scholarly journals Pseudomonas aeruginosa Oligoribonuclease Contributes to Tolerance to Ciprofloxacin by Regulating Pyocin Biosynthesis

2017 ◽  
Vol 61 (3) ◽  
Author(s):  
Fei Chen ◽  
Gukui Chen ◽  
Yiwei Liu ◽  
Yongxin Jin ◽  
Zhihui Cheng ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Transcription Initiation ◽  
Global Gene Expression ◽  
Secretion System ◽  
Negative Regulator ◽  
Content Type ◽  
Bacterial Response ◽  
Hydrolysis Of

ABSTRACTBacterial oligoribonuclease (Orn) is a conserved 3′-to-5′ exonuclease. InPseudomonas aeruginosa, it has been demonstrated that Orn plays a major role in the hydrolysis of pGpG, which is required for cyclic-di-GMP homeostasis. Meanwhile, Orn is involved in the degradation of nanoRNAs, which can alter global gene expression by serving as transcription initiation primers. Previously, we found that Orn is required for the type III secretion system and pathogenesis ofP. aeruginosa, indicating a role of Orn in the bacterial response to environmental stimuli. Here we report that Orn is required for the tolerance ofP. aeruginosato ciprofloxacin. Transcriptome analysis of anornmutant revealed the upregulation of pyocin biosynthesis genes. Mutation of genes involved in pyocin biosynthesis in the background of anornmutant restored bacterial tolerance to ciprofloxacin. We further demonstrate that the upregulation of pyocin biosynthesis genes is due to RecA-mediated autoproteolysis of PrtR, which is the major negative regulator of pyocin biosynthesis genes. In addition, the SOS response genes were upregulated in theornmutant, indicating a DNA damage stress. Therefore, our results revealed a novel role of Orn in bacterial tolerance to ciprofloxacin.

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 Derivatives of Plant Phenolic Compound Affect the Type III Secretion System of Pseudomonas aeruginosa via a GacS-GacA Two-Component Signal Transduction System

2011 ◽  
Vol 56 (1) ◽  
pp. 36-43 ◽  
Author(s):  
Akihiro Yamazaki ◽  
Jin Li ◽  
Quan Zeng ◽  
Devanshi Khokhani ◽  
William C. Hutchins ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Phenolic Compounds ◽  
Virulence Factors ◽  
Type Iii Secretion ◽  
Small Rnas ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Human Pathogen ◽  
Content Type ◽  
Type Iii

ABSTRACTAntibiotic therapy is the most commonly used strategy to control pathogenic infections; however, it has contributed to the generation of antibiotic-resistant bacteria. To circumvent this emerging problem, we are searching for compounds that target bacterial virulence factors rather than their viability.Pseudomonas aeruginosa, an opportunistic human pathogen, possesses a type III secretion system (T3SS) as one of the major virulence factors by which it secretes and translocates T3 effector proteins into human host cells. The fact that this human pathogen also is able to infect several plant species led us to screen a library of phenolic compounds involved in plant defense signaling and their derivatives for novel T3 inhibitors. Promoter activity screening ofexoS, which encodes a T3-secreted toxin, identified two T3 inhibitors and two T3 inducers ofP. aeruginosaPAO1. These compounds alterexoStranscription by affecting the expression levels of the regulatory small RNAs RsmY and RsmZ. These two small RNAs are known to control the activity of carbon storage regulator RsmA, which is responsible for the regulation of the key T3SS regulator ExsA. As RsmY and RsmZ are the only targets directly regulated by GacA, our results suggest that these phenolic compounds affect the expression ofexoSthrough the GacSA-RsmYZ-RsmA-ExsA regulatory pathway.

scholarly journals RNase E Promotes Expression of Type III Secretion System Genes in Pseudomonas aeruginosa

2019 ◽  
Vol 201 (22) ◽  
Author(s):  
Josh S. Sharp ◽  
Arne Rietsch ◽  
Simon L. Dove
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Rnase E ◽  
Content Type ◽  
Type Iii ◽  
Small Regulatory Rnas

ABSTRACT Pseudomonas aeruginosa is an important opportunistic pathogen that employs a type III secretion system (T3SS) to inject effector proteins into host cells. Using a protein depletion system, we show that the endoribonuclease RNase E positively regulates expression of the T3SS genes. We also present evidence that RNase E antagonizes the expression of genes of the type VI secretion system and limits biofilm production in P. aeruginosa. Thus, RNase E, which is thought to be the principal endoribonuclease involved in the initiation of RNA degradation in P. aeruginosa, plays a key role in controlling the production of factors involved in both acute and chronic stages of infection. Although the posttranscriptional regulator RsmA is also known to positively regulate expression of the T3SS genes, we find that RNase E does not appreciably influence the abundance of RsmA in P. aeruginosa. Moreover, we show that RNase E still exerts its effects on T3SS gene expression in cells lacking all four of the key small regulatory RNAs that function by sequestering RsmA. IMPORTANCE The type III secretion system (T3SS) is a protein complex produced by many Gram-negative pathogens. It is capable of injecting effector proteins into host cells that can manipulate cell metabolism and have toxic effects. Understanding how the T3SS is regulated is important in understanding the pathogenesis of bacteria with such systems. Here, we show that RNase E, which is typically thought of as a global regulator of RNA stability, plays a role in regulating the T3SS in Pseudomonas aeruginosa. Depleting RNase E results in the loss of T3SS gene expression as well as a concomitant increase in biofilm formation. These observations are reminiscent of the phenotypes associated with the loss of activity of the posttranscriptional regulator RsmA. However, RNase E-mediated regulation of these systems does not involve changes in the abundance of RsmA and is independent of the known small regulatory RNAs that modulate RsmA activity.

scholarly journals Novel Type III Effectors in Pseudomonas aeruginosa

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
David Burstein ◽  
Shirley Satanower ◽  
Michal Simovitch ◽  
Yana Belnik ◽  
Meital Zehavi ◽  
...  
Keyword(s):  
Machine Learning ◽  
Pseudomonas Aeruginosa ◽  
Host Cell ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Content Type ◽  
Machine Learning Classification ◽  
Type Iii ◽  
Wide Range

ABSTRACT Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes chronic and acute infections in immunocompromised patients. Most P. aeruginosa strains encode an active type III secretion system (T3SS), utilized by the bacteria to deliver effector proteins from the bacterial cell directly into the cytoplasm of the host cell. Four T3SS effectors have been discovered and extensively studied in P. aeruginosa: ExoT, ExoS, ExoU, and ExoY. This is especially intriguing in light of P. aeruginosa's ability to infect a wide range of hosts. We therefore hypothesized that additional T3SS effectors that have not yet been discovered are encoded in the genome of P. aeruginosa. Here, we applied a machine learning classification algorithm to identify novel P. aeruginosa effectors. In this approach, various types of data are integrated to differentiate effectors from the rest of the open reading frames of the bacterial genome. Due to the lack of a sufficient learning set of positive effectors, our machine learning algorithm integrated genomic information from another Pseudomonas species and utilized dozens of features accounting for various aspects of the effector coding genes and their products. Twelve top-ranking predictions were experimentally tested for T3SS-specific translocation, leading to the discovery of two novel T3SS effectors. We demonstrate that these effectors are not part of the injection structural complex and report initial efforts toward their characterization. IMPORTANCE Pseudomonas aeruginosa uses a type III secretion system (T3SS) to secrete toxic proteins, termed effectors, directly into the cytoplasm of the host cell. The activation of this secretion system is correlated with disease severity and patient death. Compared with many other T3SS-utilizing pathogenic bacteria, P. aeruginosa has a fairly limited arsenal of effectors that have been identified. This is in sharp contrast with the wide range of hosts that this bacterium can infect. The discovery of two novel effectors described here is an important step toward better understanding of the virulence and host evasion mechanisms adopted by this versatile pathogen and may provide novel approaches to treat P. aeruginosa infections.

scholarly journals Phosphatidylinositol 4,5-Bisphosphate Is a Novel Coactivator of the Pseudomonas aeruginosa Cytotoxin ExoU

2013 ◽  
Vol 81 (8) ◽  
pp. 2873-2881 ◽  
Author(s):  
Gregory H. Tyson ◽  
Alan R. Hauser
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Eukaryotic Cell ◽  
Secretion System ◽  
Phospholipase A ◽  
Effector Protein ◽  
Yeast Mutant ◽  
Membrane Phospholipids ◽  
Content Type ◽  
Ubiquitinated Proteins

ABSTRACTExoU is a potent phospholipase A2effector protein secreted by the type III secretion system ofPseudomonas aeruginosa. By cleaving plasma membrane phospholipids, it causes rapid lysis of eukaryotic cells. However, ExoU does not exhibit activity on its own but instead requires eukaryotic cell cofactors for activation. Ubiquitin and ubiquitinated proteins have been shown to activate ExoU, but previous work suggested that other cofactors are also involved. In this study, we demonstrate that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is another important coactivator of ExoU. PI(4,5)P2 works synergistically with ubiquitin to greatly enhance the phospholipase A2activity of ExoU. Distinct residues of ExoU were critical for activation by PI(4,5)P2 and by ubiquitin, indicating that these factors activate ExoU by discrete mechanisms. In support of the biological relevance of PI(4,5)P2 coactivation, a yeast mutant with reduced PI(4,5)P2 levels was less susceptible to the cytotoxic activity of ExoU. Together, these findings further elaborate the molecular mechanism of ExoU.

scholarly journals Role of Toll Interleukin-1 Receptor (IL-1R) 8, a Negative Regulator of IL-1R/Toll-Like Receptor Signaling, in Resistance to Acute Pseudomonas aeruginosa Lung Infection

2011 ◽  
Vol 80 (1) ◽  
pp. 100-109 ◽  
Author(s):  
Tania Véliz Rodriguez ◽  
Federica Moalli ◽  
Nadia Polentarutti ◽  
Moira Paroni ◽  
Eduardo Bonavita ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Interleukin 1 ◽  
Bacterial Load ◽  
Lung Infection ◽  
Negative Regulator ◽  
Current Evidence ◽  
Toll Like Receptor ◽  
Content Type ◽  
Deficient Mice

ABSTRACTToll interleukin-1 receptor (IL-1R) 8 (TIR8), also known as single Ig IL-1 receptor (IL-R)-related molecule, or SIGIRR, is a member of the IL-1R-like family, primarily expressed by epithelial cells. Current evidence suggests that TIR8 plays a nonredundant role as a negative regulatorin vivounder different inflammatory conditions that are dependent on IL-R and Toll-like receptor (TLR) activation. In the present study, we examined the role of TIR8 in innate resistance to acute lung infections caused byPseudomonas aeruginosa, a Gram-negative pathogen responsible for life-threatening infections in immunocompromised individuals and cystic fibrosis patients. We show that Tir8 deficiency in mice was associated with increased susceptibility to acuteP. aeruginosainfection, in terms of mortality and bacterial load, and to exacerbated local and systemic production of proinflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], IL-1β, and IL-6) and chemokines (CXCL1, CXCL2, and CCL2). It has been reported that host defense againstP. aeruginosaacute lung infection can be improved by blocking IL-1 since exaggerated IL-1β production may be harmful for the host in this infection. In agreement with these data, IL-1RI deficiency rescues the phenotype observed in Tir8-deficient mice: in Tir8−/−IL-1RI−/−double knockout mice we observed higher survival rates, enhanced bacterial clearance, and reduced levels of local and systemic cytokine and chemokine levels than in Tir8-deficient mice. These results suggest that TIR8 has a nonredundant effect in modulating the inflammation caused byP. aeruginosa, in particular, by negatively regulating IL-1RI signaling, which plays a major role in the pathogenesis of this infectious disease.

scholarly journals Genome Sequences of Two Pseudomonas aeruginosa Isolates with Defects in Type III Secretion System Gene Expression from a Chronic Ankle Wound Infection

2021 ◽  
Author(s):  
Sardar Karash ◽  
Robert Nordell ◽  
Egon A. Ozer ◽  
Timothy L. Yahr
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Biofilm Formation ◽  
Secretion System ◽  
Chronic Infections ◽  
Genome Sequences ◽  
Content Type ◽  
Chronic Colonization ◽  
Host Tolerance

A common feature of microorganisms that cause chronic infections is a stealthy lifestyle that promotes immune avoidance and host tolerance. During chronic colonization of cystic fibrosis (CF) patients, Pseudomonas aeruginosa acquires numerous adaptations that include reduced expression of some factors, such as motility, O antigen, and the T3SS, and increased expression of other traits, such as biofilm formation.

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

2005 ◽  
Vol 73 (7) ◽  
pp. 4263-4271 ◽  
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.

scholarly journals Mutations in the Pseudomonas aeruginosa Needle Protein GenepscFConfer Resistance to Phenoxyacetamide Inhibitors of the Type III Secretion System

2014 ◽  
Vol 58 (4) ◽  
pp. 2211-2220 ◽  
Author(s):  
Nicholas O. Bowlin ◽  
John D. Williams ◽  
Claire A. Knoten ◽  
Matthew C. Torhan ◽  
Tommy F. Tashjian ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Molecular Target ◽  
Secretion System ◽  
Host Cells ◽  
Content Type ◽  
Type Iii ◽  
Unknown Protein ◽  
Needle Protein

ABSTRACTThe type III secretion system (T3SS) is a clinically important virulence mechanism inPseudomonas aeruginosathat secretes and translocates effector toxins into host cells, impeding the host's rapid innate immune response to infection. Inhibitors of T3SS may be useful as prophylactic or adjunctive therapeutic agents to augment the activity of antibiotics inP. aeruginosainfections, such as pneumonia and bacteremia. One such inhibitor, the phenoxyacetamide MBX 1641, exhibits very responsive structure-activity relationships, including striking stereoselectivity, in its inhibition ofP. aeruginosaT3SS. These features suggest interaction with a specific, but unknown, protein target. Here, we identify the apparent molecular target by isolating inhibitor-resistant mutants and mapping the mutation sites by deep sequencing. Selection and sequencing of four independent mutants resistant to the phenoxyacetamide inhibitor MBX 2359 identified the T3SS genepscF, encoding the needle apparatus, as the only locus of mutations common to all four strains. Transfer of the wild-type and mutated alleles ofpscF, together with its chaperone and cochaperone genespscEandpscG, to a ΔpscF P. aeruginosastrain demonstrated that each of the single-codon mutations inpscFis necessary and sufficient to provide secretion and translocation that is resistant to a variety of phenoxyacetamide inhibitor analogs but not to T3SS inhibitors with different chemical scaffolds. These results implicate the PscF needle protein as an apparent new molecular target for T3SS inhibitor discovery and suggest that three other chemically distinct T3SS inhibitors interact with one or more different targets or a different region of PscF.

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