Relationship and Role of CRISPR-Cas System in Antimicrobial
发布时间：2023-01-09 12:53
空肠弯曲杆菌是一种革兰氏阴性菌,被认为是引起细菌性胃肠炎的主要原因之一。空肠弯曲杆菌是大约90%弯曲杆菌病的病因。空肠弯曲杆菌病被认为是自我限制的,感染后症状在几天内就会解决。然而,抗生素被用来治疗免疫受损的病人,特别是患有肠外感染或细菌血症的患者。抗菌药物的合理使用对保护食品用动物和人类的健康有着重要的作用,但不适当的使用可能会导致抗菌药物的耐药性。已有报导,弯曲杆菌对氟喹诺酮类、大环内酯类、β-内酰胺类和氨基糖苷类产生耐药。尤其是氟喹诺酮类和大环内酯类药物的交叉耐药与空肠弯曲菌的临床分离株有关。成簇的规律间隔短回文重复序列（CRISPR）是一种保护原核微生物免受外来遗传因素影响的适应性免疫系统。空肠弯曲杆菌有一个CRISPR-Cas免疫系统,它不仅可以识别而且可以干扰入侵者的基因组DNA。该免疫系统可分为三种类型:CRISPR-系统I型、II型和III型。CRISPR-Ⅱ型（Nmeni亚型）存在于空肠弯曲杆菌中,由Cas1、Cas2和Cas9/csn 1蛋白组成。CRISPR-cas系统可以保护细菌免受外来入侵者的影响,但是CRISPR-cas系统在增强抗菌药耐药性方面的作用仍存在...
【文章页数】：184 页
【学位级别】：博士
【文章目录】：
摘要
ABSTRACT
LIST OF ABBREVIATIONS
1.INTRODUCTION
1.1 GENERAL CHARACTERISTICS OF CAMPYLOBACTER SPECIES
1.2 EPIDEMIOLOGICAL SIGNIFICANCE
1.3 ANTIBIOTIC RESISTANCE IN CAMPYLOBACTER
1.3.1 Quinolone resistance
1.3.2 Resistance to tetracycline
1.3.3 Resistance to macrolides
1.3.4 Resistance to aminoglycosides
1.4 CRISPR-CAS SYSTEM
1.5 CRISPR-CAS SYSTEM INVOLVEMENT IN ANTIMICROBIAL RESISTANCE
1.6 THE CRISPR-CAS SYSTEM IN C.JEJUNI
1.7 RNA-SEQ BASED TRANSCRIPTOME SEQUENCING
1.8 AIMS AND OBJECTIVES OF STUDY
2.MATERIALS AND METHODS
2.1 CHEMICALS,MEDIA AND REAGENTS
2.2 INSTRUMENTS
2.3 ANTIMICROBIAL AGENTS
2.4 TEST SOLUTIONS PREPARATION
2.5 BACTERIAL STRAINS
2.6 ISOLATION AND GROWTH CONDITIONS OF CLINICAL ISOLATES OF C.JEJUNI
2.6.1 Sample collection and transport
2.6.2 Isolation and confirmation of clinical isolates of C.jejuni by PCR
2.6.3 Antimicrobial susceptibility testing of clinical isolates of C.jejuni
2.7 DETECTION OF CRISPR-SPACER FROM THE POULTRY ISOLATES OF C.JEJUNI
2.8 Recovery of C.jejuni NCTC11168 and C.coli ATCC33559
2.9 DETECTION OF CRISPR GENES EXPRESSION BY RT-QPCR AFTER EXPOSURE TO DIFFERENT ANTIMICROBIALS
2.9.1 Reverse transcription
2.9.2 Quantitative PCR
2.10 CONSTRUCTION OF CJ1523C(CAS9)DELETION MUTANT
2.10.1 Extraction of C.jejuni NCTC11168 genomic DNA
2.10.2 Vector construction
2.10.3 Construction of mutant vector
2.10.4 Fusion PCR
2.10.5 Ligation with vector or plasmid
2.10.6 Plasmid extraction
2.10.7 Construction ofΔcas9 mutant
2.10.8 Confirmation of mutant
2.11 Complementation of Δcas9 mutant strain
2.12 ANTIMICROBIAL SUSCEPTIBILITY TESTING OFΔCAS9 MUTANT STRAIN
2.13 IN VITRO RESISTANCE DEVELOPMENT AGAINST DIFFERENT ANTIMICROBIALS
2.14 DETERMINATION OF STANDARD GROWTH CURVE AND GROWTH CURVE RESPONSE AGAINST DIFFERENT ANTIMICROBIAL AGENTS
2.15 NATURAL TRANSFORMATION
2.16 RNA-SEQ BASED TRANSCRIPTOME ANALYSIS
2.16.1 RNA extraction and purification
2.16.2 RNA-Seq protocol
2.16.3 Validation of library
2.16.4 Differential gene expression analysis
2.16.5 Bioinformatics analysis
2.16.6 Validation by RT-qPCR
2.16.7 Quantitative PCR
2.17 STATISTICAL ANALYSIS
3.RESULTS
3.1 CONFIRMATION OF C.JEJUNI FROM POULTRY ISOLATES
3.2 ANTIMICROBIAL SUSCEPTIBILITY OF POULTRY ISOLATES
3.3 SPACER IDENTIFICATION AND ANALYSIS FROM THE POULTRY ISOLATES OF C.JEJUNI
3.4 CRISPR-CAS GENES EXPRESSION IN STANDARD STRAINS AFTER DRUG TREATMENT
3.5 EXTRACTED BACTERIAL DNA RESULTS
3.6 CONSTRUCTION OFΔCAS9 MUTANT VECTORS
3.6.1 Homologous arm and amplification of screening genes
3.6.2 Fragments fusion by fusion PCR
3.6.3 Verification of recombinant plasmid
3.6.4 Recombinant plasmid extraction results
3.6.5 Δcas9 Mutant validation
3.7 ROLE OF CAS9 IN ANTIMICROBIAL SUSCEPTIBILITY
3.8 ROLE OF CAS9 IN RESISTANCE DEVELOPMENT
3.9 ROLE OF CAS9 ON GROWTH UNDER DRUG EXPOSURE
3.10 ROLE OF CAS9 ON TRANSFORMATION OF RESISTANCE
3.11 RNA-SEQ BASED TRANSCRIPTOME ANALYSIS
3.11.1 RNA extraction and quality
3.11.2 Quality of raw reads
3.11.3 Differential gene analysis
3.11.4 Bioinformatics analysis
3.12 VALIDATION OF RNA-SEQ RESULTS BY RT-QPCR
4.DISCUSSION
5.SUMMARY
6.PUBLISHED REVIEW ARTICLE 1
6.1 AVERTING PHAGE ADSORPTION/RECEPTOR MUTATION
6.2 BLOCKAGE OF INVADER DNA
6.3 RESTRICTION–MODIFICATION SYSTEMS
6.4 ABORTIVE INFECTION
6.5 INTERFERENCE DURING ASSEMBLY
6.6 CRISPR:BACTERIA ADAPTIVE IMMUNE SYSTEM
6.7 COUNTER ATTACK OF INVADERS AGAINST THE CRISPR-CAS SYSTEM
6.8 FUNCTION OF ANTI-CRISPR PROTEINS EXHIBITED BY VARIOUS MECHANISMS
6.9 ANTI-CRISPR GENES IN MGES
6.10 SUMMARY
6.11 FUTURE PERSPECTIVES
7.PUBLISHED REVIEW ARTICLE 2
7.1 HISTORY OF CRISPR-CAS SYSTEM
7.2 FATE OF CRISPR SYSTEM AGAINST INVADING DNA
7.3 CLASSIFICATION OF CRISPR-CAS SYSTEM
7.3.1 Type I CRISPR system
7.3.2 Type II CRISPR system
7.3.3 Type III CRISPR system
7.4 ROLE OF CRISPR BEYOND ADAPTIVE IMMUNITY OF PROKARYOTES
7.5 THE EVOLUTION OF CRISPR SYSTEM PLAYING AS A DEFENSE
7.6 CRISPR SYSTEM AND ITS REGULATION
7.7 GENE REGULATION BY CRISPR AND ITS ROLE IN PATHOGENESIS
7.8 CRISPR-CAS SYSTEM FACILITATES EVOLUTION OF THE GENOME
7.9 CRISPR BASED APPLICATIONS
7.9.1 Genome engineering
7.9.2 Use of CRISPR-cas9 as a tool of genome editing in disease
7.9.3 Use of CRISPR-cas9 in curing human genetic diseases
7.9.4 CRISPR-cas9 potential to target diseases developed by epigenetic alterations
7.10 FUTURE DIMENSIONS
7.11 CONCLUSION
REFERENCE
APPENDIX
SEQUENCES DETAIL OF3H,KAN AND5H
SEQUENCING MAP OF RECOMBINANT PLASMID P3HKAN5H-3
M13-F primer sequencing results
M13-R primer sequencing results
W1-F primer sequencing results
W1-R primer sequencing results:
W2-R primer sequencing results:
W3-R primer sequencing results:
RAW DATA
RNA-SEQ RAW DATA
ACKNOWLEDGEMENT
CURRICULUM VITAE
【参考文献】：
期刊论文
[1]DNA甲基转移酶分类、功能及其研究进展[J]. 王志刚,吴建新. 遗传. 2009(09)
本文编号：3729219
【文章页数】：184 页
【学位级别】：博士
【文章目录】：
摘要
ABSTRACT
LIST OF ABBREVIATIONS
1.INTRODUCTION
1.1 GENERAL CHARACTERISTICS OF CAMPYLOBACTER SPECIES
1.2 EPIDEMIOLOGICAL SIGNIFICANCE
1.3 ANTIBIOTIC RESISTANCE IN CAMPYLOBACTER
1.3.1 Quinolone resistance
1.3.2 Resistance to tetracycline
1.3.3 Resistance to macrolides
1.3.4 Resistance to aminoglycosides
1.4 CRISPR-CAS SYSTEM
1.5 CRISPR-CAS SYSTEM INVOLVEMENT IN ANTIMICROBIAL RESISTANCE
1.6 THE CRISPR-CAS SYSTEM IN C.JEJUNI
1.7 RNA-SEQ BASED TRANSCRIPTOME SEQUENCING
1.8 AIMS AND OBJECTIVES OF STUDY
2.MATERIALS AND METHODS
2.1 CHEMICALS,MEDIA AND REAGENTS
2.2 INSTRUMENTS
2.3 ANTIMICROBIAL AGENTS
2.4 TEST SOLUTIONS PREPARATION
2.5 BACTERIAL STRAINS
2.6 ISOLATION AND GROWTH CONDITIONS OF CLINICAL ISOLATES OF C.JEJUNI
2.6.1 Sample collection and transport
2.6.2 Isolation and confirmation of clinical isolates of C.jejuni by PCR
2.6.3 Antimicrobial susceptibility testing of clinical isolates of C.jejuni
2.7 DETECTION OF CRISPR-SPACER FROM THE POULTRY ISOLATES OF C.JEJUNI
2.8 Recovery of C.jejuni NCTC11168 and C.coli ATCC33559
2.9 DETECTION OF CRISPR GENES EXPRESSION BY RT-QPCR AFTER EXPOSURE TO DIFFERENT ANTIMICROBIALS
2.9.1 Reverse transcription
2.9.2 Quantitative PCR
2.10 CONSTRUCTION OF CJ1523C(CAS9)DELETION MUTANT
2.10.1 Extraction of C.jejuni NCTC11168 genomic DNA
2.10.2 Vector construction
2.10.3 Construction of mutant vector
2.10.4 Fusion PCR
2.10.5 Ligation with vector or plasmid
2.10.6 Plasmid extraction
2.10.7 Construction ofΔcas9 mutant
2.10.8 Confirmation of mutant
2.11 Complementation of Δcas9 mutant strain
2.12 ANTIMICROBIAL SUSCEPTIBILITY TESTING OFΔCAS9 MUTANT STRAIN
2.13 IN VITRO RESISTANCE DEVELOPMENT AGAINST DIFFERENT ANTIMICROBIALS
2.14 DETERMINATION OF STANDARD GROWTH CURVE AND GROWTH CURVE RESPONSE AGAINST DIFFERENT ANTIMICROBIAL AGENTS
2.15 NATURAL TRANSFORMATION
2.16 RNA-SEQ BASED TRANSCRIPTOME ANALYSIS
2.16.1 RNA extraction and purification
2.16.2 RNA-Seq protocol
2.16.3 Validation of library
2.16.4 Differential gene expression analysis
2.16.5 Bioinformatics analysis
2.16.6 Validation by RT-qPCR
2.16.7 Quantitative PCR
2.17 STATISTICAL ANALYSIS
3.RESULTS
3.1 CONFIRMATION OF C.JEJUNI FROM POULTRY ISOLATES
3.2 ANTIMICROBIAL SUSCEPTIBILITY OF POULTRY ISOLATES
3.3 SPACER IDENTIFICATION AND ANALYSIS FROM THE POULTRY ISOLATES OF C.JEJUNI
3.4 CRISPR-CAS GENES EXPRESSION IN STANDARD STRAINS AFTER DRUG TREATMENT
3.5 EXTRACTED BACTERIAL DNA RESULTS
3.6 CONSTRUCTION OFΔCAS9 MUTANT VECTORS
3.6.1 Homologous arm and amplification of screening genes
3.6.2 Fragments fusion by fusion PCR
3.6.3 Verification of recombinant plasmid
3.6.4 Recombinant plasmid extraction results
3.6.5 Δcas9 Mutant validation
3.7 ROLE OF CAS9 IN ANTIMICROBIAL SUSCEPTIBILITY
3.8 ROLE OF CAS9 IN RESISTANCE DEVELOPMENT
3.9 ROLE OF CAS9 ON GROWTH UNDER DRUG EXPOSURE
3.10 ROLE OF CAS9 ON TRANSFORMATION OF RESISTANCE
3.11 RNA-SEQ BASED TRANSCRIPTOME ANALYSIS
3.11.1 RNA extraction and quality
3.11.2 Quality of raw reads
3.11.3 Differential gene analysis
3.11.4 Bioinformatics analysis
3.12 VALIDATION OF RNA-SEQ RESULTS BY RT-QPCR
4.DISCUSSION
5.SUMMARY
6.PUBLISHED REVIEW ARTICLE 1
6.1 AVERTING PHAGE ADSORPTION/RECEPTOR MUTATION
6.2 BLOCKAGE OF INVADER DNA
6.3 RESTRICTION–MODIFICATION SYSTEMS
6.4 ABORTIVE INFECTION
6.5 INTERFERENCE DURING ASSEMBLY
6.6 CRISPR:BACTERIA ADAPTIVE IMMUNE SYSTEM
6.7 COUNTER ATTACK OF INVADERS AGAINST THE CRISPR-CAS SYSTEM
6.8 FUNCTION OF ANTI-CRISPR PROTEINS EXHIBITED BY VARIOUS MECHANISMS
6.9 ANTI-CRISPR GENES IN MGES
6.10 SUMMARY
6.11 FUTURE PERSPECTIVES
7.PUBLISHED REVIEW ARTICLE 2
7.1 HISTORY OF CRISPR-CAS SYSTEM
7.2 FATE OF CRISPR SYSTEM AGAINST INVADING DNA
7.3 CLASSIFICATION OF CRISPR-CAS SYSTEM
7.3.1 Type I CRISPR system
7.3.2 Type II CRISPR system
7.3.3 Type III CRISPR system
7.4 ROLE OF CRISPR BEYOND ADAPTIVE IMMUNITY OF PROKARYOTES
7.5 THE EVOLUTION OF CRISPR SYSTEM PLAYING AS A DEFENSE
7.6 CRISPR SYSTEM AND ITS REGULATION
7.7 GENE REGULATION BY CRISPR AND ITS ROLE IN PATHOGENESIS
7.8 CRISPR-CAS SYSTEM FACILITATES EVOLUTION OF THE GENOME
7.9 CRISPR BASED APPLICATIONS
7.9.1 Genome engineering
7.9.2 Use of CRISPR-cas9 as a tool of genome editing in disease
7.9.3 Use of CRISPR-cas9 in curing human genetic diseases
7.9.4 CRISPR-cas9 potential to target diseases developed by epigenetic alterations
7.10 FUTURE DIMENSIONS
7.11 CONCLUSION
REFERENCE
APPENDIX
SEQUENCES DETAIL OF3H,KAN AND5H
SEQUENCING MAP OF RECOMBINANT PLASMID P3HKAN5H-3
M13-F primer sequencing results
M13-R primer sequencing results
W1-F primer sequencing results
W1-R primer sequencing results:
W2-R primer sequencing results:
W3-R primer sequencing results:
RAW DATA
RNA-SEQ RAW DATA
ACKNOWLEDGEMENT
CURRICULUM VITAE
【参考文献】：
期刊论文
[1]DNA甲基转移酶分类、功能及其研究进展[J]. 王志刚,吴建新. 遗传. 2009(09)
本文编号：3729219
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