Injectisome assembly primes Pseudomonas aeruginosa for Type III secretion

Many Gram-negative pathogens, including Pseudomonas aeruginosa , use a Type III Secretion System (T3SS) to intoxicate eukaryotic cells. The T3SS is an important virulence factor linked to increased morbidity and mortality in infections, yet its expression slows bacterial growth and activates innate immune receptors. T3SS genes are expressed heterogeneously, with T3SS-ON cells arising from ‘primed’ bacteria that express the T3SS transcriptional activator ExsA and respond immediately to T3SS activating signals. However, the mechanistic basis for priming is not known. ExsA is part of a complex protein-sequestration network, positively regulating its own expression as well as that of its anti-activator (ExsD), its anti-anti-activator (ExsC) and ExsC’s binding partner (ExsE). These four proteins create a bistable regulatory network. We hypothesized that transcription from a cAMP-dependent promoter upstream of ExsA could drive cells into the primed state and tested this at the single cell level. Exogenous cAMP increased the proportion of primed, ExsA-expressing cells, with whole cell cryo-electron tomography demonstrating assembly of T3SS injectisomes under these conditions. Intrastrain variation in endogenous cAMP levels correlated with strain-specific proportions of primed bacteria, while genetic manipulation of adenylate cyclases and cAMP phosphodiesterase altered primed population size. Priming occurred in planktonically cultured populations and was independent of type IV pilus assembly or retraction; however, mutation of genes in the Pil-Chp system, which regulates cAMP production, changed proportions of primed cells. This work demonstrates how endogenous and exogenous cAMP inputs into a bistable regulatory switch generate subpopulations of T3SS-primed cells poised to respond to activating signals.