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

2004 ◽  
Vol 72 (3) ◽  
pp. 1383-1390 ◽  
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.

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

Microbiology ◽  
2006 ◽  
Vol 152 (1) ◽  
pp. 143-152 ◽  
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.

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

2006 ◽  
Vol 75 (3) ◽  
pp. 1089-1098 ◽  
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.

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

2001 ◽  
Vol 69 (1) ◽  
pp. 538-542 ◽  
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.

scholarly journals MucA-Mediated Coordination of Type III Secretion and Alginate Synthesis in Pseudomonas aeruginosa

2004 ◽  
Vol 186 (22) ◽  
pp. 7575-7585 ◽  
Author(s):  
Weihui Wu ◽  
Hassan Badrane ◽  
Shiwani Arora ◽  
Henry V. Baker ◽  
Shouguang Jin
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Regulatory Networks ◽  
Protective Role ◽  
Environmental Stresses ◽  
Host Cells ◽  
Calcium Signal ◽  
Insertion Mutant ◽  
Type Iii

ABSTRACT The type III secretion system (T3SS) of Pseudomonas aeruginosa is an important virulence factor. The T3SS of P. aeruginosa can be induced by a low calcium signal or upon direct contact with the host cells. The exact pathway of signal sensing and T3SS activation is not clear. By screening a transposon insertion mutant library of the PAK strain, mutation in the mucA gene was found to cause repression of T3SS expression under both type III-inducing and -noninducing conditions. Mutation in the mucA gene is known to cause alginate overproduction, resulting in a mucoid phenotype. Alginate production responds to various environmental stresses and plays a protective role for P. aeruginosa. Comparison of global gene expression of mucA mutant and wild-type PAK under T3SS-inducing conditions confirmed the down regulation of T3SS genes and up regulation of genes involved in alginate biosynthesis. Further analysis indicated that the repression of T3SS in the mucA mutant was AlgU and AlgR dependent, as double mutants mucA/algU and mucA/algR showed normal type III expression. An algR::Gm mutant showed a higher level of type III expression, while overexpression of the algR gene inhibited type III gene expression; thus, it seems that the AlgR-regulated product inhibits the expression of the T3SS genes. It is likely that P. aeruginosa has evolved tight regulatory networks to turn off the energy-expensive T3SS when striving for survival under environmental stresses.

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

1999 ◽  
Vol 67 (10) ◽  
pp. 5530-5537 ◽  
Author(s):  
Alan R. Hauser ◽  
Joanne N. Engel
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Opportunistic Pathogen ◽  
Type Iii ◽  
Two Factors ◽  
Primary Bone ◽  
Hoechst Staining ◽  
Darby Canine Kidney ◽  
Primary Bone Marrow

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.

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

2007 ◽  
Vol 189 (6) ◽  
pp. 2203-2209 ◽  
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.

scholarly journals PsrA Is a Positive Transcriptional Regulator of the Type III Secretion System in Pseudomonas aeruginosa

2006 ◽  
Vol 74 (2) ◽  
pp. 1121-1129 ◽  
Author(s):  
D. K. Shen ◽  
D. Filopon ◽  
L. Kuhn ◽  
B. Polack ◽  
B. Toussaint
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Cell Contact ◽  
Transcriptional Level ◽  
Calcium Depletion ◽  
Type Iii ◽  
Full Activation

ABSTRACT The type III secretion system (TTSS) of Pseudomonas aeruginosa is induced in vivo upon contact with eukaryotic cells and in vitro by calcium depletion in culture medium. We have observed a previously identified protein, PsrA, necessary for full activation of TTSS gene expression in P. aeruginosa. Electrophoretic mobility shift assays showed that recombinant PsrA could bind to the exsCEBA promoter region. A mutant with a deletion in the psrA gene was constructed. Using transcriptional fusions, we demonstrated that PsrA is required for the full activation of transcription of the TTSS regulatory operon exsCEBA and effector exoS, although the deletion mutant still responded to calcium depletion, to serum, and to host cell contact. The psrA mutant showed a marked decrease in the secretion of the type III effectors and weak resistance to phagocyte-like PLB-985 cells. The defect in TTSS transcription and secretion in the psrA mutant could be complemented by expression in trans of psrA. PsrA was previously identified as a transcriptional activator of RpoS, a central regulator during stationary phase. We confirmed with our strain that RpoS has a negative effect on TTSS gene expression. Taken altogether, these results suggest that PsrA is a newly identified activator that is involved in the expression of the TTSS by enhancing the exsCEBA transcriptional level.

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

2006 ◽  
Vol 74 (7) ◽  
pp. 3880-3889 ◽  
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.

scholarly journals ExoS Controls the Cell Contact-Mediated Switch to Effector Secretion in Pseudomonas aeruginosa

2007 ◽  
Vol 190 (8) ◽  
pp. 2726-2738 ◽  
Author(s):  
Michelle Cisz ◽  
Pei-Chung Lee ◽  
Arne Rietsch
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Plasma Membrane ◽  
Host Cell ◽  
Type Iii Secretion ◽  
Host Cells ◽  
Cell Contact ◽  
Cell Plasma Membrane ◽  
Type Iii ◽  
Cell Plasma ◽  
Effector Secretion

ABSTRACT Type III secretion is used by many gram-negative bacterial pathogens to directly deliver protein toxins (effectors) into targeted host cells. In all cases, secretion of effectors is triggered by host cell contact, although the mechanism is unclear. In Pseudomonas aeruginosa, expression of all type III secretion-related genes is up-regulated when secretion is triggered. We were able to visualize this process using a green fluorescent protein reporter system and to use it to monitor the ability of bacteria to trigger effector secretion on cell contact. Surprisingly, the action of one of the major type III secreted effectors, ExoS, prevented triggering of type III secretion by bacteria that subsequently attached to cells, suggesting that triggering of secretion is feedback regulated. Evidence is presented that translocation (secretion of effectors across the host cell plasma membrane) of ExoS is indeed self-regulated and that this inhibition of translocation can be achieved by either of its two enzymatic activities. The translocator proteins PopB, PopD, and PcrV are secreted via the type III secretion system and are required for pore formation and translocation of effectors across the host cell plasma membrane. Here we present data that secretion of translocators is in fact not controlled by calcium, implying that triggering of effector secretion on cell contact represents a switch in secretion specificity, rather than a triggering of secretion per se. The requirement for a host cell cofactor to control effector secretion may help explain the recently observed phenomenon of target cell specificity in both the Yersinia and P. aeruginosa type III secretion systems.

scholarly journals 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

2017 ◽  
Vol 61 (11) ◽  
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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