病原细菌中转移酶固有耐药组的发现及其进化研究

Pathogenic bacteria with intrinsic resistance to antibiotics are a worrisome health problem. Intrinsic antibiotic resistance of bacterial pathogens is involved in the cellular impermeability and activity of efflux pumps. However, few reports refer to intrinsic resistance of transferase in pathogens. Previous works show that the transferase genes located in conservative chromosome loci have the resistance phenotype in some pathogen, which remind us that there exists a new intrinsic resistome. Based on the preceding studies, this project will apply bioinformatic methods and find intrinsic resistance genes of transferase by alignment of sequenced genome of pathogens and known resistance genes of transferases and nucleotide diversity analysis; study the resistance activities of the intrinsic transferase genes in vitro and in vivo; identify the function of the intrinsic resistance gene of transferase by determination of the product structure of enzyme reaction. Simultaneously, we will explore the evolution and origin of the intrinsic resistance genes of transferase by analysis of evolutionary relationship with konwn resistance genes of transferases located in mobile elements. Furthermore, we will perform directed evolution of intrinsic resistance genes of transferases and change their resistance activities for other types of antibiotics and predict the evolution of intrinsic resistome of transferases. Accomplishment of these works will not only be able to reveal that the transferase genes located in conservative chromosomal loci are one of the source of intrinsic resistome, expand and deepen the understanding of intrinsic resistance, but also provide a theoretical basis for prediction of resistance development, prevention and treatment of clinical infections and new drugs development.

病原细菌的固有耐药是一个与人类健康密切相关的重要问题，涉及细胞膜渗透和外排泵等功能，而有关病原菌中转移酶固有耐药的研究却鲜有报道。前期工作显示位于某些病原菌染色体上位置保守的转移酶基因具有耐药表型，提示了新的固有耐药组的存在。为此，本项目拟采用生物信息学方法，通过与已知转移酶耐药基因库的比对及核苷酸多态性分析，发现转移酶固有耐药基因；研究其体外表达活性及对原菌的耐药贡献；通过鉴定固有转移酶酶促反应产物结构，确定该酶的修饰功能；分析转移酶固有耐药基因与已知转移酶耐药基因的进化关系，探讨固有转移酶耐药基因的进化及起源；定向进化转移酶固有耐药基因，改变其底物谱或获得新的抗性，预测转移酶固有耐药组的进化。本项目的完成不仅可以揭示位于染色体上位置保守的转移酶基因是固有耐药组的来源之一，将拓宽和加深对固有耐药的认识，并且对临床预测耐药、防治感染和新药研发提供理论依据。

本项目主要研究病原菌的固有耐药，而细菌耐药性由获得性耐药和固有耐药组成。固有耐药是细菌对某类抗生素的天然耐药，由固有耐药基因决定。固有耐药基因是指存在于某类细菌染色体上位置保守的与耐药相关的一类基因。固有耐药基因的发现可以为新药研制提供药物作用靶标，并且固有耐药基因可以通过移动元件的捕获而成为获得性耐药的来源。目前，对细菌固有耐药的研究还比较欠缺。在本项目的研究中，我们在不动杆菌包括临床常见的鲍曼不动杆菌（Acinetobacter baumanii, IIa）、皮特不动杆菌（A. pittii, IIa）、吉伦伯不动杆菌（A. gyllenbergii, IIc）、小不动杆菌（A. parvus, IIc）中发现了固有的新型氨基糖苷腺苷转移酶ANT(3")-II。在坦都不动杆菌（A. tandoii）、鲁迪不动杆菌（A. rudis）、吉伦伯不动杆菌（A. gyllenbergii）、吉洛不动杆菌（A. guillouiae）和波西米亚不动杆菌（A. bohemicus）中发现了5个新型的碳青霉烯水解D型β-内酰胺酶（OXA）家族。在临床病原菌铜绿假单胞菌中发现了新型的氨基糖苷磷酸转移酶APH，以及在致病菌副溶血弧菌中发现了新型氯霉素乙酰转移酶type C CAT（CATC）。进一步的研究显示，ant(3")-II基因可以在不动杆菌中进行频繁的水平转移，并且这种水平转移的方式是同源重组、而非移动元件介导的。新型blaOXAs基因在鲍曼不动杆菌中表达时对碳青霉烯类抗生素（美罗培兰、亚胺培兰、厄他培南）有明显的耐药表型，同时也对青霉素和阿莫西林有高耐药性，并进一步揭示了不动杆菌是OXA的储存库。新型aph基因在原宿主铜绿假单胞菌中不表达，而在大肠杆菌中表达，并具有明显的耐药表型，表明了其对原宿主是隐性的耐药基因。我们还发现了CATC在副溶血弧菌中存在广泛的变异体，这些天然变体具有明显差异的氯霉素耐药水平，显示了固有耐药基因的进化多样性。这些固有耐药基因的发现及其耐药机制的解析为我们深入认识固有耐药以及防治相关病原菌导致的耐药性奠定了理论基础。