Injectisome assembly primes Pseudomonas aeruginosa for type III secretion

Area of Science:

• Microbiology
• Bacterial Pathogenesis
• Molecular Biology

Background:

• Type III secretion systems (T3SS) are crucial virulence factors in Gram-negative pathogens like *Pseudomonas aeruginosa*, enabling toxin delivery into host cells.
• T3SS expression incurs a fitness cost, slowing bacterial growth and increasing susceptibility to immune detection, leading to heterogeneous expression within populations.
• A subset of bacteria, termed 'primed,' express the T3SS transcriptional activator ExsA and are poised for immediate T3SS activation, but the mechanism of priming is unknown.

Purpose of the Study:

• To investigate the mechanistic basis of bacterial priming for T3SS expression.
• To determine if cyclic AMP (cAMP) signaling influences the transition to a primed state.
• To elucidate the role of cAMP in regulating the ExsA activator and T3SS injectisome assembly.

Main Methods:

• Single-cell analysis to assess the impact of exogenous cAMP on bacterial priming.
• Whole-cell cryo-electron tomography to visualize T3SS injectisome formation.
• Correlation analysis of endogenous cAMP levels with primed bacterial proportions and genetic manipulation of cAMP levels.

Main Results:

• Exogenous cAMP significantly increased the proportion of primed, ExsA-expressing *Pseudomonas aeruginosa* cells.
• Cryo-electron tomography confirmed T3SS injectisome assembly in cAMP-stimulated primed cells.
• Endogenous cAMP levels varied between strains and correlated with the proportion of primed bacteria; manipulating cAMP levels altered this proportion.

Conclusions:

• cAMP acts as a key input signal that drives bacteria into a primed state for T3SS expression.
• This cAMP-mediated priming involves the bistable regulatory network controlling ExsA.
• The study reveals how cAMP signaling generates subpopulations of bacteria ready to deploy T3SS virulence factors.