细菌毒力基因筛选方法研究进展

doi:10.16431/j.cnki.1671-7236.2025.12.033

摘要/Abstract

摘要： 细菌感染是全球人和动物健康的重大挑战，致病菌携带有多种毒力基因，在感染过程中发挥各种独特的作用，从而实现在宿主的敌对环境中生存与繁殖。随着细菌耐药性的不断加剧，以及“超级细菌”的出现，使得细菌感染变得难以治疗，因此，利用有效的筛选方法发现新的细菌毒力基因，解析其致病机制，提供新的疫苗和药物靶点显得尤为迫切。笔者通过Sci-Hub、Science Driect、谷歌学术、Web of Science、PubMed、中国知网等文献检索网站，对细菌毒力基因相关文献进行了系统检索与分类整合。重点从基因突变、体内表达技术、组学、基因编辑及转座子技术等方面对筛选细菌毒力基因的方法及其应用进行了详尽阐述，以期为筛选细菌新的毒力基因提供思路和理论依据。

关键词: 细菌; 毒力基因; 筛选方法; 应用

Abstract: Bacterial infections pose a significant challenge to the health of humans and animals globally.Pathogenic bacteria carry multiple virulence genes that play various unique roles during infection,enabling them to survive and reproduce in the hostile environment of the host.With the increasing prevalence of antibiotic resistance and the emergence of “superbugs”,bacterial infections have become increasingly difficult to treat.Therefore,it is urgent to discover new bacterial virulence genes using effective screening methods,to elucidate their pathogenic mechanisms,and provide new vaccine and drug targets.This article systematically reviews and categorizes relevant literature on bacterial virulence genes using literature search websites such as Sci-Hub,ScienceDirect,Google Scholar,Web of Science,PubMed,and China National Knowledge Infrastructure (CNKI).It focuses on screening methods for bacterial virulence genes and their applications,particularly in the gene mutation,in vivo expression techniques,omics,gene editing and transposon bioinfoemation,aiming to provide insights and a theoretical foundation for the discovery of new bacterial virulence genes.

Key words: bacteria; virulence genes; screening methods; application

中图分类号:

S852.61

常璐, 刘蒙达, 孙翔翔, 南文龙, 胡莉萍, 刘建柱, 张皓博, 樊晓旭. 细菌毒力基因筛选方法研究进展[J]. 中国畜牧兽医, 2025, 52(12): 5891-5900.

CHANG Lu, LIU Mengda, SUN Xiangxiang, NAN Wenlong, HU Liping, LIU Jianzhu, ZHANG Haobo, FAN Xiaoxu. Advance in Screening Methods for Bacterial Virulence Genes[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(12): 5891-5900.

参考文献

[1] GBD 2019 ANTIMICROBIAL RESISTANCE COLLABORATORS.Global mortality associated with 33 bacterial pathogens in 2019:A systematic analysis for the Global Burden of Disease Study 2019[J].Lancet，2022，400(10369):2221-2248.
[2] 刘利，胡勇，丛延广.细菌毒力基因的筛选技术研究进展[J].医学综述，2011，17(12):1786-1788.
LIU L，HU Y，CONG Y G.Reserch progress on filtration technologies of bacterial virulence gene[J].Medical Recapitulate，2011，17(12):1786-1788.(in Chinese)
[3] XIE S T,DING L J,HUANG F Y,et al.VFG-Chip:A high-throughput qPCR microarray for profiling virulence factor genes from the environment[J].Environment International，2023，172:107761.
[4] HAUTEFORT I，HINTON J C.Measurement of bacterial gene expression in vivo[J].Philosophical Transactions of the Royal Society of London.Series B，Biological Sciences，2000，355(1397):601-611.
[5] 陈师勇，莫照兰，张振冬，等.细菌毒力基因体内表达检测技术研究进展[J].遗传，2005，27(3):505-511.
CHEN S Y，MO Z L，ZHANG Z D，et al.Research progress in detection technologies of bacterial virulence gene expression in vivo[J].Hereditas，2005，27(3):505-511.(in Chinese)
[6] HENSEL M，SHEA J E，GLEESON C，et al.Simultaneous identification of bacterial virulence genes by negative selection[J].Science，1995，269(5222):400-403.
[7] JUNG K W，LEE K T，BAHN Y S.A Signature-tagged mutagenesis (STM)-based murine-infectivity assay for Cryptococcus neoformans[J].Journal of Microbiology，2020，58(10):823-831.
[8] 王玉飞，王恒樑，黄留玉.转座子相关技术在微生物功能基因组研究中的应用[J].生物技术通讯，2006，3:405-408.
WANG Y F，WANG H L，HUANG L Y.Transposon-based strategies for microbial functional genomics[J].Letters in Biotechnology，2006，3:405-408.(in Chinese)
[9] 姚潇，黄留玉，杨伯伦，等.一种体内研究病原体致病机理的新方法——信号标签诱变技术[J].遗传，2002，24(6):721-726.
YAO X，HUANG L Y，YANG B L，et al.A novel approach to study pathogenesis of pathogens in vivo—Signature-tagged mutagenesis[J].Hereditas，2002，24(6):721-726.(in Chinese)
[10] LEHOUX D E，SANSCHAGRIN F，KUKAVICA-IBRULJ I，et al.Identification of novel pathogenicity genes by PCR signature-tagged mutagenesis and related technologies[J].Methods in Molecularbiology，2004，266:289-304.
[11] AUTRET N，CHARBIT A.Lessons from signature-tagged mutagenesis on the infectious mechanisms of pathogenic bacteria[J].FEMS Microbiology Reviews，2005，29(4):703-717.
[12] KUKAVICA-IBRULJ I，LEVESQUE R C.Signature-tagged mutagenesis[J].Methods in Molecular Biology，2014，1149:541-554.
[13] 袁佳杰，胡涵奎，余旭平.应用信号标签诱变技术检测细菌体内毒力基因的研究进展[J].上海畜牧兽医通讯，2008，4:20-21.
YUAN J J，HU H K，YU X P.Research progress in the detection of bacterial virulence genes in vivo by using signature-tagged mutagenesis technology[J]. Shanghai Journal of Animal Husbandry and Veterinary Medicine，2008，4:20-21.(in Chinese)
[14] MAHAN M J，SLAUCH J M，MEKALANOS J J.Selection of bacterial virulence genes that are specifically induced in host tissues[J].Science，1993，259(5095):686-688.
[15] 吴苗苗，黄明明，陈友梅，等.细菌体内诱生抗原筛选技术的研究进展[J].动物医学进展，2011，32(4):90-94.
WU M M，HUANG M M，CHEN Y M，et al.Advance in screening technologies of in vivo induced antigen of bacteria[J].Progress in Veterinary Medicine，2011，32(4):90-94.(in Chinese)
[16] JACKSON R W，GIDDENS S R.Development and application of in vivo expression technology (IVET) for analysing microbial gene expression in complex environments[J].Infectious Disorders Drug Targets，2006，6(3):207-240.
[17] MERRELL D S，CAMILLI A.Detection and analysis of gene expression during infection by in vivo expression technology[J].Philosophical Transactions of the Royal Society of London.Series B，Biological Sciences，2000，355(1397):587-599.
[18] 黎庶，胡福泉.病原微生物的体内诱导基因及相关研究方法简介[J].免疫学杂志，2011，27(4):350-354.
LI S，HU F Q.Technologies for in vivo induced gene induce[J].Immunological Journal，2011，27(4):350-354.(in Chinese)
[19] MAO W，ZHANG S，SUN J，et al.Identification of Haemophilus parasuis genes uniquely expressed during infection using in vivo-induced antigen technology[J].Veterinary Microbiology，2020，243:108650.
[20] HU Y，HUANG J，LI Q，et al.Use of in vivo-induced antigen technology to identify in vivo-expressed genes of Campylobacter jejuni during human infection[J].Journal of Microbiology and Biotechnology，2014，24(3):363-370.
[21] 李书光，张娜，王金良，等.应用体内诱导抗原技术筛选病原菌体内表达基因的研究进展[J].中国畜牧兽医，2012，39(7):87-89.
LI S G，ZHANG N，WANG J L，et al.Overview of identification of Ivi genes uniquely expressed during the infection with pathogenic bacteria using IVIAT[J].China Animal Husbandry & Veterinary Medicine，2012，39(7):87-89.(in Chinese)
[22] HANDFIELD M,PROGULSKE-FOX A，HILLMAN J D.In vivo induced genes in human diseases[J].Periodontology 2000，2005，38:123-134.
[23] ROLLINS S M，PEPPERCORN A,HANG L,et al.In vivo induced antigen technology (IVIAT)[J].Cellular Microbiology，2005，7(1):1-9.
[24] WENG T，CHEN G，LI N，et al.Identifying the in vivo-induced antigenic genes is a strategy to develop DNA vaccine against Nocardia seriolae in hybrid snakehead (Channa maculata ♀×Channa argus ♂)[J].Fish & Shellfish Immunology，2024，147:109410.
[25] 张念章，储岳峰，贺英，等.筛选细菌毒力基因方法的研究进展[J].中国兽医学报，2011，31(2):288-291.
ZHANG N Z，CHU Y F，HE Y，et al.Advance on methods of screening bacterial virulence genes[J].Chinese Journal of Veterinary Science，2011，31(2):288-291.(in Chinese)
[26] NAIDOO N，ZISHIRI O T.Comparative genomics analysis and characterization of Shiga toxin-producing Escherichia coli O157∶H7 strains reveal virulence genes，resistance genes，prophages and plasmids[J].BMC Genomics，2023，24(1):791.
[27] VIEIRA A A，PICCOLI B C，Y CASTRO T R，et al.Pipeline validation for the identification of antimicrobial-resistant genes in carbapenem-resistant Klebsiella pneumoniae[J].Scientific Reports，2023，13(1):15189.
[28] VANCAMP P J，PRASATH V B S，HASLAM D B，et al.MGS2AMR:A gene-centric mining of metagenomic sequencing data for pathogens and their antimicrobial resistance profile[J].Microbiome，2023，11(1):223.
[29] BALAJI A，KILLE B，KAPPELL A D，et al.SeqScreen:Accurate and sensitive functional screening of pathogenic sequences via ensemble learning[J].Genome Biology，2022，23(1):133.
[30] JIANG Y，HU X，FAN S，et al.RVFScan predicts virulence factor genes and hypervirulence of the clinical metagenome[J].Briefings in Bioinformatics，2023，24(6):bbad403.
[31] ARANGO-ARGOTY G，GARNER E，PRUDEN A，et al.DeepARG:A deep learning approach for predicting antibiotic resistance genes from metagenomic data[J].Microbiome，2018，6(1):23.
[32] BALLOUX F，BRONSTAD B O，VAN D L，et al.From theory to practice:Translating whole-genome sequencing (WGS) into the clinic[J].Trends in Microbiology，2018，26(12):1035-1048.
[33] SENGUPTA S，AZAD R K.Leveraging comparative genomics to uncover alien genes in bacterial genomes[J].Microbial Genomics，2023，9(1):mgen000939.
[34] 曹培丽.禽多杀性巴氏杆菌基因组重测序、转录组分析及其毒力基因鉴定[D].北京：中国农业科学院，2017.
CAO P L.Genome resequencing，transcriptome analysis and virulence gene identification of avian Pasteurella multocida[D].Beijing:Chinese Academy of Agricultural Sciences，2017.(in Chinese)
[35] WANG Z，GERSTEIN M，SNYDER M.RNA-Seq:A revolutionary tool for transcriptomics[J].Nature Reviews.Genetics，2009，10(1):57-63.
[36] POUNTAIN A W，JIANG P,YAO T，et al.Transcription-replication interactions reveal bacterial genome regulation[J].Nature,2024，626(7999):661-669.
[37] ZHANG Z，WU S，STENOIEN D L，et al.High-throughput proteomics[J].Annual Review of Analytical Chemistry(Palo Alto Calif)，2014，7:427-454.
[38] CHEN Y，LI E M，XU L Y.Guide to metabolomics analysis:A bioinformatics workflow[J].Metabolites,2022，12(4)：357.
[39] MIELKO K A，JABO AN＇G SKI S J，MILCZEWSKA J，et al.Metabolomic studies of Pseudomonas aeruginosa[J].World Bournal of Microbiology & Biotechnology，2019，35(11):178.
[40] 郑佳佳，薛松，杨晴晴，等.细菌半乳糖代谢通路及对细菌毒力的影响研究进展[J].微生物学报，2025，65(1):17-28.
ZHENG J J，XUE S，YANG Q Q，et al.Research progress on bacterial galactose metabolism pathway and its influence on bacterial virulence[J].Acta Microbiologica Sinica，2025，65(1):17-28.(in Chinese)
[41] BARRANGOU R，FREMAUX C，DEVEAU H，et al.CRISPR provides acquired resistance against viruses in prokaryotes[J].Science，2007，315(5819):1709-1712.
[42] 闫晓敏，李文泽，龚高，等.CRISPR/Cas9基因编辑技术在羊育种功能基因验证中的应用研究进展[J].黑龙江畜牧兽医，2024，1:29-35.
YAN X M，LI W Z，GONG G，et al.Research progress in application of CRISPR/Cas9 gene editing technology in functional gene verification in sheep breeding[J]. Heilongjiang Animal Science and Veterinary Medicine，2024，1:29-35.(in Chinese)
[43] 陈林，徐光明，王家乡，等.CRISPR/Cas9基因编辑技术在家禽中应用的研究进展[J].中国畜牧杂志，2023，59(7):1-8.
CHEN L，XU G M，WANG J X，et al.Research progress in the application of CRISPR/Cas9 gene editing technology in poultry[J]. Chinese Journal of Animal Science，2023，59(7):1-8.(in Chinese)
[44] SHARMA N，DAS A，NAIR A V，et al.CRISPR-Cas system positively regulates virulence of Salmonella enterica serovar Typhimurium[J].Gut Pathogens，2024，16(1):63.
[45] SAMPSON T R，WEISS D S.CRISPR-Cas systems:New players in gene regulation and bacterial physiology[J].Frontiers in Cellular Infection Microbiology，2014，4:37.
[46] BARRANGOU R，MARRAFFINI L A.CRISPR-Cas systems:Prokaryotes upgrade to adaptive immunity[J].Molecular Cell，2014，54(2):234-244.
[47] GUO C,MA X,GAO F,et al.Off-target effects in CRISPR/Cas9 gene editing[J].Frontiers in Bioengineering and Biotechnology,2023，11:1143157.
[48] AMITAI G，SOREK R.CRISPR-Cas adaptation:Insights into the mechanism of action[J].Nature Reviews，2016，14(2):67-76.
[49] HOU M，SUN S，FENG Q，et al.Genetic editing of the virulence gene of Escherichia coli using the CRISPR system[J].PeerJ，2020，8:e8881.
[50] 朱锦祥.基于转座子突变技术的毕赤酵母必须基因鉴定和功能基因筛选[D].上海：华东理工大学，2018.
ZHU J X.Identification of essential genes and screening of functional genes in Pichia pastoris based on transposon mutation technology[D].Shanghai:East China University of Science and Technology，2018.(in Chinese)
[51] YIN F,HU Y,BU Z,et al.Genome-wide identification of genes critical for in vivo fitness of multi-drug resistant porcine extraintestinal pathogenic Escherichia coli by transposon-directed insertion site sequencing using a mouse infection model[J].Virulence，2023,14(1):2158708.
[52] 魏立帆.利用转座子插入突变文库研究杀鱼爱德华氏菌代谢和毒力互作机制[D].上海：华东理工大学，2019.
WEI L F.Research on the interaction mechanism between metabolism and virulence of Edwardsiella piscicida using transposon insertion mutation library[D].Shanghai:East China University of Science and Technology，2019.(in Chinese)
[53] WARNER I A，KOK W J，MARTINELLI N，et al.Microbial Primer:Transposon directed insertion site sequencing (TraDIS):A high throughput method for linking genotype to phenotype[J].Microbiology (Reading)，2023，169(11):001385.
[54] VAN O T，CAMILLI A.Transposon insertion sequencing:A new tool for systems-level analysis of microorganisms[J].Nature Reviews.Microbiology，2013，11(7):435-442.
[55] GOODMAN A L，MCNULTY N P，ZHAO Y，et al.Identifying genetic determinants needed to establish a human gut symbiont in its habitat[J].Cell Host Microbe，2009，6(3):279-289.
[56] VAN O T，BODI K L，CAMILLI A.Tn-Seq:High-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms[J].Nature Methods，2009，6(10):767-772.
[57] LANGRIDGE G C，PHAN M D，TURNER D J，et al.Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants[J].Genome Research，2009，19(12):2308-2316.
[58] GAWRONSKI J D，WONG S M，GIANNOUKOS G，et al.Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung[J].Proceedings of the National Academy of Sciences of the United States of America，2009，106(38):16422-16427.
[59] GANDOUZI I,TERTIS M,CERNAT A,et al.A nanocomposite based on reduced graphene and gold nanoparticles for highly sensitive electrochemical detection of Pseudomonas aeruginosa through its virulence factors[J].Materials (Basel),2019，12(7):1180.
[60] RASHID J I A,KANNAN V,AHMAD M H,et al.An electrochemical sensor based on gold nanoparticles-functionalized reduced graphene oxide screen printed electrode for the detection of pyocyanin biomarker in Pseudomonas aeruginosa infection[J].Materials Science and Engineering.C, Materials for Biogical Applications 2020，120:111625.
[61] SANTIVERI R C，SISMAET J H D，KIMANI M，et al.Electrochemical detection of Pseudomonas aeruginosa in polymicrobial environments[J].Chemistry Select，2018，3(11):2926-2930.
[62] DRIEHUIS E，CLEVERS H.CRISPR/Cas9 genome editing and its applications in organoids[J].American Journal of Physiology.Gastrointestinal and Liver Physiology,2017，312(3):G257-G265.
[63] AGUIRRE GARCIA M,HILLION K,CAPPELIER J M，et al.Intestinal organoids:New tools to comprehend the virulence of bacterial foodborne pathogens[J].Foods，2022，11(1):108.
[64] XIANG T,WANG J,LI H L.Current applications of intestinal organoids:A review[J].Stem Cell Research & Therapy，2024，15(1):155.
[65] BOUFFI C,WIKENHEISER-BROKAMP K A,CHATURVEDI P，et al.In vivo development of immune tissue in human intestinal organoids transplanted into humanized mice[J].Nature Biotechnology，2023，41(6):824-831.
[66] YIN Y,ZHOU D.Organoid and enteroid modeling of Salmonella infection[J].Frontiers in Cellular and Infection Microbiology，2018，8:102.