Application of Genomic Approaches to Bacterial Pathogenesis and Mechanisms of Antimicrobial Resistance

基因组方法在细菌发病机制和抗菌素耐药性机制中的应用

• 批准号: 10692209
• 负责人: John Dekker
• 金额: $ 186.75万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10692209
• 关键词: Acute; Alleles; Amino Acids; Animals; Antibiotic Resistance; Antibiotic Therapy; Antibiotic susceptibility; Antibiotics; Antimicrobial Resistance; Antimicrobial susceptibility; Area; Bacteremia; Bacteroides fragilis; Bioreactors; Bordetella; Burkholderia; Ceftazidime; Cell Line; Centers for Disease Control and Prevention (U.S.); Chromosomes; Citric Acid Cycle; Clinical; Collaborations; Collection; Combinatorics; Communication; Complex; Consequentialism; Cryoelectron Microscopy; DNA; DNA Methylation; DNA Polymerase III; DNA Replication Proofreading; DNA biosynthesis; Development; Dipeptides; Elements; Environment; Enzymes; Episome; Event; Evolution; Gene Expression; Gene Mutation; Genes; Genetic; Genetic Drift; Genetic Transcription; Genome; Genomic approach; Genomics; Gluconeogenesis; Goals; Host Defense; Human; Immunocompromised Host; In Vitro; Individual; Infection; Link; Mediating; Mediator of activation protein; Metabolic; Metabolism; Methylation; Methyltransferase; Methyltransferase Gene; Mismatch Repair; Mismatch Repair Deficiency; Modern Medicine; Morbidity - disease rate; Multi-Drug Resistance; Mutation; Mutation Spectra; National Institute of Diabetes and Digestive and Kidney Diseases; Nature; Organism; Pathogenesis; Pathway interactions; Patients; Pattern; Phenotype; Phylogenetic Analysis; Physiological; Physiology; Plasmids; Prevalence; Proteins; Pseudomonas aeruginosa; Pump; Resistance; Structure; System; Systems Biology; Tazobactam; Testing; Treatment Protocols; United States National Institutes of Health; Work; Zoonoses; antibiotic resistant infections; attributable mortality; base; chronic infection; clinical center; design; efflux pump; emerging pathogen; epigenome; exhaustion; experimental study; fitness; genome sequencing; human microbiota; in vivo; infectious disease treatment; member; metabolic phenotype; metabolome; metabolomics; methylome; mortality; nanopore; novel; pathogen; pathogenic bacteria; reconstitution; reconstruction; resilience; resistance gene; resistance mechanism; screening; transcriptome; transcriptome sequencing; transcriptomics; trend; whole genome

MDR gram-negative bacterial pathogens undergo selection and evolution in the natural context of antibiotic treatment in a human host, though important features of host context are often not included in studies of AMR. Additionally, other features underlying bacterial resilience in the context of infection including the ability to evade host defenses often synergize with specific AMR mechanisms and consequently have linked evolutionary relationships. Our work employs a systems biology approach to study the evolutionary mechanisms by which resistance - defined broadly to include resistance to antibiotics and to host defenses - emerges in the natural context of host infection. Current work is organized around three primary projects: Project 1: Mechanisms by which mismatch repair (MMR) deficiencies can facilitate rapid evolution of antimicrobial resistance in P. aeruginosa. P. aeruginosa is an important pathogen responsible for significant nosocomial morbidity and mortality. We previously demonstrated that evolved MMR deficiencies may be dynamically exploited by P. aeruginosa to facilitate rapid acquisition of mutations mediating resistance to two critical broad-spectrum antibiotics, ceftazidime-avibactam (CZA), and ceftolozane-tazobactam (C/T), in the context of acute clinical infection (Khil et al, mBio, 2019). Then, using a combination of directed in vitro microevolution and high throughput genomic and transcriptomic analysis, we characterized the detailed mutational and transcriptional events underlying the development of CZA resistance in wild type (WT) and MMR-deficient P. aeruginosa and identified a number of potential novel resistance mechanisms unique to the MMR deficient isolates. Work performed in 2022 included screening a number of potential novel resistance genes identified in the study above through the introduction of mutations into a common genetic background using a two allele exchange system followed by antibiotic susceptibility testing. This work identified the MexVW efflux pump as a previously unappreciated mediator of resistance to CZA and C/T (Dulanto Chiang et al, submitted 2022). Ongoing work seeks to characterize the structure and function of this pump. RNA-seq experiments were performed to characterize the expression of the mexV and mexW genes, and strategies were devised to express the MexV and MexW proteins in cell lines for purification to facilitate cryo-EM structural studies; purification and reconstitution of the complex in collaboration with Susan Buchanan's lab in NIDDK is ongoing. Other work in 2022 has involved initiating a comprehensive genomic study of antimicrobial resistance in a 20-year collection of P. aeruginosa bacteremia isolates (350) from the NIH Clinical Center. This work is focusing particularly on mechanisms and targets that we have defined in our separate in vitro evolution work above, and for evidence of hypermutation. Potential novel targets will be cloned into the isogenic lab strain system for further study. This collection will also allow us to look historically at changes in the prevalence of different resistance mechanisms, including examining for MexVW associated mutations. All isolates will undergo genomic sequencing and characterization of antimicrobial resistance mechanisms. The genomes of the first 50 of the isolates have been sequenced and assembled and sequencing of the remainder is ongoing. Project 2: In vivo evolution of an emerging zoonotic pathogen Bordetella hinzii in an immunocompromised host. This project applies system biology approaches, including genomics, transcriptomics, and metabolomics to understanding the adaptive evolution in the emerging pathogen Bordetella hinzii following presumptive zoonotic transfer from an animal reservoir to an individual with IL-12RB1 deficiency. Initial work (Launay et al, Nature Communications, 2021) demonstrated that a mutation in the DNA Pol III epsilon proofreading subunit resulted in a replicative DNA proofreading deficiency and drove genetic divergence among the isolates over the course of 45 months of persistent infection. Within these proofreading-deficient lineages, secondary compound hypermutators with complex alterations in mutational spectra emerged and dominated clinical cultures for a period of 12 months, demonstrating their superior in vivo fitness. Evidence of mutational targeting and positive selection was present in multiple sequential enzymes of the tricarboxylic acid cycle and gluconeogenesis pathways, suggesting specialized metabolic adaptation to the host environment. To study the transcriptional landscape of adaptation in these isolates, >100 transcriptomes were sequenced with Illumina short reads and a subset underwent nanopore-based direct long read RNA sequencing. Work completed during FY 2022 focused on detailed metabolic phenotyping of the entire set of isolates and computational integration with large scale transcriptional mapping to characterize how the metabolome was reprogrammed during host adaptation in terms of underlying gene expression and mutations in individual enzymes. The preliminary finding from this work is that the efficiency of amino acid and dipeptide transport/metabolism appears to have been substantially modified during the course of host adaptation. Current ongoing experiments are designed to quantify the fitness consequences of these adaptations in physiologically appropriate media environments using a bioreactor. A second project to study within-host evolution and adaptation of Burkholderia vietnamiensis isolates from another patient with IL-12RB1 deficiency has been initiated and will be pursued in parallel with the B. hinzii analysis. Genomic sequencing of 180 B. vietnamiensis isolates has been completed for this project and genomic analysis of host adaptation is ongoing. Project 3: Comprehensive whole genome sequencing and genomic analysis of a historical collection of clinical Bacteroides fragilis group (BFG) isolates spanning decades. Members of the BFG are important constituents of the human microbiota, but they can also behave as significant pathogens in certain contexts. Historically, antimicrobial susceptibility patterns in BFG isolates were largely predictable, allowing effective use of empiric treatment regimens. Alarming increases in AMR have recently necessitated reconsideration of empiric strategies. To understand the genomic basis of these AMR trends, we have initiated an effort to sequence a large group of clinical BFG isolates spanning a period of five decades. Previous involved long-read nanopore-based sequencing of 386 BFG genomes facilitating end-to-end contiguous assemblies of chromosomes, episomes, and plasmids. Detailed phylogenetic reconstruction and exhaustive annotation of AMR elements in both genome and plasmids has been performed. Work completed in FY 2022 involved using nanopore sequencing in combination with recently developed computational approaches to characterize the 6mA, 5mC, and 4mC methylomes of 260 BFG isolates selected on the basis of AMR phenotype. This work has revealed that single BFG species harbor hundreds of DNA methylation motifs, with most individual motif combinations occurring uniquely in single isolates, implying immense unsampled combinatoric diversity within BFG epigenomes. Additionally, we have refined existing computational approaches to mine methylase genes from the sequenced genomes and identified more than 6000 methyltransferase genes within the genome set, explaining this profound diversity of methylation motifs. Many of the observed methylated motifs are located adjacent to, or within, the gene bodies of AMR genes. Further work will study whether AMR phenotypes are regulated by methylation.

MDR革兰氏阴性细菌病原体在人类宿主的抗生素处理自然背景下经历选择和进化，尽管宿主环境的重要特征通常不包括在AMR研究中。此外，在感染的背景下，包括逃避宿主防御能力的细菌韧性的其他特征通常与特定的AMR机制协同作用，因此与进化关系联系起来。我们的工作采用系统生物学方法来研究抗药性（包括对抗生素的抗药性和宿主防御）的进化机制 - 在宿主感染的自然背景下出现。当前的工作是围绕三个主要项目组织的： 项目1：不匹配修复（MMR）缺陷可以促进铜绿假单胞菌中抗菌抗性的快速进化的机制。铜绿假单胞菌是负责明显的医院发病率和死亡率的重要病原体。我们先前证明，铜绿假单胞菌可能会动态利用进化的MMR缺乏症，以促进对两种关键抗药性介导的突变，从而介导两种关键的宽光谱抗生素，头孢兹兹兹省 - 阿acavibactam（CZA）和ceftolozane-tazobactam（C/T）（C/T）（C/T）（C/T）（C/T）。然后，使用定向的体外微进化和高吞吐量基因组和转录组分析的组合，我们表征了野生型（WT）中CZA耐药性发展的详细突变和转录事件，以及MMR缺陷型P. p. aeruginosa，并确定了潜在的新型阻力机制独特的MMR隔离率的许多潜在的新型阻力机制。 2022年进行的工作包括筛选上述研究中通过两个等位基因交换系统将突变引入常见遗传背景中的许多潜在的新型抗性基因，然后进行抗生素敏感性测试。这项工作将MEXVW外排泵确定为先前未认可的对CZA和C/T的抗性介体（Dulanto Chiang等人，提交了2022年）。正在进行的工作旨在表征该泵的结构和功能。进行了RNA-Seq实验以表征MEXV和MEXW基因的表达，并设计了在细胞系中表达MEXV和MEXW蛋白的策略以纯化以促进冷冻EM结构研究；与苏珊·布坎南（Susan Buchanan）在NIDDK的实验室合作的纯化和重建正在进行中。 2022年的其他工作涉及从NIH临床中心收集20年的铜绿假单胞菌分离株（350），对抗菌耐药性进行了全面的基因组研究。这项工作特别关注我们在上面的单独的体外进化工作中定义的机制和目标，并提供了超称的证据。潜在的新靶标将被克隆到Isogenic Lab菌株系统中，以进行进一步研究。该集合还将使我们能够从历史上研究不同抗药性机制的发生率的变化，包括检查MEXVW相关突变。所有分离株将接受基因组测序和抗菌耐药机制的表征。已经对分离株的前50种基因组进行了测序并组装，其余的测序也在进行中。 项目2：在免疫功能低下的宿主中，新兴的人畜共患病原体hinzii的体内进化。该项目采用系统生物学方法，包括基因组学，转录组学和代谢组学来理解从动物储层到患有IL-12RB1缺乏症个体的个体的人们的人畜共患病后，新兴病原体hinzii的适应性演变。最初的工作（Launay等人，自然通信，2021）表明，DNA Polii epsilon校对亚基的突变导致复制性DNA校对缺乏症，并在45个月的持续感染过程中脱离了分离株的遗传差异。在这些校对缺陷的谱系中，出现了突变光谱中具有复杂变化的次级化合物高血压剂，并以12个月的形式占据了临床培养，表明其在体内适应性优越。突变靶向和阳性选择的证据存在于三羧酸周期的多种顺序酶和糖异生途径中，这表明对宿主环境的专门代谢适应。为了研究这些分离株适应性的转录景观，用Illumina简短读数对> 100个转录组进行了测序，并进行了基于纳米孔的直接长读RNA测序。 在2022财年完成的工作重点是整个分离株和计算整合的详细代谢表型，并具有大规模转录映射，以表征在宿主适应过程中如何根据基因表达和单个酶中的突变对代谢组进行重编程。这项工作的初步发现是，在宿主适应过程中，氨基酸和二肽传输/代谢的效率似乎已实质性改变。当前正在进行的实验旨在使用生物反应器在生理上适当的培养基环境中量化这些适应性的适应性后果。已经开始了第二个研究伯克霍尔德越南人越南人分离株的项目，从另一名IL-12RB1缺乏症患者中分离出来，并将与Hinzii分析并行进行。为该项目完成了180 B.越南分离株的基因组测序，并且宿主适应的基因组分析正在进行中。 项目3：跨越数十年的临床细菌片（BFG）分离株的历史集合的全面基因组测序和基因组分析。 BFG的成员是人类微生物群的重要成分，但在某些情况下它们也可以作为重要的病原体。从历史上看，BFG分离株中的抗菌敏感性模式在很大程度上是可以预测的，从而有效地使用了经验治疗方案。 AMR的令人震惊的增加需要重新考虑经验策略。为了了解这些AMR趋势的基因组基础，我们开始努力对跨越五十年的大量临床BFG分离株进行测序。以前涉及386个BFG基因组的长阅读基于纳米孔的测序，促进染色体，插发体和质粒的端到端连续组件。已经进行了详细的系统发育重建和基因组和质粒中AMR元素的详尽注释。 在2022财年完成的工作涉及使用纳米孔测序与最近开发的计算方法结合使用，以表征基于AMR表型选择的260 BFG分离株的6MA，5MC和4MC甲基组。这项工作表明，单个BFG物种具有数百种DNA甲基化基序，大多数单独的基序组合都是在单个分离株中独特出现的，这意味着BFG表观基因瘤中巨大的未采样组合多样性。此外，我们从测序的基因组中完善了现有的计算方法来挖掘甲基酶基因，并确定了基因组集合中的6000多个甲基转移酶基因，从而解释了甲基化基序的多样性。许多观察到的甲基化基序位于AMR基因的基因体或内部。进一步的工作将研究AMR表型是否通过甲基化调节。