Pseudomonas aeruginosa Cystic Fibrosis Isolates Induce Rapid, Type III Secretion-Dependent, but ExoU-Independent, Oncosis of Macrophages and Polymorphonuclear Neutrophils

ABSTRACT Pseudomonas aeruginosa, an opportunistic pathogen responsible most notably for severe infections in cystic fibrosis (CF) patients, utilizes the type III secretion system for eukaryotic cell intoxication. The CF clinical isolate CHA shows toxicity towards human polymorphonuclear neutrophils (PMNs) which is dependent on the type III secretion system but independent of the cytotoxin ExoU. In the present study, the cytotoxicity of this strain toward human and murine macrophages was demonstrated. In low-multiplicity infections (multiplicity of infection, 10), approximately 40% of the cells die within 60 min. Analysis of CHA-infected cells by transmission electron microscopy, DNA fragmentation assay, and Hoechst staining revealed the hallmarks of oncosis: cellular and nuclear swelling, disintegration of the plasma membrane, and absence of DNA fragmentation. A panel of 29P. aeruginosa CF isolates was screened for type III system genotype, protein secretion profile, and cytotoxicity toward PMNs and macrophages. This study showed that six CF isolates were able to induce rapid ExoU-independent oncosis on phagocyte cells.

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Cell Death of Human Polymorphonuclear Neutrophils Induced by a Pseudomonas aeruginosa Cystic Fibrosis Isolate Requires a Functional Type III Secretion System

Infection and Immunity ◽

10.1128/iai.67.11.6164-6167.1999 ◽

1999 ◽

Vol 67 (11) ◽

pp. 6164-6167 ◽

Cited By ~ 72

Author(s):

D. Dacheux ◽

I. Attree ◽

C. Schneider ◽

B. Toussaint

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Polymorphonuclear Neutrophils ◽

Bactericidal Action ◽

Cellular Death ◽

Type Iii ◽

Cystic Fibrosis Isolate

ABSTRACT With a coincubation model incorporating Pseudomonas aeruginosa and human polymorphonuclear neutrophils (PMNs), a cystic fibrosis (CF) P. aeruginosa isolate has been shown to resist the bactericidal action of PMNs and to induce their cellular death. An isogenic mutant of this CF isolate in which the type III secretion system was rendered nonfunctional was unable to induce cellular death of PMNs.

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Activation of the Pseudomonas aeruginosa Type III Secretion System Requires an Intact Pyruvate Dehydrogenase aceAB Operon

Infection and Immunity ◽

10.1128/iai.70.7.3973-3977.2002 ◽

2002 ◽

Vol 70 (7) ◽

pp. 3973-3977 ◽

Cited By ~ 65

Author(s):

Denis Dacheux ◽

Olivier Epaulard ◽

Arjan de Groot ◽

Benoit Guery ◽

Rozen Leberre ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Transcriptional Activation ◽

Pyruvate Dehydrogenase ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Polymorphonuclear Neutrophils ◽

Type Iii ◽

New Genes ◽

Cystic Fibrosis Isolate

ABSTRACT Pseudomonas aeruginosa clinical cystic fibrosis isolate CHA was mutagenized with Tn5Tc to identify new genes involved in type III secretion system (TTSS)-dependent cytotoxicity toward human polymorphonuclear neutrophils. Among 25 mutants affected in TTSS function, 14 contained the insertion at different positions in the aceAB operon encoding the PDH-E1 and -E2 subunits of pyruvate dehydrogenase. In PDH mutants, no transcriptional activation of TTSS genes in response to calcium depletion occurred. Expression in trans of ExsA restored TTSS function and cytotoxicity.

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Expression of ExsA in trans Confers Type III Secretion System-Dependent Cytotoxicity on Noncytotoxic Pseudomonas aeruginosa Cystic Fibrosis Isolates

Infection and Immunity ◽

10.1128/iai.69.1.538-542.2001 ◽

2001 ◽

Vol 69 (1) ◽

pp. 538-542 ◽

Cited By ~ 55

Author(s):

Denis Dacheux ◽

Ina Attree ◽

Bertrand Toussaint

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Transcriptional Activation ◽

Type Iii Secretion ◽

Ex Vivo ◽

Type Iii Secretion System ◽

Secretion System ◽

Type Iii ◽

In Trans

ABSTRACT Twelve Pseudomonas aeruginosa cystic fibrosis isolates that are not able to exert a type III secretion system (TTSS)-dependent cytotoxicity towards phagocytes have been further studied. The strains, although possessing TTSS genes and exsA, which encodes a positive regulator of the TTSS regulon, showed no transcriptional activation of the exsCBA regulatory operon. The expression of exsA in trans restored the in vitro secretion of TTSS proteins and ex vivo cytotoxicity.

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Alveolar Response to Pseudomonas aeruginosa: Role of the Type III Secretion System

Infection and Immunity ◽

10.1128/iai.73.7.4263-4271.2005 ◽

2005 ◽

Vol 73 (7) ◽

pp. 4263-4271 ◽

Cited By ~ 35

Author(s):

F. Ader ◽

R. Le Berre ◽

K. Faure ◽

P. Gosset ◽

O. Epaulard ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Lung Injury ◽

Type Iii Secretion ◽

A549 Cells ◽

Weight Ratio ◽

Type Iii Secretion System ◽

Secretion System ◽

Polymorphonuclear Neutrophils ◽

Type Iii

ABSTRACT The type III secretion system (TTSS) is a specialized cytotoxin-translocating apparatus of gram-negative bacteria which is involved in lung injury, septic shock, and a poor patient outcome. Recent studies have attributed these effects mainly to the ExoU effector protein. However, few studies have focused on the ExoU-independent pathogenicity of the TTSS. For the present study, we compared the pathogenicities of two strains of Pseudomonas aeruginosa in a murine model of acute lung injury. We compared the CHA strain, which has a functional TTSS producing ExoS and ExoT but not ExoU, to an isogenic mutant with an inactivated exsA gene, CHA-D1, which does not express the TTSS at all. Rats challenged with CHA had significantly increased lung injury, as assessed by the wet/dry weight ratio for the lungs and the protein level in bronchoalveolar lavage fluid (BALF) at 12 h, compared to those challenged with CHA-D1. Consistent with these findings, the CHA strain was associated with increased in vitro cytotoxicity on A549 cells, as assessed by the release of lactate dehydrogenase. CHA was also associated at 12 h with a major decrease in polymorphonuclear neutrophils in BALF, with a proinflammatory response, as assessed by the amounts of tumor necrosis factor alpha and interleukin-1β, and with decreased bacterial clearance from the lungs, ultimately leading to an increased mortality rate. These results demonstrate that the TTSS has a major role in P. aeruginosa pathogenicity independent of the role of ExoU. This report underscores the crucial roles of ExoS and ExoT or other TTSS-related virulence factors in addition to ExoU.

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Pseudomonas aeruginosa Type III Secretion System Virulotypes and Their Association with Clinical Features of Cystic Fibrosis Patients

Infection and Drug Resistance ◽

10.2147/idr.s273759 ◽

2020 ◽

Vol Volume 13 ◽

pp. 3771-3781

Author(s):

Edilene do Socorro Nascimento Falcão Sarges ◽

Yan Corrêa Rodrigues ◽

Ismari Perini Furlaneto ◽

Marcos Vinicios Hino de Melo ◽

Giulia Leão da Cunha Brabo ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Clinical Features ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Type Iii

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RplI interacts with 5’ UTR of exsA to repress its translation and type III secretion system in Pseudomonas aeruginosa

PLoS Pathogens ◽

10.1371/journal.ppat.1010170 ◽

2022 ◽

Vol 18 (1) ◽

pp. e1010170

Author(s):

Dan Wang ◽

Xinxin Zhang ◽

Liwen Yin ◽

Qi Liu ◽

Zhaoli Yu ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Large Subunit ◽

Type Iii Secretion System ◽

Opportunistic Pathogen ◽

Secretion System ◽

Mobility Shift ◽

Subunit Protein ◽

Type Iii ◽

Real Time Quantitative Pcr

Pseudomonas aeruginosa is an important opportunistic pathogen capable of causing variety of infections in humans. The type III secretion system (T3SS) is a critical virulence determinant of P. aeruginosa in the host infections. Expression of the T3SS is regulated by ExsA, a master regulator that activates the expression of all known T3SS genes. Expression of the exsA gene is controlled at both transcriptional and posttranscriptional levels. Here, we screened a P. aeruginosa transposon (Tn5) insertional mutant library and found rplI, a gene coding for the ribosomal large subunit protein L9, to be a repressor for the T3SS gene expression. Combining real-time quantitative PCR (qPCR), western blotting and lacZ fusion assays, we show that RplI controls the expression of exsA at the posttranscriptional level. Further genetic experiments demonstrated that RplI mediated control of the exsA translation involves 5’ untranslated region (5’ UTR). A ribosome immunoprecipitation assay and qPCR revealed higher amounts of a 24 nt fragment from exsA mRNA being associated with ribosomes in the ΔrplI mutant. An interaction between RplI and exsA mRNA harboring its 24 nt, but not 12 nt, 5’ UTR was confirmed by RNA Gel Mobility Shift and Microscale Thermophoresis assays. Overall, this study identifies the ribosomal large subunit protein L9 as a novel T3SS repressor that inhibits ExsA translation in P. aeruginosa.

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Identification of Small Molecules Blocking the Pseudomonas aeruginosa Type III Secretion System Protein PcrV

Biomolecules ◽

10.3390/biom11010055 ◽

2021 ◽

Vol 11 (1) ◽

pp. 55

Author(s):

Charlotta Sundin ◽

Michael Saleeb ◽

Sara Spjut ◽

Liena Qin ◽

Mikael Elofsson

Keyword(s):

Pseudomonas Aeruginosa ◽

Host Cell ◽

Type Iii Secretion ◽

Bacterial Pathogen ◽

Type Iii Secretion System ◽

Eukaryotic Cell ◽

Secretion System ◽

Adaptive Resistance ◽

Type Iii ◽

Relationship Analysis

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that employs its type III secretion system (T3SS) during the acute phase of infection to translocate cytotoxins into the host cell cytoplasm to evade the immune system. The PcrV protein is located at the tip of the T3SS, facilitates the integration of pore-forming proteins into the eukaryotic cell membrane, and is required for translocation of cytotoxins into the host cell. In this study, we used surface plasmon resonance screening to identify small molecule binders of PcrV. A follow-up structure-activity relationship analysis resulted in PcrV binders that protect macrophages in a P. aeruginosa cell-based infection assay. Treatment of P. aeruginosa infections is challenging due to acquired, intrinsic, and adaptive resistance in addition to a broad arsenal of virulence systems such as the T3SS. Virulence blocking molecules targeting PcrV constitute valuable starting points for development of next generation antibacterials to treat infections caused by P. aeruginosa.

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