A versatile dual-color bacterial reporter system highlights two distinct Pseudomonas aeruginosa type 3 secretion system intracellular populations

ABSTRACT Pseudomonas aeruginosa is known to infect both phagocytic and non-phagocytic host cells, where it exhibits a complex intracellular lifestyle strategy for host survival that is governed by the expression and regulation of its type III secretion system (T3SS). To better understand such complexities in the context of niche bacterial populations in P. aeruginosa and other gram-negatives, here we construct and characterize a novel and versatile dual-reporter plasmid, Constitutive GFP, Variable mScarlet-I (pCG-VmS), whose utility proves useful for longitudinal assays of transcriptional reporter activity in vitro, ex vivo, and in whole animals. As an added utility, we show that pCG-VmS can also be used for translational fusions to monitor bacterial cellular protein localization. We then exploit pCG-VmS to visualize P. aeruginosa T3SS expression in a human corneal cell line that identifies a distinct intracellular “quiescent” population, where the T3SS is never expressed. An ex vivo murine eye infection model is then used to verify the existence of the two ‘T3SS-on” versus ‘T3SS-off’ expression profiles in infected tissues. In sum, pCG-VmS allows for novel spatiotemporal insights into P. aeruginosa protein localization and infection routes and can be tailored to other plasmid backbones amenable to different gram-negative bacteria.IMPORTANCEIn this study, we develop a novel, well-optimized fluorescent dual-reporter system, pCG-VmS, which can be used for both transcriptional monitoring and protein localization. We demonstrate its use with a range of in vitro and in vivo experiments with Pseudomonas aeruginosa and whole-animal imaging of squid colonization with Vibrio fischeri. After showing pCG-VmS efficacy and versatility, we use the plasmid to simultaneously visualize the naturally occurring type-3 secretion system (T3SS) active (T3SS-on) and “quiescent” (T3SS-off) populations during intracellular infection of a human corneal epithelial cell line and the murine eye. Taken together, pCG-VmS is well-balanced in its optical traits and can be used for a variety of both in vitro and in vivo studies to help separate and further interrogate difficult niche spatiotemporal bacterial populations.