]
[10] Wang, X., Ren, Y., Li, J., Ji, Z., Chen, F. and Wang, X. (2021) Identification of the 14-3-3 β/α-A Protein as a Novel Maternal Peptidoglycan-Binding Protein That Protects Embryos of Zebrafish against Bacterial Infections. Developmental & Comparative Immunology, 114, Article 103867. [Google Scholar] [CrossRef] [PubMed]
[11] Bai, W., Zhang, Z., Tian, W., He, X., Ma, Y., Zhao, Y., et al. (2010) Toxicity of Zinc Oxide Nanoparticles to Zebrafish Embryo: A Physicochemical Study of Toxicity Mechanism. Journal of Nanoparticle Research, 12, 1645-1654. [Google Scholar] [CrossRef] 
[12] Ni, S., Zhou, Y., Song, L., Chen, Y., Wang, X., Du, X., et al. (2021) ELAVL1a Is an Immunocompetent Protein That Protects Zebrafish Embryos from Bacterial Infection. Communications Biology, 4, Article No. 251. [Google Scholar] [CrossRef] [PubMed]
[13] Hammond-Weinberger, D.R. and ZeRuth, G.T. (2020) Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae. Journal of Visualized Experiments, 155, 1-9. [Google Scholar] [CrossRef] 
[14] Gong, C., He, J., Guo, D., Zhang, L., Shi, Z. and Wang, X. (2023) Identification of Zebrafish GIGYF2 Presents in Egg/Embryo as an Antibacterial Protein. Fish & Shellfish Immunology, 140, Article 108957. [Google Scholar] [CrossRef] [PubMed]
[15] Livak, K.J. and Schmittgen, T.D. (2001) Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods, 25, 402-408. [Google Scholar] [CrossRef] [PubMed]
[16] Wang, X., Du, X., Li, H. and Zhang, S. (2016) Identification of the Zinc Finger Protein ZRANB2 as a Novel Maternal Lipopolysaccharide-Binding Protein That Protects Embryos of Zebrafish against Gram-Negative Bacterial Infections. Journal of Biological Chemistry, 291, 4019-4034. [Google Scholar] [CrossRef] [PubMed]
[17] Guo, D., Wang, H., He, J., Zhang, L., Liu, L. and Wang, X. (2024) Two Novel Antimicrobial Peptides P33-57 and Mp168-187 from Zebrafish Showing Potent Antibacterial Activities. Fish & Shellfish Immunology, 154, Article 109950. [Google Scholar] [CrossRef] [PubMed]
[18] Wang, P., Zhang, X., Zheng, X., Gao, J., Shang, M., Xu, J., et al. (2022) Folic Acid Protects against Hyperuricemia in C57BL/6J Mice via Ameliorating Gut–kidney Axis Dysfunction. Journal of Agricultural and Food Chemistry, 70, 15787-15803. [Google Scholar] [CrossRef] [PubMed]
[19] Chen, X., Shen, X., Liu, S., Li, W., Wang, H., Li, J., et al. (2025) A C-Type Lectin Hclec1 with Dual Function of Immunology and Mineralization from the Freshwater Oyster (Hyriopsis cumingii Lea). Frontiers in Immunology, 15, Article 1530732. [Google Scholar] [CrossRef] [PubMed]
[20] Duan, Y., Liu, Q., Wang, Y., Zhang, J. and Xiong, D. (2018) Impairment of the Intestine Barrier Function in Litopenaeus Vannamei Exposed to Ammonia and Nitrite Stress. Fish & Shellfish Immunology, 78, 279-288. [Google Scholar] [CrossRef] [PubMed]
[21] Waghu, F.H., Barai, R.S., Gurung, P. and Idicula-Thomas, S. (2015) CAMPR3: A Database on Sequences, Structures and Signatures of Antimicrobial Peptides: Table 1. Nucleic Acids Research, 44, D1094-D1097. [Google Scholar] [CrossRef] [PubMed]
[22] Wang, X., Ren, Y., Gong, C., Chen, Y., Ge, X., Kong, J., et al. (2021) 40S Ribosomal Protein S18 Is a Novel Maternal Peptidoglycan-Binding Protein That Protects Embryos of Zebrafish from Bacterial Infections. Developmental & Comparative Immunology, 125, Article 104212. [Google Scholar] [CrossRef] [PubMed]
[23] Luo, X.Y., Hu, C.M., Yin, Q., et al. (2024) Dual‐Mechanism Peptide SR25 Has Broad Antimicrobial Activity and Potential Application for Healing Bacteria‐Infected Diabetic Wounds. Advanced Science, 11, Article 2401793. [Google Scholar] [CrossRef] [PubMed]
[24] Bai, Y., Zhang, W., Zheng, W., Meng, X., Duan, Y., Zhang, C., et al. (2024) A 14-Amino Acid Cationic Peptide Bolespleenin334-347 from the Marine Fish Mudskipper Boleophthalmus Pectinirostris Exhibiting Potent Antimicrobial Activity and Therapeutic Potential. Biochemical Pharmacology, 226, Article 116344. [Google Scholar] [CrossRef] [PubMed]
[25] Zhu, D., Chen, F., Chen, Y., Peng, H. and Wang, K. (2021) The Long-Term Effect of a Nine Amino-Acid Antimicrobial Peptide As-Hepc3(48-56) against Pseudomonas Aeruginosa with No Detectable Resistance. Frontiers in Cellular and Infection Microbiology, 11, Article 752637. [Google Scholar] [CrossRef] [PubMed]
[26] Li, Y., Yu, S., Weng, P., et al. (2023) Purification and Antimicrobial Mechanism of a Novel Bacteriocin Produced by Lactiplantibacillus plantarum FB-2. LWT, 185, Article 115123.
[27] Gong, Z., Ju, B., Wang, X., He, J., Wan, H., Sudha, P.M., et al. (2001) Green Fluorescent Protein Expression in Germ-Line Transmitted Transgenic Zebrafish under a Stratified Epithelial Promoter from Keratin8. Developmental Dynamics, 223, 204-215. [Google Scholar] [CrossRef] [PubMed]
[28] Martorana, M.L., Tawk, M., Lapointe, T., Barre, N., Imboden, M., Joulie, C., et al. (2001) Zebrafish Keratin 8 Is Expressed at High Levels in the Epidermis of Regenerating Caudal Fin. The International Journal of Developmental Biology, 45, 449-452. [Google Scholar] [CrossRef] 
[29] Moll, R., Divo, M. and Langbein, L. (2008) The Human Keratins: Biology and Pathology. Histochemistry and Cell Biology, 129, 705-733. [Google Scholar] [CrossRef] [PubMed]
[30] Zhou, Y., Chen, L., Hao, S., Cao, X. and Ni, S. (2024) Zebrafish ANGPT4, Member of Fibrinogen-Related Proteins, Is an LTA-, LPS-and PGN-Binding Protein with a Bacteriolytic Activity. Fish & Shellfish Immunology, 147, Article 109451. [Google Scholar] [CrossRef] [PubMed]
[31] 张昊, 牛海涛, 李改瑞, 等. 抗菌肽分子结构对其活性的影响[J]. 中国抗生素杂志, 2010, 35(12): 892-897.
[32] Han, R. and Wang, S. (2022) Mechanisms of Antimicrobial Peptides as Characterized by Solid-State NMR. Magnetic Resonance Letters, 2, 119-129. [Google Scholar] [CrossRef] 
[33] Priyadarshini, D., Ivica, J., Separovic, F. and de Planque, M.R.R. (2022) Characterisation of Cell Membrane Interaction Mechanisms of Antimicrobial Peptides by Electrical Bilayer Recording. Biophysical Chemistry, 281, Article 106721. [Google Scholar] [CrossRef] [PubMed]
[34] Margalho, L.P., Feliciano, M.D., Silva, C.E., Abreu, J.S., Piran, M.V.F. and Sant'Ana, A.S. (2020) Brazilian Artisanal Cheeses Are Rich and Diverse Sources of Nonstarter Lactic Acid Bacteria Regarding Technological, Biopreservative, and Safety Properties—Insights through Multivariate Analysis. Journal of Dairy Science, 103, 7908-7926. [Google Scholar] [CrossRef] [PubMed]