细菌细胞周期及其关键蛋白的单细菌水平多参数定量关系研究

Pathogen infection poses the greatest threat to global health, and the continuous emergence of antibiotic resistance demands the development of novel classes of antibiotics. Antibiotics targeting at bacterial cell division and cell cycle proteins may lead to promising antibacterial countermeasures. However, the inherent stochasticity of cell cycle and division processes renders it difficulty to quantitative study the relationship between bacterial cell division and cell cycle proteins by using ensemble averaged approaches. Taking advantage of the high sensitivity, rapid speed, and multiparameter and quantitative analysis capability of the high sensitivity flow cytometry (HSFCM) developed in our laboratory, we aim to establish an in situ and high throughput platform for the comprehensive study of bacterial cell division status and cell cycle proteins through the integration with CRISPR/Cas9 gene editing system and biarsenical tetrasysteine motif for protein labeling. This can be achieved by the simultaneous measurement of the light scattering and multiple fluorescence signals emitted from single bacterial cells as they transit individually through the highly focused laser beam. Statistical distribution of cell cycle protein can be determined quickly by analyzing thousands of individual cells within a couple of minutes, revealing the heterogeneous expression pattern that is otherwise masked by the ensemble analysis. With dual fluorescence labeling of chromosome DNA and cell cycle protein, this platform can be used to study the relationship between cell cycle proteins and cell division for a better understanding of the fundamental roles of cell cycle proteins in bacterial cell cycle progression. Meanwhile, synthetic biology method will be employed to precisely regulate the expression of targeting cell cycle proteins. So that, how the cell cycle proteins regulate and coordinate DNA replication and cell cycle progression can be monitored. This newly established method will reveal the intrinsic expression heterogeneity of cell cycle proteins and facilitate the correlation with cell cycle status in a single bacterial cell. Thus, this method will provide an advanced tool for the study of signal transduction and molecular mechanisms of bacterial cell division and for the development of new strategies for designing novel drugs against pathogenic bacteria and antimicrobial resistance.

细胞周期是生命的核心过程，细菌细胞周期及其关键蛋白是研发新型作用机制抗菌药物的潜在靶标。本项目拟利用实验室自行研制的超高灵敏流式检测装置的灵敏、快速、多参数定量分析的独特性能，结合CRISPR/Cas基因编辑技术及双砷染料－四半胱氨酸体系的蛋白标记优点，通过对单个细菌散射光和多色荧光信号的同时检测，发展原位、高通量的细菌细胞周期及其关键蛋白的单细菌水平研究方法，研究细菌细胞周期关键蛋白与细胞周期的关系，为揭示细胞周期关键蛋白在细菌细胞周期不同阶段的作用机理提供先进的实验手段。引入合成生物学技术，通过精确定量调控目标蛋白的表达，探索细胞周期关键蛋白如何调控细胞内DNA复制与细胞周期进程。本研究将建立的单细菌水平研究方法能快速揭示细胞周期关键蛋白的表达异质性并实现其与细胞周期的单细菌水平关联，对于揭示细菌如何在分子水平上调控细胞周期的作用机理和研发对抗致病菌及细菌耐药的新型策略具有重要价值。

细胞周期是生命的核心过程，将DNA复制和细菌细胞周期关键蛋白结合有望全面理解细菌细胞周期机制。本项目通过对单个细菌散射光和多色荧光信号的同时检测，发展原位、高通量的细菌细胞周期及其关键蛋白的单细菌水平研究方法；研究细菌细胞周期关键蛋白与细胞周期的关系，考察细胞周期关键蛋白在细菌细胞周期不同阶段的作用机理；定量调控目标蛋白的表达，探索细胞周期关键蛋白如何调控细胞内DNA复制与细胞周期进程。在单细菌水平实现细胞周期关键蛋白的表达异质性与细胞周期的关联。取得的重要进展如下：.1) 构建细胞周期关键蛋白多肽原位标记系统。成功对与细胞分裂，形态维持以及DNA复制事件相关的三个关键蛋白FtsZ，MreB 和 DnaA原位构建四半胱氨酸标签，实现细胞周期关键蛋白的单细菌水平检测。.2) 考察了关键蛋白在一个细胞周期不同阶段B，C，D的表达情况，发现三种关键蛋白在一个慢细胞周期里的表达量以各自功能依赖的方式变化。.3) 建立单细菌水平双荧光同时考察细菌细胞周期关键蛋白与DNA含量关系的流式分析方法。首次发现发现关键蛋白 FtsZ，MreB和DnaA表达与DNA含量存在不相关和正相关的关系由细菌所含的染色体数目决定。.4) 通过对染色体数目从1到16拷贝的10种不同营养培养基中生长的细菌的总蛋白和细胞内平均染色体复制起始点数目相关联，首次发现了满足快慢生长的生长规律，首次指出偏离SMK生长定律的内在节点。.5) 分别通过转录和翻译的抑制和染色体数目的扰动实现关键蛋白表达与DNA含量关系的转换，证明染色体数目是细胞周期的关键转折的决定因素。.6) 构建MreB-Flag/DnaA-TC的基因组双突变菌株，通过免疫荧光标记FtsZ、MreB，双砷染料标记DnaA，再结合DNA染色建立细菌细胞周期及多个关键蛋白同时的表征方法。.7) 此外，通过单细菌水平研究解析了抗毒素蛋白在细菌应激中的调控作用。（ACS Chemical Biology., 2019, 14, 2859–2866）.8) 建立了单细菌水平蛋白－蛋白相互作用定量检测新方法。（Talanta 2021, 233: 122549）.9) 运用噬菌体“鸡尾酒”策略实现了多种致病菌的同时、定量检测。（Anal. Chim. Acta. 2021, 1166: 338596）