Construction And Application Of The Light-controlled Two-component System In Pseudomonas Aeruginosa

Optogenetics is an emerging technique allows targeted,fast control of precisely defined events in biological systems with light.Compared with traditional methods,optogenetics has numerous advantages,such as high spatial temporal resolution,and low toxic effects.Nowadays,optogenetic tools are gaining wide acceptance in biological research,and have been proven to be extremely useful for neural activity monitoring,genome editing,bioprinting,and drug delivering.One key part of basic microbiology is pathogenic bacteria research,because infections caused by pathogens are critical threats to human health.Therefore,it will be of great significance to develop and establish new methods for the study of bacterial pathogenicity.In this study,we developed a new optogenetic system for the precise spatiotemporal modulation of infection signaling cascade in Pseudomonas aeruginosa(P.aeruginosa).Moreover,we introduced our system into Caenorhabditis elegans(C.elegans)host model to replenish the toolkit of host-pathogen research.We first engineered a light-regulated system based on the GacS-GacA two-component system(TCS)in P.aeruginosa.The global regulatory GacS-GacA TCS plays a leading role in governing the transition between planktonic and biofilm modes of lifestyle,as well as acute and chronic modes of infection.We replaced the sensor module of GacS with LOV photoreceptor to change input signal specificity,and therefore produced the photoactivatable derivatives of GacS named YGS24.The light-responsive YGS24 kinase can reversibly activate the cognate response regulator GacA by phosphotransfer.C.elegans-P.aeruginosa pathogenesis model was introduced for the study of host-pathogen interactions.The results showed that YGS24 has the ability to stimulate the pathogenicity of P.aeruginosa in both slow-killing model and paralysis-killing model upon blue-light exposure.Furthermore,using a combination of microfluidics and microscopy techniques,we successfully induced the pathogenicity of P.aeruginosa in C.elegans with spatial resolution.In conclusion,we provided a novel light-mediated YGS24-GacA TCS which expands the optogenetic toolkit.When tested in host models,this optogenetic system enables the light-dependent pathogenicity switch of bacteria cells and correspondingly tunes the susceptibility of C.elegans to P.aeruginosa-mediated killing.This useful optogenetic tool will help understanding pathogenic mechanism and interactions between host and pathogen,and will thereby expedite the development of innovative therapeutics.