oppCDF基因缺失对鼠伤寒沙门菌致病性的影响

doi:10.16431/j.cnki.1671-7236.2024.08.029

摘要/Abstract

摘要： 【目的】沙门菌是常见的人畜共患病原菌,可引发多种食源性疾病,鼠伤寒沙门菌是沙门菌的重要血清型之一,具有重要的公共卫生研究意义。本试验旨在研究转运蛋白相关基因oppCDF对鼠伤寒沙门菌生物特性的影响,为新型沙门菌减毒疫苗的制备提供理论基础。【方法】本试验通过比较鼠伤寒沙门菌野生株及已成功构建的oppCDF基因缺失株的生化特性、遗传稳定性、耐药性、黏附性、侵袭力、半数致死量(LD₅₀)及小鼠肝脏、脾脏载菌量,分析oppCDF基因与鼠伤寒沙门菌生物特性之间的关系。【结果】oppC、oppD和oppF基因分别缺失后鼠伤寒沙门菌生化特性未发生明显改变且能稳定遗传,表明oppCDF基因表型未发生变化;与野生株STM LT2相比,基因缺失株STM LT2ΔoppC对红霉素变为敏感、氯霉素敏感性降低,STM LT2ΔoppD对头孢氨苄敏感性降低,STM LT2ΔoppF对复方新诺明敏感性增强,3株基因缺失株均对氨苄西林不敏感。3株基因缺失株LD₅₀检测结果显示,与野生株STM LT2相比,STM LT2ΔoppC毒力下降,但STM LT2ΔoppD、STM LT2ΔoppF毒力分别上升1.6和8.6倍;STM LT2ΔoppC的黏附性显著降低(P＜0.05),侵袭力降低,定植力极显著下降(P＜0.01);STM LT2ΔoppD的黏附性、侵袭力降低,但定植力极显著上升(P＜0.01);STM LT2ΔoppF的黏附性显著降低P＜0.05),但侵袭力显著增强P＜0.05),定植力极显著上升(P＜0.01)。【结论】oppC基因缺失可降低鼠伤寒沙门菌的致病性,但oppD、oppF基因缺失可增强鼠伤寒沙门菌的致病性,以上结果为沙门菌的致病机制研究提供了理论基础。

关键词: 鼠伤寒沙门菌; oppCDF基因; 生物学特性; 致病性

Abstract: 【Objective】 Salmonella is a common zoonotic pathogen that could cause a variety of foodborne diseases,and Salmonella Typhimurium is one of the important serotypes of Salmonella,which had important public health research significance.The purpose of this study was to study the effect of transporter-related gene oppCDF on the biological characteristics of Salmonella Typhimurium,and to provide a theoretical basis for the preparation of a new attenuated vaccine against Salmonella.【Method】 In this study,the relationship between oppCDF gene and the biological characteristics of Salmonella Typhimurium was analyzed by comparing the biochemical characteristics,genetic stability,drug resistance,adhesion,invasiveness,half lethal dose (LD₅₀) of Salmonella Typhimurium wild-type strain and the successfully constructed oppCDF gene deletion strains,as well as the bacterial load capacity of mouse liver and spleen.【Result】 The results showed that the biochemical characteristics of Salmonella Typhimurium with oppC,oppD and oppF genes deletion did not change significantly and could be inherited stably,indicating that the phenotype of oppCDF gene did not change.Compared with wild-type strain STM LT2,gene deletion strain STM LT2ΔoppC became susceptible to erythromycin,and the sensitivity of chloramphenicol decreased.STM LT2ΔoppD was less sensitive to cephalosporin,STM LT2ΔoppF was more sensitive to compound sulfamethoxazole,and all three gene deletion strains were insensitive to ampicillin.The LD₅₀ of the three gene deletion strains showed that,compared with wild-type strain STM LT2,the virulence of STM LT2ΔoppC decreased,but the virulence of STM LT2ΔoppD and STM LT2ΔoppF increased by 1.6 and 8.6 times,respectively.Compared with wild-type strain STM LT2,the adhesion of STM LT2ΔoppC was significantly reduced (P＜0.05),the invasiveness was reduced,and the colonization capacity was extremely significantly reduced (P＜0.01). The adhesion and invasiveness of STM LT2ΔoppD were decreased,but the colonization was extremely significantly increased (P＜0.01). And the adhesion of STM LT2ΔoppF was significantly decreased (P＜0.05),but the invasiveness and the colonization was extremely significantly increased (P＜0.01).【Conclusion】 oppC gene deletion could reduce the pathogenicity of Salmonella Typhimurium,but oppD and oppF genes deletion could enhance the pathogenicity of Salmonella Typhimurium,and the above results provided a theoretical basis for the study of the pathogenicity of Salmonella.

Key words: Salmonella Typhimurium; oppCDF gene; biological characteristics; pathogenicity

中图分类号:

S852.61

叶景芬, 张家莉, 陈世雄, 杨婉, 武绍碧, 杨琦. oppCDF基因缺失对鼠伤寒沙门菌致病性的影响[J]. 中国畜牧兽医, 2024, 51(8): 3519-3527.

YE Jingfen, ZHANG Jiali, CHEN Shixiong, YANG Wan, WU Shaobi, YANG Qi. Effects of oppCDF Gene Delection on the Pathogenicity of Salmonella Typhimurium[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(8): 3519-3527.

参考文献

[1] 唐宁,殷茵,任慧英,等.沙门氏菌高效价宽谱噬菌体的分离纯化及生物学特性测定[J].中国畜牧兽医,2021,48(7):2577-2583. TANG N,YIN Y,REN H Y,et al.Isolation,purification and detection of biological characteristics of a high-titer and wide-spectrum Salmonella phage[J]. China Animal Husbandry & Veterinary Medicine,2021,48(7):2577-2583.(in Chinese)
[2] 周南龙,丁永辉,李天森.鼠伤寒沙门菌毒力基因SptP的原核表达及生物信息学分析[J].中国畜牧兽医,2022,49(12):4734-4744. ZHOU N L,DING Y H,LI T S.Prokaryotic expression and bioinformatics analysis of Salmonella Typhimurium virulence gene SptP[J].China Animal Husbandry & Veterinary Medicine,2022,49(12):4734-4744.(in Chinese)
[3] 沈幸玲,丁康宁,王幽丛,等.火炭母缓解鼠伤寒沙门菌致小鼠肝损伤的机制研究[J].中国畜牧兽医,2022,49(7):2768-2777. SHEN X L,DING K N,WANG Y C,et al.Study on the mechanism of Polygonum chinense L.alleviating liver injury induced by Salmonella Typhimurium in mice[J].China Animal Husbandry & Veterinary Medicine,2022,49(7):2768-2777.(in Chinese)
[4] TANAKA K J,PINKETT H W.Oligopeptide-binding protein from nontypeable Haemophilus influenzae has ligand-specific sites to accommodate peptides and heme in the binding pocket[J].Journal of Biological Chemistry,2019,294(3):1070-1082.
[5] LEE E M,AHN S H,PARK J H,et al.Identification of oligopeptide permease (opp) gene cluster in Vibrio fluvialis and characterization of biofilm production by oppA knockout mutation[J].FEMS Microbiology Letters,2004,240(1):21-30.
[6] DETMERS F J,LANFERMEIJER F C,POOLMAN B.Peptides and atp binding cassette peptide transporters[J]. Research of Microbiology,2001,152(3-4):245-258.
[7] LAZAZZERA B A.The intracellular function of extracellular signaling peptides[J].Peptides,2001,22(10):1519-1527.
[8] COULTER S N,SCHWAN W R,NG E Y,et al. Staphylococcus aureus genetic loci impacting growth and survival in multiple infection environments[J]. Molecular Microbiology,1998,30(2):393-404.
[9] SASSETTI C M,RUBIN E J.Genetic requirements for mycobacterial survival during infection[J].Proceedings of the National Academy of Sciences of the United States of America,2003,100(22):12989-12994.
[10] PARK J T,RAYCHAUDHURI D,LI H,et al.Mppa,a periplasmic binding protein essential for import of the bacterial cell wall peptide L-alanyl-γ-D-glutamyl-meso-diaminopimelate[J].Journal of Bacteriology,1998,180(5):1215-1223.
[11] 张家莉.沙门菌oppCDF基因缺失株的构建及初步应用分析[D].贵阳:贵州大学,2023. ZHANG J L.Construction and preliminary application of oppCDF gene deletion strain of Salmonella[D].Guiyang:Guizhou University,2023.(in Chinese)
[12] 段世宇.鼠伤寒沙门菌Ⅲ型分泌系统相关基因缺失菌株的构建及初步应用[D].贵阳:贵州大学,2022. DUAN S Y.Construction and preliminary application of Salmonella Typhimurium type Ⅲ secretion system related gene deletion strain[D].Guiyang:Guizhou University,2022.(in Chinese)
[13] ALPUCHE A C,SWANSON J A,LOOMIS W P,et al.Salmonella Typhimurium activates virulence gene transcription within acidified macrophage phagosomes[J].Proceedings of the National Academy of Sciences of the United States of America,1992,89(21):10079-10083.
[14] HUANG L,HU J,SU Y,et al.Identification and characterization of three Vibrio alginolyticus non-coding RNAs involved in adhesion,chemotaxis,and motility processes[J].Frontiers in Cellular Infection Microbiology,2015,5:56.
[15] CROXEN M A,FINLAY B B.Molecular mechanisms of Escherichia coli pathogenicity[J].Nature Reviews Microbiology,2010,8(1):26-38.
[16] GAHRING L C,HEFFRON F,FINLAY B B,et al.Invasion and replication of Salmonella Typhimurium in animal cells[J].Infection and Immunity,1990,58(2):443-448.
[17] GRIFFITHS N M,HIRST B H,SIMMONS N L.Active intestinal secretion of the fluoroquinolone antibacterials ciprofloxacin,norfloxacin and pefloxacin;A common secretory pathway?[J].The Journal of Pharmacology and Experimental Therapeutics,1994,269(2):496-502.
[18] PANDREA I V,CARRIERE V,BARBAT A,et al.Postmitotic differentiation of colon carcinoma Caco-2 cells does not prevent reentry in the cell cycle and tumorigenicity[J]. Experimental and Molecular Pathology,2000,69(1):37-45.
[19] LIU W,HUANG L,SU Y,et al.Contributions of the oligopeptide permeases in multistep of Vibrio alginolyticus pathogenesis[J].MicrobiologyOpen,2017,6(5):e00511.
[20] SAMEN U,GOTTSCHALK B,EIKMANNS B J,et al.Relevance of peptide uptake systems to the physiology and virulence of Streptococcus agalactiae[J].Journal of Bacteriology,2004,186(5):1398-1408.
[21] LIU Y H,YOO B B,HWANG C A,et al.Involvement of a putative ATP-Binding Cassette (ABC) involved in manganese transport in virulence of Listeria monocytogenes[J].PLoS One,2022,17(5):e0268924.
[22] SAIF N A,HASHEM Y A,AMIN H M,et al. In silico and in vitro investigation of the distribution and expression of key genes in the fucose operon of Escherichia coli[J].Microorganisms,2023,11(5):1265.
[23] PICKARD J M,MAURICE C F,KINNEBREW M A,et al.Rapid fucosylation of intestinal epithelium sustains host-commensal symbiosis in sickness[J].Nature,2014,514(7524):638-641.
[24] BADÍA J,ROS J,AGUILAR J.Fermentation mechanism of fucose and rhamnose in Salmonella Typhimurium and Klebsiella pneumoniae[J].Journal of Bacteriology,1985,161(1):435-437.