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Construction of the rfaH Gene Deletion Strain in Salmonella anatum and Study of Its Biological Characteristics |
SONG Ya-Wen1, GAO Yu-Jie1, ZHANG Xiao-Hui1, WANG Wen-Xin1, GE Tong-Xin1, ZHAI Rui-Dong1, SONG Hou-Hui1, CHENG Chang-Yong1, LI Ai-Yun2,*, HAN Yue1,* |
1 College of Animal Science and Technology/College of Veterinary Medicine/Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province/Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics/Advanced Technology, Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management/China-Australia Joint Laboratory for Animal Health Big Data Analytics, Zhejiang A&F University, Hangzhou 311300, China; 2 Women's Hospital School of Medicine Zhejiang University, Hangzhou 310006, China |
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Abstract Salmonella anatum is a major pathogen that causes salmonellosis in ducks (Anatidae). rfaH encodes the transcriptional anti-termination factor RfaH, which is essential for bacterial transcription, infection, and other biological functions. This study investigated the effect of the rfaH gene on various biological aspects of S. anatum, including growth, motility, biofilm formation, and cell infection. To deeply understand the biological roles of the rfaH gene in S. anatum, an rfaH gene deletion strain ΔrfaH and the complement strain CΔrfaH derived from the S. anatum isolate strain SA01 were generated using Red homologous recombination technology. The sequencing results showed that the rfaH gene deletion strain ΔrfaH and the complement strain CΔrfaH of SA01 were successfully constructed. After the deletion of the rfbH gene, the growth rate remained unchanged, yet the O-antigen synthesis was impaired, leading to a significant decrease in bacterial motility, an enhancement in biofilm formation, an increased adhesion and invasion capacity against DF-1 cells, and a weakened proliferation ability in RAW264.7 cells. The findings indicated that the rfaH gene was involved in bacterial motility, biofilm formation, and cell infection. This study establishes a solid platform for future research on the biological functions of the rfaH gene and the development of Salmonella-based delivery vectors for vaccine applications.
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Received: 07 February 2024
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Corresponding Authors:
* yuehan@zafu.edu.cn; 5194015@zju.edu.cn
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[1] 齐静, 王守荣, 李兰波, 等. 2014. 山东省肉鸭沙门氏菌的分离鉴定及敏感抗菌药物的筛选[J]. 中国畜牧兽医, 41(7): 216-220. (Qi J, Wang S R, Li L B, et al.2014. Isolation and identification of duck Salmonella and screening of sensitive antimicrobials in Shandong province[J]. China Animal Husbandry & Veterinary Medicine, 41(7): 216-220) [2] 沈子城, 孟闯, 谢添, 等. 2023. 重组减毒沙门氏菌疫苗候选株研究进展[J]. 中国禽业导刊, 40(5): 45-51. (Shen Z C, Meng C, Xie T, et al.2023. The research progress on recombinant attenuated Salmonella vaccine candidates[J]. Guide to Chinese Poultry, 40(5): 45-51.) [3] 臧金刚. 2024. 鸭沙门氏菌病的诊断及中西医防治[J]. 养殖与饲料, 23(4): 102-104. (Zang J G.2024. Diagnosis of salmonellosis in ducks and its prevention and treatment by Chinese and western medicine[J]. Animals Breeding and Feed, 23(4): 102-104.) [4] Adzitey F, Huda N, Ali G R.2012. Prevalence and antibiotic resistance of Campylobacter, Salmonella, and L. monocytogenes in ducks: A review[J]. Foodborne Pathogens and Disease, 9(6): 498-505. [5] Artsimovitch I, Landick R.2002. The transcriptional regulator RfaH stimulates RNA chain synthesis after recruitment to elongation complexes by the exposed nontemplate DNA strand[J]. Cell, 109(2): 193-203. [6] Bailey M J A, Hughes C, Koronakis V.2003. RfaH and the ops element, components of a novel system controlling bacterial transcription elongation[J]. Molecular Microbiology, 26(5): 845-851. [7] Conn A B, Diggs S, Tam T K, et al.2019. Two old dogs, one new trick: A review of RNA polymerase and ribosome interactions during transcription-translation coupling[J]. International Journal of Molecular Sciences, 20(10): 2595. [8] Coutermarsh S L, Broadway K M, Scharf B E, et al.2017. Effect of Salmonella enterica serovar Typhimurium VNP20009 and VNP20009 with restored chemotaxis on 4T1 mouse mammary carcinoma progression[J]. Oncotarget, 8(20): 33601-33613. [9] Datsenko K A, Wanner B L.2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[J]. Proceedings of the National Academy of Sciences of the USA, 97(12): 6640-6645. [10] Foley S L, Johnson T J, Ricke S C, et al.2013. Salmonella pathogenicity and host adaptation in chicken-associated serovars[J]. Microbiology and Molecular Biology Reviews, 77(4): 582-607. [11] Gómez-Baltazar A, Vázquez-Garcidueñas M S, Larsen J, et al.2019. Comparative stress response to food preservation conditions of ST19 and ST213 genotypes of Salmonella enterica serotype Typhimurium[J]. Food Microbiology, 82: 303-315. [12] Guo L, Dai H, Feng S, et al.2023a. Contribution of GalU to biofilm formation, motility, antibiotic and serum resistance, and pathogenicity of Salmonella typhimurium[J]. Frontiers in Cellular and Infection Microbiology, 13: 1149541. [13] Guo L L, Xiao T A, Wu L Q, et al.2023b. Comprehensive profiling of serotypes, antimicrobial resistance and virulence of Salmonella isolates from food animals in China, 2015-2021[J]. Frontiers in Microbiology, 14: 1133241 [14] Hitchcock P J, Brown T M.1983. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels[J]. Journal of Bacteriology, 154(1): 269-277. [15] Jazeela K, Chakraborty A, Karunasagar I, et al.2020. Nontyphoidal Salmonella: A potential anticancer agent[J]. Journal of Applied Microbiology, 128(1): 2-14. [16] Klein G, Wieczorek A, Szuster M, et al.2021. Checkpoints that regulate balanced biosynthesis of lipopolysaccharide and its essentiality in Escherichia coli[J]. International Journal of Molecular Sciences, 23(1): 189. [17] Li R, Lai J, Wang Y, et al.2013. Prevalence and characterization of Salmonella species isolated from pigs, ducks and chickens in Sichuan province, China[J]. International Journal of Food Microbiology, 163(1): 14-18. [18] Lindberg A A, Hellerqvist C G.1980. Rough mutants of Salmonella typhimurium: Immunochemical and structural analysis of lipopolysaccharides from rfaH mutants[J]. Journal of General Microbiology, 116(1): 25-32. [19] Maldonado R F, Sa-Correia I, Valvano M A.2016. Lipopolysaccharide modification in gram-negative bacteria during chronic infection[J]. FEMS Microbiology Reviews, 40(4): 480-493. [20] Nagy G, Danino V, Dobrindt U, et al.2006. Down-regulation of key virulence factors makes the Salmonella enterica serovar Typhimurium rfaH mutant a promising live-attenuated vaccine candidate[J]. Infection and Immunity, 74(10): 5914-5925. [21] Nagy G b, Dobrindt U, Hacker J, et al.2004. Oral Immunization with an rfaH mutant elicits protection against Salmonellosis in mice[J]. Infection and Immunity, 72(7): 4297-4301. [22] Simm R, Lusch A, Kader A, et al.2007. Role of EAL-containing proteins in multicellular behavior of Salmonella enterica serovar Typhimurium[J]. Journal of Bacteriology, 189(9): 3613-3623. [23] Tang T, Gao Q, Barrow P, et al.2015. Development and evaluation of live attenuated Salmonella vaccines in newly hatched duckings[J]. Vaccine, 33(42): 5564-5571. [24] Wang B, Gumerov V M, Andrianova E P, et al.2020. Origins and molecular evolution of the NusG paralog RfaH[J]. mBio, 11(5): e02717-20 [25] Wang L, Jensen S, Hallman R, et al.1998. Expression of the O antigen gene cluster is regulated by RfaH through the JUMPstart sequence[J]. FEMS Microbiology Letters, 165(1): 201-206. |
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