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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.

SUBMITTER: Lombardi C

PROVIDER: S-EPMC6455054 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Lombardi Charlotte C Tolchard James J Bouillot Stephanie S Signor Luca L Gebus Caroline C Liebl David D Fenel Daphna D Teulon Jean-Marie JM Brock Juliane J Habenstein Birgit B Pellequer Jean-Luc JL Faudry Eric E Loquet Antoine A Attrée Ina I Dessen Andréa A Job Viviana V

Frontiers in microbiology 20190329

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 Pseudomona ...[more]

PMID: 31001211

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Project description:Herein we describe a pathogenic role for the Pseudomonas aeruginosa type three secretion system (T3SS) needle tip complex protein, PcrV, in causing lung endothelial injury. We first established a model in which P. aeruginosa wild type strain PA103 caused pneumonia-induced sepsis and distal organ dysfunction. Interestingly, a PA103 derivative strain lacking its two known secreted effectors, ExoU and ExoT [denoted PA103 (?U/?T)], also caused sepsis and modest distal organ injury whereas an isogenic PA103 strain lacking the T3SS needle tip complex assembly protein [denoted PA103 (?PcrV)] did not. PA103 (?U/?T) infection caused neutrophil influx into the lung parenchyma, lung endothelial injury, and distal organ injury (reminiscent of sepsis). In contrast, PA103 (?PcrV) infection caused nominal neutrophil infiltration and lung endothelial injury, but no distal organ injury. We further examined pathogenic mechanisms of the T3SS needle tip complex using cultured rat pulmonary microvascular endothelial cells (PMVECs) and revealed a two-phase, temporal nature of infection. At 5-hours post-inoculation (early phase infection), PA103 (?U/?T) elicited PMVEC barrier disruption via perturbation of the actin cytoskeleton and did so in a cell death-independent manner. Conversely, PA103 (?PcrV) infection did not elicit early phase PMVEC barrier disruption. At 24-hours post-inoculation (late phase infection), PA103 (?U/?T) induced PMVEC damage and death that displayed an apoptotic component. Although PA103 (?PcrV) infection induced late phase PMVEC damage and death, it did so to an attenuated extent. The PA103 (?U/?T) and PA103 (?PcrV) mutants grew at similar rates and were able to adhere equally to PMVECs post-inoculation indicating that the observed differences in damage and barrier disruption are likely attributable to T3SS needle tip complex-mediated pathogenic differences post host cell attachment. Together, these infection data suggest that the T3SS needle tip complex and/or another undefined secreted effector(s) are important determinants of P. aeruginosa pneumonia-induced lung endothelial barrier disruption.

Dataset Information

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

Publications

Structural and Functional Characterization of the Type Three Secretion System (T3SS) Needle of <i>Pseudomonas aeruginosa</i>.

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