Structural and Functional Characterization of the Type Three Secretion System (T3SS) Needle of Pseudomonas aeruginosa

Abstract

The type three secretion system (T3SS) is a macromolecular protein nano-syringe used by different bacterial pathogens to inject effectors into host cells. The extracellular part of the syringe is a needle-like filament formed by the polymerization of a 9-kDa protein whose structure and proper localization on the bacterial surface are key determinants for efficient toxin injection. Here, we combined in vivo , in vitro , and in silico approaches to characterize the Pseudomonas aeruginosa T3SS needle and its major component PscF. Using a combination of mutagenesis, phenotypic analyses, immunofluorescence, proteolysis, mass spectrometry, atomic force microscopy, electron microscopy, and molecular modeling, we propose a model of the P. aeruginosa needle that exposes the N-terminal region of each PscF monomer toward the outside of the filament, while the core of the fiber is formed by the C-terminal helix. Among mutations introduced into the needle protein PscF, D76A, and P47A/Q54A caused a defect in the assembly of the needle on the bacterial surface, although the double mutant was still cytotoxic on macrophages in a T3SS-dependent manner and formed filamentous structures in vitro . These results suggest that the T3SS needle of P. aeruginosa displays an architecture that is similar to that of other bacterial needles studied to date and highlight the fact that small, targeted perturbations in needle assembly can inhibit T3SS function. Therefore, the T3SS needle represents an excellent drug target for small molecules acting as virulence blockers that could disrupt pathogenesis of a broad range of bacteria.

Advances in Understanding of the Copper Homeostasis in Pseudomonas aeruginosa

“…T3SS is an injection apparatus used by P. aeruginosa to infuse effector proteins into host cells [ 56 , 57 , 58 ]. The large number of genes (about 36) involved in regulating and encoding for T3SS demonstrates the importance and effort carried out by P. aeruginosa to maintain this virulence factor [ 58 ].…”

High prevalence of atypical virulotype and genetically diverse background among Pseudomonas aeruginosa isolates from a referral hospital in the Brazilian Amazon

“…So far, only four effector exotoxins encoded by the exoS, exoU, exoT and exoY genes have been identified, which are variably present and expressed by P. aeruginosa strains. ExoS and ExoU are associated to invasive and cytotoxic phenotypes, respectively, and rarely concomitant detected, while ExoT and ExoY demonstrate few cytotoxic effects and are encoded by most of strains [8,[14][15][16][17]. Furthermore, survey of cytotoxic/exoU + virulotype has been highly recommended due to the impact of this exotoxin in patient's mortality, especially in isolates from high risk settings, such as ICUs [18,19].…”

Genome-Scale Screening of Vaccine Candidates Against Pseudomonas Aeruginosa

“…Another possible reason could be that the MBP tag (~ 40 kDa) affects the folding of the target antigen. To date, the conserved residues D76, P47, and Q54 are essential for the folding and polymerization of PscF, which ensures the needle structure of the T3SS of PA [32]. However, the folding and polymerization of PscF are not correct after fusion with MBP, which results in little protection.…”