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In the absence of effector proteins, the Pseudomonas aeruginosa type three secretion system needle tip complex contributes to lung injury and systemic inflammatory responses.

ABSTRACT: 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.

SUBMITTER: Audia JP

PROVIDER: S-EPMC3842252 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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In the absence of effector proteins, the Pseudomonas aeruginosa type three secretion system needle tip complex contributes to lung injury and systemic inflammatory responses.

Audia Jonathon P JP Lindsey Ashley S AS Housley Nicole A NA Ochoa Courtney R CR Zhou Chun C Toba Michie M Oka Masahiko M Annamdevula Naga S NS Fitzgerald Meshann S MS Frank Dara W DW Alvarez Diego F DF

PloS one 20131127 11

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 (ΔU/ΔT)], also caused sepsis and modest distal organ injury whereas an isogeni ...[more]

PMID: 24312357

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Project description:The Pseudomonas aeruginosa type III secretion system (T3SS) needle comprised of multiple PscF subunits is essential for the translocation of effector toxins into human cells, facilitating the establishment and dissemination of infection. Mutations in the pscF gene provide resistance to the phenoxyacetamide (PhA) series of T3SS inhibitory chemical probes. To better understand PscF functions and interactions with PhA, alleles of pscF with 71 single mutations altering 49 of the 85 residues of the encoded protein were evaluated for their effects on T3SS phenotypes. Of these, 37% eliminated and 63% maintained secretion, with representatives of both evenly distributed across the entire protein. Mutations in 14 codons conferred a degree of PhA resistance without eliminating secretion, and all but one were in the alpha-helical C-terminal 25% of PscF. PhA-resistant mutants exhibited no cross-resistance to two T3SS inhibitors with different chemical scaffolds. Two mutations caused constitutive T3SS secretion. The pscF allele at its native locus, whether wild type (WT), constitutive, or PhA resistant, was dominant over other pscF alleles expressed from nonnative loci and promoters, but mixed phenotypes were observed in chromosomal ΔpscF strains with both WT and mutant alleles at nonnative loci. Some PhA-resistant mutants exhibited reduced translocation efficiency that was improved in a PhA dose-dependent manner, suggesting that PhA can bind to those resistant needles. In summary, these results are consistent with a direct interaction between PhA inhibitors and the T3SS needle, suggest a mechanism of blocking conformational changes, and demonstrate that PscF affects T3SS regulation, as well as carrying out secretion and translocation.IMPORTANCEP. aeruginosa effector toxin translocation into host innate immune cells is critical for the establishment and dissemination of P. aeruginosa infections. The medical need for new anti-P. aeruginosa agents is evident by the fact that P. aeruginosa ventilator-associated pneumonia is associated with a high mortality rate (40 to 69%) and recurs in >30% of patients, even with standard-of-care antibiotic therapy. The results described here confirm roles for the PscF needle in T3SS secretion and translocation and suggest that it affects regulation, possibly by interaction with T3SS regulatory proteins. The results also support a model of direct interaction of the needle with PhA and suggest that, with further development, members of the PhA series may prove useful as drugs for P. aeruginosa infection.

Dataset Information

In the absence of effector proteins, the Pseudomonas aeruginosa type three secretion system needle tip complex contributes to lung injury and systemic inflammatory responses.

Publications

In the absence of effector proteins, the Pseudomonas aeruginosa type three secretion system needle tip complex contributes to lung injury and systemic inflammatory responses.

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