单细菌表型的高通量表征和控制

Characterization and control of bacterial phenotypes at the single cell level

致病菌在感染宿主的过程中需要精确调控自身的基因表达,从而完成对宿主的入侵、适应等过程,例如形成生物被膜,所以对病原菌在生物被膜形成过程中表型、行为的定量分析是联系其基因特定表达与感染性疾病如何发生的纽带,具有重大科学意义.本文首先综述了如何通过发展一些高通量的原位表征方法去量化分析病原菌在单细胞层次的表型,其中包括细菌运动行为、黏附行为和社会行为相关的表型,并讨论了如何在单细菌水平上去理解这些表型在生物被膜发展过程中扮演的角色.之后,简单介绍如何通过引入光遗传学技术结合定量工程生物学的方法去控制这些与感染性疾病发生密切相关的表型,如控制细菌的黏附、生物被膜的形成、细菌在宿主内的定殖,以及细菌在宿主内感染模式的转变等.

Microbial biofilms comprise surface-associated, multicellular, highly structured matrix-enclosed, morphologicallycomplex microbial communities. The subject of biofilms has received much attention, in part owing to scientificcommunities’ acknowledgements that biofilm formation of pathogen can cause many acute or chronic diseases that usuallycannot be treated by antibiotic therapies, such as Pseudomonas aeruginosa, one of the highly prevalent opportunistichuman pathogens, that is the leading cause of morbidity and mortality in immunocompromised patients and in patientssuffering from cystic fibrosis (CF). We have learned that biofilm-forming bacteria are phenotypically distinct from those ofplanktonically grown cells, because they express genes in a pattern differs profoundly from that of their free-swimming,planktonic counterparts. Therefore, understanding the roles played by bacterial behaviors and phenotypes in thedevelopment of biofilms is an emerging link to disease pathogenesis.In the first half of this review, we present how to develop characteristic approaches by using high-throughputmicroscopical techniques together with automatic image-processing methods and customized microfluidic system to in situvisualize bacterial cells and investigate their behaviors at the single cell scale. We mainly focused on the pathogen of P.aeruginosa and provided an overview of recent related progress using those approaches in characterization of bacterialphenotypes at the single cell level in the process of biofilm formation, especially in areas of bacterial twitching motility,attachment phenotypes, social behaviors and architecture of biofilms. For instance, by monitoring single-cell motilitybehaviors, researchers observed single-cell twitching chemotaxis in developing biofilms and revealed that cells canmodulate twitching motility behaviors to survive or thrive in slowly changing and locally heterogeneous naturalenvironments.We then referred to optogenetics, which is a technology that allows targeted, fast control of precisely defined events inbiological systems by expressing exogenous genes coding for light-sensitive proteins. In optogenetics, light as inducer canbe applied more precisely in the concentration, time and space dimensions than traditional effectors such as chemicalmolecules. In the field of microbiology, different light responsive sensors, such as UV, blue, green, red and far-redtranscriptional regulation systems, have been engineered, and application of these optogenetic systems enables littleperturbations and unprecedented spatiotemporal resolution in controlling bacterial behaviors that can reveal new insightsinto biological function. For example, researchers have used blue light-switchable gene regulation system to optogeneticcontrol of gene expression to spatiotemporally manipulate biofilm formation and pattern cells with high-resolution. Thesestudies provide the ability to grow structured biofilms, with applications toward an improved understanding of naturalbiofilm communities, as well as the engineering of living biomaterials and bottom-up approaches to microbial consortiadesign. In addition, optogenetics enables characterization of bacterial gene circuit dynamics with optically programmedgene expression signals, which could advance understanding of gene circuit dynamics and cell signaling pathways. We alsoproposed that the development of optogenetics in bacteriology is limited by the requirement of engineered light-sensitiveproteins for specific regulation networks and new methods for manipulation of living bacterial cells at the single-cell level.This review concludes with some expectations on the foreseeable future application of those single-cell analysisapproaches in biofilms-related fields, and a discussion of innovative anti-biofilm strategies that are inspired from thestudies of characterization and control of single-cell phenotypes.