Engineering Gac/Rsm Signaling Cascade for Optogenetic Induction of the Pathogenicity Switch in Pseudomonas aeruginosa

Abstract

Bacterial pathogens operate by tightly controlling the pathogenicity to facilitate invasion and survival in host. While small molecule inducers can be designed to modulate pathogenicity to perform studies of pathogen−host interaction, these approaches, due to the diffusion property of chemicals, may have unintended, or pleiotropic effects that can impose limitations on their use. By contrast, light provides superior spatial and temporal resolution. Here, using optogenetics we reengineered GacS of the opportunistic pathogen Pseudomonas aeruginosa, signal transduction protein of the global regulatory Gac/Rsm cascade which is of central importance for the regulation of infection factors. The resultant protein (termed YGS24) displayed significant lightdependent activity of GacS kinases in Pseudomonas aeruginosa. When introduced in the Caenorhabditis elegans host systems, YGS24 stimulated the pathogenicity of the Pseudomonas aeruginosa strain PAO1 in a brain−heart infusion and of another strain, PA14, in slow killing media progressively upon blue-light exposure. This optogenetic system provides an accessible way to spatiotemporally control bacterial pathogenicity in defined hosts, even specific tissues, to develop new pathogenesis systems, which may in turn expedite development of innovative therapeutics.

Light-regulated gene expression in Bacteria: Fundamentals, advances, and perspectives

“…Analogous to the YF1 design (Möglich et al, 2009), the activity of the Pseudomonas aeruginosa GacS SHK was put under light control by exchanging its sensor domain for the LOV module from B. subtilis YtvA (Cheng et al, 2021). Use of the PATCHY method (Ohlendorf et al, 2016) facilitated the exploration of multiple SHK designs that differed in the length and sequence of the linker between the LOV photosensor and histidine-kinase effector modules.…”

“…Another study used optogenetics to control by light the virulence of P. aeruginosa via the YGS24:GacA TCS (Cheng et al, 2021). Blue light promoted the transcription of two endogenous small regulatory RNAs (sRNA) which in turn relieved translational repression of several virulence factors e.g., components of the secretion system.…”

“…Doing so enhanced the bacterial pathogenicity and caused the killing of the animals. The ability to control pathogenicity of bacteria inside animals paves the way towards kinetic and mechanistic studies of the infection process (Cheng et al, 2021).…”

Red-Light-Induced Genetic System for Control of Extracellular Electron Transfer

“…2−7 Optogenetic circuits have been utilized to control gene expression to regulate many microbial processes, such as biochemical production, 8−11 biofilm formation, 12,13 and bacterial infection. 14 Although optogenetics has been implemented in model bacteria, such as Escherichia coli, 6,12 Bacillus subtilis, 15 and Pseudomonas aeruginosa, 14,16 there is still a need to extend these systems to other hosts for specific microbial processes.…”

Programming the lifestyles of engineered bacteria for cancer therapy

“…Using optogenetics, we developed a genetic circuit in engineered bacteria that allows dynamic manipulation of bacterial lifestyles (planktonic, biofilm and lysis lifestyle) to precisely control the process of bacterial adhesion, colonization and drug release, with near-infrared (NIR) light in the BMCT process. The deep tissue penetration of NIR enables spatio-temporal and non-invasive control of the genetic circuit [ 39 , 40 ] and is widely used to trigger certain behaviors of bacteria in vivo [ 41 , 42 ]. In addition, the lighting power density (LPD) of NIR used to program the lifestyles of engineered bacteria H017 shows a reduction of 3 orders of magnitude compared with that of bacteria-based photothermal therapy (PTT) [ 43 , 44 ], which enables widespread clinical use outside of dermatological indications.…”