CRISPR系统对鲍曼不动杆菌生物膜形成、耐药、抗噬菌体能力的调控作用研究

Acinetobacter baumannii have rather serious problem in drug resistance. They could also form into biofilms which would further increase the difficulty in treatment while easily causing infection outbreaks and promoting the spread of drug resistance. Thus the pathgen is a health threat worldwide. We previously found a negative correlation between the ability of drug resistance and biofilm formation in Acinetobacter baumannii, suggesting a common regulatory mechanism to protect the cells under pressure. CRISPR system is found mainly distributed in strong biofilm formers, and related genes expressed higher in biofilm condition. These results suggest that CRISPR systemmight be involved in the formation and survival of biofilms. To date, CRISPR system has rarely been studied in Acinetobacter baumannii. It has been reported that CRISPR system is involved in the regulation of drug and phage resistance in other genera, but the biological significance varies with species and evolutionary process. Some studies have predicted through mathematical models that CRISPR system can improve the resistance of biofilm to bacteriophage, and proposed a possible treatment plan based on the models. However, there is still no experimental data regarding this question. In this study, we plan to knockout cas1, coding CAS1 which involved in the recognition and integration of new sequences in the stage of immune adaptation, to analyze the biological role of CRISPR system in the regulation of biofilm formation, drug resistance and anti-phage ability, in the aim of providing new theoretical and technical support for the treatment of biofilm-related infections.

鲍曼不动杆菌耐药问题严重，而且能够形成生物膜，进一步增加治疗难度的同时极易引起感染暴发，促进耐药性的传播，是全球范围的健康威胁。本项目组发现鲍曼不动杆菌耐药性和生物膜形成能力负相关，提示存在共同的调控机制以维持压力下生存。CRISPR系统主要分布在生物膜形成能力强的菌株中，相关基因在生物膜中高表达，可能参与生物膜的形成和生存过程。CRISPR系统在鲍曼不动杆菌中研究较少，有报道在其他菌属中参与耐药性和抗噬菌体的调控，但生物学意义因种属和进化过程差异而不同。对于生物膜，有研究通过数学模型预测CRISPR系统可提高生物膜对噬菌体的抵抗力，并根据模型提出了可能的治疗方案，但尚无实验数据支持。本课题拟选择参与免疫功能适应阶段新序列识别和整合的CAS1蛋白编码基因cas1为切入点，分析CRISPR系统参与生物膜形成、耐药、抗噬菌体能力调控的生物学作用，为生物膜感染治疗提供新角度的理论和技术支持。

鲍曼不动杆菌耐药问题严重，而且能够形成生物膜，进一步增加治疗难度的同时极易引起感染暴发，促进耐药性的获得和传播，给感染防控造成极大挑战。前期研究发现，鲍曼不动杆菌生物膜与耐药能力负相关，提示存在共同的调控机制。CRISPR系统有可能在其中发挥作用，但它在鲍曼不动杆菌和生物膜中的研究都较少。本课题在前期研究基础上，选择CRISPR系统中参与免疫功能适应阶段新序列识别和整合的CAS1蛋白编码基因cas1为切入点，对其进行基因敲除，分析其参与生物膜形成、耐药、抗噬菌体能力调控的生物学作用。主要内容及结果包括：1. 构建鲍曼不动杆菌cas1基因敲除株。2. 观察cas1基因缺失对鲍曼不动杆菌形态、生物膜和耐药能力的影响，发现cas1敲除株菌落大且低平，褶皱增多，生物膜形成能力更强，但是在游离状态下对7种抗生素耐药性下降。形成生物膜后野生株耐药能力增强幅度高于cas1敲除株，对青霉素类、头孢类和碳青霉烯类抗生素尤其突出。3. 筛选全国多地污水，从中分离出8株新的鲍曼不动杆菌噬菌体，裂解谱分析总共能裂解90株菌中的21株，最佳感染复数在0.0001-1之间，最适pH为pH7-9。4. 比较野生株和cas1敲除株噬菌体转导效率，结果无显著差异。5. 考察在噬菌体压力下CRISPR系统获取新间隔序列的情况，序列比对发现实验菌株的间隔序列与筛选到的噬菌体基因组无片段匹配。噬菌体对宿主反复侵染后，在野生株和cas1敲除株中均未观察到CRISPR系统获得新的间隔序列。6. 比较野生株和cas1敲除株所形成的生物膜抗噬菌体能力差异，发现噬菌体对宿主生物膜的破坏能力不同，噬菌体A、S9等仅能少量破坏生物膜，而噬菌体S1株能将生物膜完全清除。本课题结果验证了前期提出的假设，进一步说明CRISPR系统能够帮助维持鲍曼不动杆菌正常的菌落形态，而且参与生物膜状态下耐药能力增强的调控。但对于外源基因水平转移效率未观察到显著的差异。研究结果为促进鲍曼不动杆菌感染的预防和治疗工作，研究其他重要病原菌的生物膜与耐药，以及使用噬菌体防治生物膜感染提供了新的理论支持。