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 The RNA Helicase DeaD Stimulates ExsA Translation To Promote Expression of the Pseudomonas aeruginosa Type III Secretion System

2015 ◽  
Vol 197 (16) ◽  
pp. 2664-2674 ◽  
Author(s):  
Peter J. Intile ◽  
Grant J. Balzer ◽  
Matthew C. Wolfgang ◽  
Timothy L. Yahr
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Rna Helicase ◽  
Secretion System ◽  
Extrinsic Factors ◽  
Content Type ◽  
Type Iii ◽  
Dead Box

ABSTRACTThePseudomonas aeruginosatype III secretion system (T3SS) is a primary virulence factor important for phagocytic avoidance, disruption of host cell signaling, and host cell cytotoxicity. ExsA is the master regulator of T3SS transcription. The expression, synthesis, and activity of ExsA is tightly regulated by both intrinsic and extrinsic factors. Intrinsic regulation consists of the well-characterized ExsECDA partner-switching cascade, while extrinsic factors include global regulators that alterexsAtranscription and/or translation. To identify novel extrinsic regulators of ExsA, we conducted a transposon mutagenesis screen in the absence of intrinsic control. Transposon disruptions within gene PA2840, which encodes a homolog of theEscherichia coliRNA-helicase DeaD, significantly reduced T3SS gene expression. Recent studies indicate thatE. coliDeaD can promote translation by relieving inhibitory secondary structures within target mRNAs. We report here that PA2840, renamed DeaD, stimulates ExsA synthesis at the posttranscriptional level. Genetic experiments demonstrate that the activity of anexsAtranslational fusion is reduced in adeaDmutant. In addition,exsAexpression intransfails to restore T3SS gene expression in adeaDmutant. We hypothesized that DeaD relaxes mRNA secondary structure to promoteexsAtranslation and found that altering the mRNA sequence ofexsAor the nativeexsAShine-Dalgarno sequence relieved the requirement for DeaDin vivo. Finally, we show that purified DeaD promotes ExsA synthesis usingin vitrotranslation assays. Together, these data reveal a novel regulatory mechanism forP. aeruginosaDeaD and add to the complexity of global regulation of T3SS.IMPORTANCEAlthough members of the DEAD box family of RNA helicases are appreciated for their roles in mRNA degradation and ribosome biogenesis, an additional role in gene regulation is now emerging in bacteria. By relaxing secondary structures in mRNAs, DEAD box helicases are now thought to promote translation by enhancing ribosomal recruitment. We identify here an RNA helicase that plays a critical role in promoting ExsA synthesis, the central regulator of thePseudomonas aeruginosatype III secretion system, and provide additional evidence that DEAD box helicases directly stimulate translation of target genes. The finding that DeaD stimulatesexsAtranslation adds to a growing list of transcriptional and posttranscriptional regulatory mechanisms that control type III gene expression.

scholarly journals Killing from the inside: Intracellular role of T3SS in the fate ofPseudomonas aeruginosawithin macrophages revealed bymgtCandoprFmutants

2018 ◽  
Author(s):  
Preeti Garai ◽  
Laurence Berry ◽  
Malika Moussouni ◽  
Sophie Bleves ◽  
Anne-Béatrice Blanc-Potard
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Gene Expression Pattern ◽  
Type Iii Secretion System ◽  
Cell Types ◽  
Cell Lysis ◽  
Secretion System ◽  
Intracellular Bacteria ◽  
Type Iii

AbstractWhile considered solely an extracellular pathogen, increasing evidence indicates thatPseudomonas aeruginosaencounters intracellular environment in diverse mammalian cell types, including macrophages. In the present study, we have deciphered the intramacrophage fate of wild-typeP. aeruginosaPAO1 strain by live and electron microscopy.P. aeruginosafirst resided in phagosomal vacuoles and subsequently could be detected in the cytoplasm, indicating phagosomal escape of the pathogen, a finding also supported by vacuolar rupture assay. The intracellular bacteria could eventually induce cell lysis. Two bacterial factors, MgtC and OprF, recently identified to be important for survival ofP. aeruginosain macrophages, were found to be involved in bacterial escape from the phagosome as well as cell lysis caused by intracellular bacteria. Strikingly, type III secretion system (T3SS) genes ofP. aeruginosawere down-regulated within macrophages in bothmgtCandoprFmutants. Concordantly, cyclic di-GMP (c-di-GMP) level was increased in both mutants, providing a clue for negative regulation of T3SS inside macrophages. Consistent with the phenotypes and gene expression pattern ofmgtCandoprFmutants, a T3SS mutant(ΔpscN)exhibited defect in phagosomal escape and macrophage lysis driven by internalized bacteria. Importantly, these effects appeared to be largely dependent on the ExoS effector, in contrast with the known T3SS-dependent, but ExoS independent, cytotoxicity caused by extracellularP. aeruginosatowards macrophages. Hence, our work highlights T3SS and ExoS, whose expression is modulated by MgtC and OprF, as key players in the intramacrophage life ofP. aeruginosa, allowing internalized bacteria to evade macrophages.Author summaryThe ability of professional phagocytes to ingest and kill microorganisms is central to host defense andPseudomonas aeruginosahas developed mechanisms to avoid being killed by phagocytes. While considered an extracellular pathogen,P. aeruginosahas been reported to be engulfed by macrophages in animal models. Here, we visualized the fate ofP. aeruginosawithin cultured macrophages, revealing macrophage lysis driven by intracellularP. aeruginosa. Two bacterial factors, MgtC and OprF, recently discovered to be involved in the intramacrophage survival ofP. aeruginosa, appeared to play role in this cytotoxicity caused by intracellular bacteria. We provided evidence that type III secretion system (T3SS) gene expression is lowered intracellularly inmgtCandoprFmutants. We further showed that intramacrophageP. aeruginosauses its T3SS, specifically the ExoS effector, to promote phagosomal escape and cell lysis. We thus describe a transient intramacrophage stage ofP. aeruginosathat could contribute to bacterial dissemination.

scholarly journals Pseudomonas aeruginosa Magnesium Transporter MgtE Inhibits Type III Secretion System Gene Expression by Stimulating rsmYZ Transcription

2017 ◽  
Vol 199 (23) ◽  
Author(s):  
Shubham Chakravarty ◽  
Cameron N. Melton ◽  
Adam Bailin ◽  
Timothy L. Yahr ◽  
Gregory G. Anderson
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Gene Transcription ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Content Type ◽  
Type Iii ◽  
Magnesium Transporter

ABSTRACT Pseudomonas aeruginosa causes numerous acute and chronic opportunistic infections in humans. One of its most formidable weapons is a type III secretion system (T3SS), which injects powerful toxins directly into host cells. The toxins lead to cell dysfunction and, ultimately, cell death. Identification of regulatory pathways that control T3SS gene expression may lead to the discovery of novel therapeutics to treat P. aeruginosa infections. In a previous study, we found that expression of the magnesium transporter gene mgtE inhibits T3SS gene transcription. MgtE-dependent inhibition appeared to interfere with the synthesis or function of the master T3SS transcriptional activator ExsA, although the exact mechanism was unclear. We now demonstrate that mgtE expression acts through the GacAS two-component system to activate rsmY and rsmZ transcription. This event ultimately leads to inhibition of exsA translation. This inhibitory effect is specific to exsA as translation of other genes in the exsCEBA operon is not inhibited by mgtE. Moreover, our data reveal that MgtE acts solely through this pathway to regulate T3SS gene transcription. Our study reveals an important mechanism that may allow P. aeruginosa to fine-tune T3SS activity in response to certain environmental stimuli. IMPORTANCE The type III secretion system (T3SS) is a critical virulence factor utilized by numerous Gram-negative bacteria, including Pseudomonas aeruginosa, to intoxicate and kill host cells. Elucidating T3SS regulatory mechanisms may uncover targets for novel anti-P. aeruginosa therapeutics and provide deeper understanding of bacterial pathogenesis. We previously found that the magnesium transporter MgtE inhibits T3SS gene transcription in P. aeruginosa. In this study, we describe the mechanism of MgtE-dependent inhibition of the T3SS. Our report also illustrates how MgtE might respond to environmental cues, such as magnesium levels, to fine-tune T3SS gene expression.

scholarly journals An in vivo inducible gene of Pseudomonas aeruginosa encodes an anti-ExsA to suppress the type III secretion system

2004 ◽  
Vol 54 (2) ◽  
pp. 307-320 ◽  
Author(s):  
Un-Hwan Ha ◽  
Jaewha Kim ◽  
Hassan Badrane ◽  
Jinghua Jia ◽  
Henry V. Baker ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Type Iii ◽  
Inducible Gene

scholarly journals Use of the Galleria mellonella Caterpillar as a Model Host To Study the Role of the Type III Secretion System in Pseudomonas aeruginosa Pathogenesis

2003 ◽  
Vol 71 (5) ◽  
pp. 2404-2413 ◽  
Author(s):  
Sachiko Miyata ◽  
Monika Casey ◽  
Dara W. Frank ◽  
Frederick M. Ausubel ◽  
Eliana Drenkard
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Galleria Mellonella ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Triple Mutant ◽  
Type Iii ◽  
Wax Moth

ABSTRACT Nonvertebrate model hosts represent valuable tools for the study of host-pathogen interactions because they facilitate the identification of bacterial virulence factors and allow the discovery of novel components involved in host innate immune responses. In this report, we determined that the greater wax moth caterpillar Galleria mellonella is a convenient nonmammalian model host for study of the role of the type III secretion system (TTSS) in Pseudomonas aeruginosa pathogenesis. Based on the observation that a mutation in the TTSS pscD gene of P. aeruginosa strain PA14 resulted in a highly attenuated virulence phenotype in G. mellonella, we examined the roles of the four known effector proteins of P. aeruginosa (ExoS, ExoT, ExoU, and ExoY) in wax moth killing. We determined that in P. aeruginosa strain PA14, only ExoT and ExoU play a significant role in G. mellonella killing. Strain PA14 lacks the coding sequence for the ExoS effector protein and does not seem to express ExoY. Moreover, using ΔexoU ΔexoY, ΔexoT ΔexoY, and ΔexoT ΔexoU double mutants, we determined that individual translocation of either ExoT or ExoU is sufficient to obtain nearly wild-type levels of G. mellonella killing. On the other hand, data obtained with a ΔexoT ΔexoU ΔexoY triple mutant and a ΔpscD mutant suggested that additional, as-yet-unidentified P. aeruginosa components of type III secretion are involved in virulence in G. mellonella. A high level of correlation between the results obtained in the G. mellonella model and the results of cytopathology assays performed with a mammalian tissue culture system validated the use of G. mellonella for the study of the P. aeruginosa TTSS.

scholarly journals Developing Cyclic Peptomers as Broad-Spectrum Gram negative Bacterial Type III Secretion System Inhibitors

2020 ◽  
Author(s):  
Hanh N. Lam ◽  
Tannia Lau ◽  
Adam Lentz ◽  
Jessica Sherry ◽  
Alejandro Cabrera-Cortez ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Chlamydia Trachomatis ◽  
Broad Spectrum ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Type Iii ◽  
Bacterial Type

ABSTRACTAntibiotic resistant bacteria are an emerging global health threat. New antimicrobials are urgently needed. The injectisome type III secretion system (T3SS), required by dozens of Gram-negative bacteria for virulence but largely absent from non-pathogenic bacteria, is an attractive antimicrobial target. We previously identified synthetic cyclic peptomers, inspired by the natural product phepropeptin D, that inhibit protein secretion through the Yersinia Ysc and Pseudomonas aeruginosa Psc T3SSs, but do not inhibit bacterial growth. Here we describe identification of an isomer, 4EpDN, that is two-fold more potent (IC50 4 μM) than its parental compound. Furthermore, 4EpDN inhibited the Yersinia Ysa and the Salmonella SPI-1 T3SSs, suggesting that this cyclic peptomer has broad efficacy against evolutionarily distant injectisome T3SSs. Indeed, 4EpDN strongly inhibited intracellular growth of Chlamydia trachomatis in HeLa cells, which requires the T3SS. 4EpDN did not inhibit the unrelated Twin arginine translocation (Tat) system, nor did it impact T3SS gene transcription. Moreover, although the injectisome and flagellar T3SSs are evolutionarily and structurally related, the 4EpDN cyclic peptomer did not inhibit secretion of substrates through the Salmonella flagellar T3SS, indicating that cyclic peptomers broadly but specifically target the injestisome T3SS. 4EpDN reduced the number of T3SS basal bodies detected on the surface of Y. enterocolitica, as visualized using a fluorescent derivative of YscD, an inner membrane ring with low homology to flagellar protein FliG. Collectively, these data suggest that cyclic peptomers specifically inhibit the injectisome T3SS from a variety of Gram-negative bacteria, possibly by preventing complete T3SS assembly.IMPORTANCETraditional antibiotics target both pathogenic and commensal bacteria, resulting in a disruption of the microbiota, which in turn is tied to a number of acute and chronic diseases. The bacterial type III secretion system (T3SS) is an appendage used by many bacterial pathogens to establish infection, but is largely absent from commensal members of the microbiota. In this study, we identify a new derivative of the cyclic peptomer class of T3SS inhibitors. These compounds inhibit the T3SS of the nosocomial ESKAPE pathogen Pseudomonas aeruginosa and enteropathogenic Yersinia and Salmonella. The impact of cyclic peptomers is specific to the T3SS, as other bacterial secretory systems are unaffected. Importantly, cyclic peptomers completely block replication of Chlamydia trachomatis, the causative agent of genital, eye, and lung infections, in human cells, a process that requires the T3SS. Therefore, cyclic peptomers represent promising virulence blockers that can specifically disarm a broad spectrum of Gram-negative pathogens.

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