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 Discovery of Plant Phenolic Compounds That Act as Type III Secretion System Inhibitors or Inducers of the Fire Blight Pathogen, Erwinia amylovora

2013 ◽  
Vol 79 (18) ◽  
pp. 5424-5436 ◽  
Author(s):  
Devanshi Khokhani ◽  
Chengfang Zhang ◽  
Yan Li ◽  
Qi Wang ◽  
Quan Zeng ◽  
...  
Keyword(s):  
Phenolic Compounds ◽  
Virulence Factors ◽  
Type Iii Secretion ◽  
Fire Blight ◽  
Erwinia Amylovora ◽  
Fluorescent Protein ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Content Type ◽  
Type Iii

ABSTRACTErwinia amylovoracauses a devastating disease called fire blight in rosaceous plants. The type III secretion system (T3SS) is one of the important virulence factors utilized byE. amylovorain order to successfully infect its hosts. By using a green fluorescent protein (GFP) reporter construct combined with a high-throughput flow cytometry assay, a library of phenolic compounds and their derivatives was studied for their ability to alter the expression of the T3SS. Based on the effectiveness of the compounds on the expression of the T3SS pilus, the T3SS inhibitors 4-methoxy-cinnamic acid (TMCA) and benzoic acid (BA) and one T3SS inducer,trans-2-(4-hydroxyphenyl)-ethenylsulfonate (EHPES), were chosen for further study. Both the T3SS inhibitors (TMCA and BA) and the T3SS inducer (EHPES) were found to alter the expression of T3SS through the HrpS-HrpL pathway. Additionally, TMCA altered T3SS expression through thersmBEa-RsmAEasystem. Finally, we found that TMCA and BA weakened the hypersensitive response (HR) in tobacco by suppressing the T3SS ofE. amylovora. In our study, we identified phenolic compounds that specifically targeted the T3SS. The T3SS inhibitor may offer an alternative approach to antimicrobial therapy by targeting virulence factors of bacterial pathogens.

scholarly journals YbeY controls the type III and type VI secretion systems and biofilm formation through RetS in Pseudomonas aeruginosa

2020 ◽  
Author(s):  
Yushan Xia ◽  
Congjuan Xu ◽  
Dan Wang ◽  
Yuding Weng ◽  
Yongxin Jin ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Biofilm Formation ◽  
Small Rnas ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
Chronic Infections ◽  
Type Iii ◽  
Type Vi Secretion

YbeY is a highly conserved RNase in bacteria and plays essential roles in the maturation of 16S rRNA, regulation of small RNAs (sRNAs) and bacterial responses to environmental stresses. Previously, we verified the role of YbeY in rRNA processing and ribosome maturation in Pseudomonas aeruginosa and demonstrated YbeY-mediated regulation of rpoS through a sRNA ReaL. In this study, we demonstrate that mutation of the ybeY gene results in upregulation of the type III secretion system (T3SS) genes as well as downregulation of the type VI secretion system (T6SS) genes and reduction of biofilm formation. By examining the expression of the known sRNAs in P. aeruginosa, we found that mutation of the ybeY gene leads to downregulation of the small RNAs RsmY/Z that control the T3SS, the T6SS and biofilm formation. Further studies revealed that the reduced levels of RsmY/Z are due to upregulation of retS. Taken together, our results reveal the pleiotropic functions of YbeY and provide detailed mechanisms of YbeY-mediated regulation in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa causes a variety of acute and chronic infections in humans. The type III secretion system (T3SS) plays an important role in acute infection and the type VI secretion system (T6SS) and biofilm formation are associated with chronic infections. Understanding of the mechanisms that control the virulence determinants involved in acute and chronic infections will provide clues for the development of effective treatment strategies. Our results reveal a novel RNase mediated regulation on the T3SS, T6SS and biofilm formation in P. aeruginosa.

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 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 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.

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 A Proteolytic Complex Targets Multiple Cell Wall Hydrolases inPseudomonas aeruginosa

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Disha Srivastava ◽  
Jin Seo ◽  
Binayak Rimal ◽  
Sung Joon Kim ◽  
Stephanie Zhen ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Bacterial Cells ◽  
Uncharacterized Protein ◽  
Content Type ◽  
Type Iii ◽  
Cross Links ◽  
Carboxy Terminal

ABSTRACTCarboxy-terminal processing proteases (CTPs) occur in all three domains of life. In bacteria, some of them have been associated with virulence. However, the precise roles of bacterial CTPs are poorly understood, and few direct proteolytic substrates have been identified. One bacterial CTP is the CtpA protease ofPseudomonas aeruginosa, which is required for type III secretion system (T3SS) function and for virulence in a mouse model of acute pneumonia. Here, we have investigated the function of CtpA inP. aeruginosaand identified some of the proteins it cleaves. We discovered that CtpA forms a complex with a previously uncharacterized protein, which we have named LbcA (lipoproteinbinding partner ofCtpA). LbcA is required for CtpA activityin vivoand promotes its activityin vitro. We have also identified four proteolytic substrates of CtpA, all of which are uncharacterized proteins predicted to cleave the peptide cross-links within peptidoglycan. Consistent with this, actpAnull mutant was found to have fewer peptidoglycan cross-links than the wild type and grew slowly in salt-free medium. Intriguingly, the accumulation of just one of the CtpA substrates was required for some ΔctpAmutant phenotypes, including the defective T3SS. We propose that LbcA-CtpA is a proteolytic complex in theP. aeruginosacell envelope, which controls the activity of several peptidoglycan cross-link hydrolases by degrading them. Furthermore, based on these and other findings, we suggest that many bacterial CTPs might be similarly controlled by partner proteins as part of a widespread mechanism to control peptidoglycan hydrolase activity.IMPORTANCEBacterial carboxy-terminal processing proteases (CTPs) are widely conserved and have been associated with the virulence of several species. However, their roles are poorly understood, and few direct substrates have been identified in any species.Pseudomonas aeruginosais an important human pathogen in which one CTP, known as CtpA, is required for type III secretion system function and for virulence. This work provides an important advance by showing that CtpA works with a previously uncharacterized binding partner to degrade four substrates. These substrates are all predicted to hydrolyze peptidoglycan cross-links, suggesting that the CtpA complex is an important control mechanism for peptidoglycan hydrolysis. This is likely to emerge as a widespread mechanism used by diverse bacteria to control some of their peptidoglycan hydrolases. This is significant, given the links between CTPs and virulence in several pathogens and the importance of peptidoglycan remodeling to almost all bacterial cells.

scholarly journals Type III Secretion of ExoU Is Critical during Early Pseudomonas aeruginosa Pneumonia

mBio ◽  
2013 ◽  
Vol 4 (2) ◽  
Author(s):  
Heather A. Howell ◽  
Latania K. Logan ◽  
Alan R. Hauser
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Animal Models ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Therapeutic Interventions ◽  
Bacterial Survival ◽  
Content Type ◽  
Type Iii ◽  
Early Expression

ABSTRACTThePseudomonas aeruginosatype III secretion system has been associated with poor outcomes in both animal models and human patients. Despite a large number of studies exploring the regulation of type III secretionin vitro, little is known about the timing of secretion during mammalian infection. Here we demonstrate that theexoUgene, which encodes the highly cytotoxic type III effector ExoU, is induced early during acuteP. aeruginosapneumonia. Immunofluorescence microscopy indicated that the amount of ExoU protein in the lung also increased over time. The importance of early expression was examined using a strain ofP. aeruginosawith inducible production of ExoU. Delays in expression as short as 3 h led to reduced bacterial burdens in the lungs of mice and improved survival. Our results demonstrate that early expression ofexoUis critical to bacterial survival during pneumonia and suggest that therapeutic interventions that delay ExoU secretion for even short periods of time may be efficacious.IMPORTANCEPseudomonas aeruginosais a major contributor to the large numbers of health care-associated infections occurring annually, particularly for immunocompromised patients. Although this organism possesses many virulence factors, the type III secretion system plays an especially important role in both animal models and humans. This system forms a needle-like apparatus that injects toxins directly into eukaryotic cells. The most toxic protein secreted by this molecular machine is ExoU, which causes rapid cell death. In this study, we demonstrated thatexoUwas expressed and ExoU was produced early during acute pneumonia in a mouse model. Delaying expression ofexoUby as little as 3 h enhanced clearance of bacteria and survival of infected mice. Our findings highlight the importance of understanding the regulation of virulence factor expression during infection when designing therapeutic strategies to inhibit the toxic effects of these proteins.

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