Pseudomonas aeruginosa Induces Type-III-Secretion-Mediated Apoptosis of Macrophages and Epithelial Cells

ABSTRACT Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that is cytotoxic towards a variety of eukaryotic cells. To investigate the effect of this bacterium on macrophages, we infected J774A.1 cells and primary bone-marrow-derived murine macrophages with the P. aeruginosa strain PA103 in vitro. PA103 caused type-III-secretion-dependent killing of macrophages within 2 h of infection. Only a portion of the killing required the putative cytotoxin ExoU. By three criteria, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assays, cytoplasmic nucleosome assays, and Hoechst staining, the ExoU-independent but type-III-secretion-dependent killing exhibited features of apoptosis. Extracellular bacteria were capable of inducing apoptosis, and some laboratory and clinical isolates of P. aeruginosa induced significantly higher levels of this form of cell death than others. Interestingly, HeLa cells but not Madin-Darby canine kidney cells were susceptible to type-III-secretion-mediated apoptosis under the conditions of these assays. These findings are consistent with a model in which the P. aeruginosa type III secretion system transports at least two factors that kill macrophages: ExoU, which causes necrosis, and a second, as yet unidentified, effector protein, which induces apoptosis. Such killing may contribute to the ability of this organism to persist and disseminate within infected patients.

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Effect of Metabolic Imbalance on Expression of Type III Secretion Genes in Pseudomonas aeruginosa

Infection and Immunity ◽

10.1128/iai.72.3.1383-1390.2004 ◽

2004 ◽

Vol 72 (3) ◽

pp. 1383-1390 ◽

Cited By ~ 84

Author(s):

Arne Rietsch ◽

Matthew C. Wolfgang ◽

John J. Mekalanos

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Cell Contact ◽

Insertion Mutant ◽

Insertion Mutation ◽

Type Iii ◽

Transposon Insertion ◽

Metabolic Imbalance ◽

Cell Expression

ABSTRACT The type III secretion system is a dedicated machinery used by many pathogens to deliver toxins directly into the cytoplasm of a target cell. Expression and secretion of the type III effectors are triggered by cell contact. In Pseudomonas aeruginosa and Yersinia spp., expression can be triggered in vitro by removing calcium from the medium. The mechanism underlying either mode of regulation is unclear. Here we characterize a transposon insertion mutant of P. aeruginosa PAO1 that displays a marked defect in cytotoxicity. The insertion is located upstream of several genes involved in histidine utilization and impedes the ability of PAO1 to intoxicate eukaryotic cells effectively in a type III-dependent fashion. This inhibition depends on the presence of histidine in the medium and appears to depend on the excessive uptake and catabolism of histidine. The defect in cytotoxicity is mirrored by a decrease in exoS expression. Other parameters such as growth or piliation are unaffected. The cytotoxicity defect is partially complemented by an insertion mutation in cbrA that also causes overexpression of cbrB. The cbrAB two-component system has been implicated in sensing and responding to a carbon-nitrogen imbalance. Taken together, these results suggest that the metabolic state of the cell influences expression of the type III regulon.

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Interactions between effector proteins of the Pseudomonas aeruginosa type III secretion system do not significantly affect several measures of disease severity in mammals

Microbiology ◽

10.1099/mic.0.28368-0 ◽

2006 ◽

Vol 152 (1) ◽

pp. 143-152 ◽

Cited By ~ 12

Author(s):

Ciara M. Shaver ◽

Alan R. Hauser

Keyword(s):

Pseudomonas Aeruginosa ◽

Disease Severity ◽

Type Iii Secretion ◽

Disease Process ◽

Effector Proteins ◽

Host Cells ◽

Secreted Proteins ◽

Secretion Systems ◽

Type Iii

The effector proteins of the type III secretion systems of many bacterial pathogens act in a coordinated manner to subvert host cells and facilitate the development and progression of disease. It is unclear whether interactions between the type-III-secreted proteins of Pseudomonas aeruginosa result in similar effects on the disease process. We have previously characterized the contributions to pathogenesis of the type-III-secreted proteins ExoS, ExoT and ExoU when secreted individually. In this study, we extend our prior work to determine whether these proteins have greater than expected effects on virulence when secreted in combination. In vitro cytotoxicity and anti-internalization activities were not enhanced when effector proteins were secreted in combinations rather than alone. Likewise in a mouse model of pneumonia, bacterial burden in the lungs, dissemination and mortality attributable to ExoS, ExoT and ExoU were not synergistically increased when combinations of these effector proteins were secreted. Because of the absence of an appreciable synergistic increase in virulence when multiple effector proteins were secreted in combination, we conclude that any cooperation between ExoS, ExoT and ExoU does not translate into a synergistically significant enhancement of disease severity as measured by these assays.

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Pseudolipasin A Is a Specific Inhibitor for Phospholipase A2 Activity of Pseudomonas aeruginosa Cytotoxin ExoU

Infection and Immunity ◽

10.1128/iai.01184-06 ◽

2006 ◽

Vol 75 (3) ◽

pp. 1089-1098 ◽

Cited By ~ 37

Author(s):

Vincent T. Lee ◽

Stefan Pukatzki ◽

Hiromi Sato ◽

Eriya Kikawada ◽

Anastasia A. Kazimirova ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Specific Inhibitor ◽

Effector Proteins ◽

Host Cells ◽

Cytotoxic Effects ◽

Type Iii ◽

Secretion Pathway ◽

Phospholipase A2 Activity

ABSTRACT A number of bacterial pathogens utilize the type III secretion pathway to deliver effector proteins directly into the host cell cytoplasm. Certain strains of Pseudomonas aeruginosa associated with acute infections express a potent cytotoxin, exoenzyme U (ExoU), that is delivered via the type III secretion pathway directly into contacting host cells. Once inside the mammalian cell, ExoU rapidly lyses the intoxicated cells via its phospholipase A2 (PLA2) activity. A high-throughput cell-based assay was developed to screen libraries of compounds for those capable of protecting cells against the cytotoxic effects of ExoU. A number of compounds were identified in this screen, including one group that blocks the intracellular activity of ExoU. In addition, these compounds specifically inhibited the PLA2 activity of ExoU in vitro, whereas eukaryotic secreted PLA2 and cytosolic PLA2 were not inhibited. This novel inhibitor of ExoU-specific PLA2 activity, named pseudolipasin A, may provide a new lead for virulence factor-based therapeutic design.

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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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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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Presence or Absence of Lipopolysaccharide O Antigens Affects Type III Secretion by Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.01839-06 ◽

2007 ◽

Vol 189 (6) ◽

pp. 2203-2209 ◽

Cited By ~ 37

Author(s):

D. K. Augustin ◽

Y. Song ◽

M. S. Baek ◽

Y. Sawa ◽

G. Singh ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Strong Association ◽

Cellular Damage ◽

Mortality And Morbidity ◽

Type Iii ◽

O Antigen ◽

Mode Of Life

ABSTRACT Pseudomonas aeruginosa is one of the major causative agents of mortality and morbidity in hospitalized patients due to a multiplicity of virulence factors associated with both chronic and acute infections. Acute P. aeruginosa infection is primarily mediated by planktonic bacteria expressing the type III secretion system (TTSS), a surface-attached needle-like complex that injects cytotoxins directly into eukaryotic cells, causing cellular damage. Lipopolysaccharide (LPS) is the principal surface-associated virulence factor of P. aeruginosa. This molecule is known to undergo structural modification (primarily alterations in the A- and B-band O antigen) in response to changes in the mode of life (e.g., from biofilm to planktonic). Given that LPS exhibits structural plasticity, we hypothesized that the presence of LPS lacking O antigen would facilitate eukaryotic intoxication and that a correlation between the LPS O-antigen serotype and TTSS-mediated cytotoxicity would exist. Therefore, strain PAO1 (A+ B+ O-antigen serotype) and isogenic mutants with specific O-antigen defects (A+ B−, A− B+, and A− B−) were examined for TTSS expression and cytotoxicity. A strong association existed in vitro between the absence of the large, structured B-band O antigen and increased cytotoxicity of these strains. In vivo, all three LPS mutant strains demonstrated significantly increased lung injury compared to PAO1. Clinical strains lacking the B-band O antigen also demonstrated increased TTSS secretion. These results suggest the existence of a cooperative association between LPS O-antigen structure and the TTSS in both laboratory and clinical isolates of P. aeruginosa.

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Type III Secretion-Dependent Modulation of Innate Immunity as One of Multiple Factors Regulated by Pseudomonas aeruginosa RetS

Infection and Immunity ◽

10.1128/iai.01891-05 ◽

2006 ◽

Vol 74 (7) ◽

pp. 3880-3889 ◽

Cited By ~ 27

Author(s):

Irandokht Zolfaghar ◽

David J. Evans ◽

Reza Ronaghi ◽

Suzanne M. J. Fleiszig

Keyword(s):

Pseudomonas Aeruginosa ◽

Innate Immunity ◽

Type Iii Secretion ◽

Bacterial Colonization ◽

Published Data ◽

Wild Type ◽

Type Iii ◽

Severity Scores ◽

Peripheral Ring

ABSTRACT Mutation of retS (rtsM) of Pseudomonas aeruginosa strain PA103 reduces its virulence in both ocular and respiratory murine models of infection. In vitro, retS mutants exhibit loss of the ExsA-regulated type III secretion system (TTSS), reduced twitching motility, and a decrease in association with, invasion of, and survival within corneal epithelial cells. In addition, transcription of multiple other virulence genes is positively and negatively affected by retS mutation. Since our published data show that ExoU and ExoT, the two TTSS effectors encoded by strain PA103, each confer virulence in this corneal model, we hypothesized that loss of virulence of retS mutants follows loss of type III secretion. Corneal pathology, bacterial colonization, and phagocyte infiltration were compared for wild-type PA103, retS mutants, and various TTSS mutants after infection with ∼106 CFU bacteria. Results showed that either a retS or an exsA (TTSS) mutation delayed disease progression, as illustrated by reduced severity scores and colonization levels during the first 48 h postinfection. Surprisingly, retS mutant infections then became more severe than those involving exsA mutants. By day 7, colonization levels of retS mutants even surpassed those of wild-type bacteria (more than twofold, P = 0.028). Although retS mutants caused more severe opacification of central corneas than both the wild type and the exsA mutants, neither mutant caused the peripheral ring opacity commonly associated with wild-type infection, suggesting that the TTSS was involved. Histological experiments with retS and various TTSS mutants showed that ring opacification required ExoU but not ExoT and that it consisted of dense polymorphonuclear phagocyte infiltration at the corneal periphery and the absence of any cell type in the central cornea. These data suggest that these P. aeruginosa TTSS effectors have different effects on innate immunity and that RetS influences virulence beyond its effects on the TTSS.

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Impact of Type III Secretion Effectors and of Phenoxyacetamide Inhibitors of Type III Secretion on Abscess Formation in a Mouse Model of Pseudomonas aeruginosa Infection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01202-17 ◽

2017 ◽

Vol 61 (11) ◽

Cited By ~ 22

Author(s):

Bryan J. Berube ◽

Katherine R. Murphy ◽

Matthew C. Torhan ◽

Nicholas O. Bowlin ◽

John D. Williams ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Mouse Model ◽

Type Iii Secretion ◽

Abscess Formation ◽

Host Cells ◽

Block Type ◽

Content Type ◽

Type Iii

ABSTRACT Pseudomonas aeruginosa is a leading cause of intra-abdominal infections, wound infections, and community-acquired folliculitis, each of which may involve macro- or microabscess formation. The rising incidence of multidrug resistance among P. aeruginosa isolates has increased both the economic burden and the morbidity and mortality associated with P. aeruginosa disease and necessitates a search for novel therapeutics. Previous work from our group detailed novel phenoxyacetamide inhibitors that block type III secretion and injection into host cells in vitro. In this study, we used a mouse model of P. aeruginosa abscess formation to test the in vivo efficacy of these compounds against the P. aeruginosa type III secretion system (T3SS). Bacteria used the T3SS to intoxicate infiltrating neutrophils to establish abscesses. Despite this antagonism, sufficient numbers of functioning neutrophils remained for proper containment of the abscesses, as neutrophil depletion resulted in an increased abscess size, the formation of dermonecrotic lesions on the skin, and the dissemination of P. aeruginosa to internal organs. Consistent with the specificity of the T3SS-neutrophil interaction, P. aeruginosa bacteria lacking a functional T3SS were fully capable of causing abscesses in a neutropenic host. Phenoxyacetamide inhibitors attenuated abscess formation and aided in the immune clearance of the bacteria. Finally, a P. aeruginosa strain resistant to the phenoxyacetamide compound was fully capable of causing abscess formation even in the presence of the T3SS inhibitors. Together, our results further define the role of type III secretion in murine abscess formation and demonstrate the in vivo efficacy of phenoxyacetamide inhibitors in P. aeruginosa infection.

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

Infection and Immunity ◽

10.1128/iai.68.5.2916-2924.2000 ◽

2000 ◽

Vol 68 (5) ◽

pp. 2916-2924 ◽

Cited By ~ 107

Author(s):

Denis Dacheux ◽

Bertrand Toussaint ◽

Marceline Richard ◽

Guy Brochier ◽

Jacques Croize ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Dna Fragmentation ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Opportunistic Pathogen ◽

Eukaryotic Cell ◽

Secretion System ◽

Polymorphonuclear Neutrophils ◽

Type Iii

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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Secretion of Flagellar Proteins by the Pseudomonas aeruginosa Type III Secretion-Injectisome System

Journal of Bacteriology ◽

10.1128/jb.00030-15 ◽

2015 ◽

Vol 197 (12) ◽

pp. 2003-2011 ◽

Cited By ~ 21

Author(s):

Dilek Ince ◽

Fayyaz S. Sutterwala ◽

Timothy L. Yahr

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Bacterial Species ◽

Opportunistic Pathogen ◽

Host Cells ◽

Inflammasome Activation ◽

Content Type ◽

Type Iii ◽

Amino Terminal ◽

Flagellar Proteins

ABSTRACTThe opportunistic pathogenPseudomonas aeruginosautilizes an injectisome-type III secretion system (injectisome-T3SS) to elicit cytotoxicity toward epithelial cells and macrophages. Macrophage killing results from the cytotoxic properties of the translocated effector proteins (ExoS, ExoT, ExoU, and ExoY) and inflammasome-mediated induction of pyroptosis. Inflammasome activation can occur following Nlrc4-mediated recognition of cytosolic translocated flagellin (FliC). In the present study, we demonstrate that FliC is a secretion substrate of both the injectisome- and flagellum-associated T3SSs. Molecular analyses indicate that the first 20 amino-terminal residues of FliC are sufficient for secretion by the injectisome-T3SS and that the first 100 residues are sufficient for translocation of FliC into host cells. Although maximal inflammasome activation requires FliC, activation can also occur in the absence of FliC. This prompted us to examine whether other flagellar components might also be translocated into cells to elicit inflammasome activation. Indeed, we find that the flagellar cap (FliD), hook-associated (FlgK and FlgL), hook (FlgE), and rod (FlgE) proteins are secretion substrates of the injectisome-T3SS. None of these proteins, however, result in increased inflammasome activation when they are overexpressed in afliCmutant and appear to be translocated into host cells. While a role in inflammasome activation has been excluded, these data raise the possibility that flagellar components, which are highly conserved between different bacterial species, trigger other specific host responses from the extracellular milieu or contribute to the pathogenesis ofP. aeruginosa.IMPORTANCEThe inflammasome is a host defense mechanism that recognizes invading bacteria and triggers an inflammatory immune response. The opportunistic pathogenP. aeruginosaproduces both inflammasome agonists and antagonists. In this study, we demonstrate that overexpression of an agonist suppresses the activity of an antagonist, thereby resulting in inflammasome activation. Since the relative expression levels of agonists and antagonists likely vary between strains, these differences could be important predictors of whether a particularP. aeruginosastrain elicits inflammasome activation.

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