Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes.
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SUBMITTER: Ventre I
PROVIDER: S-EPMC1324988 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. In addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, we compared the transcriptional profile generated using DNA microarrays of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAOΔampR. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought, with the deletion of ampR influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Other virulence mechanisms including biofilm formation and QS-regulated acute virulence factors are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the microarray data. Further, using a Caenorhabditis elegans model, we demonstrate that a functional AmpR is required for P. aeruginosa pathogenicity. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. Further, we show differential regulation of other transcriptional regulators and sigma factors by AmpR, accounting for the extensive AmpR regulon. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating biofilm formation, a chronic infection phenotype. Unraveling this complex regulatory circuit will provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors in response to antibiotic exposure.
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Project description:UnlabelledDuring initial colonization and chronic infection, pathogenic bacteria encounter distinct host environments. Adjusting gene expression accordingly is essential for the pathogenesis. Pseudomonas aeruginosa has evolved complicated regulatory networks to regulate different sets of virulence factors to facilitate colonization and persistence. The type III secretion system (T3SS) and motility are associated with acute infections, while biofilm formation and the type VI secretion system (T6SS) are associated with chronic persistence. To identify novel regulatory genes required for pathogenesis, we screened a P. aeruginosa transposon (Tn) insertion library and found suhB to be an essential gene for the T3SS gene expression. The expression of suhB was upregulated in a mouse acute lung infection model, and loss of suhB resulted in avirulence. Suppression of T3SS gene expression in the suhB mutant is linked to a defective translation of the T3SS master regulator, ExsA. Further studies demonstrated that suhB mutation led to the upregulation of GacA and its downstream small RNAs, RsmY and RsmZ, triggering T6SS expression and biofilm formation while inhibiting the T3SS. Our results demonstrate that an in vivo-inducible gene, suhB, reciprocally regulates genes associated with acute and chronic infections and plays an essential role in the pathogenesis of P. aeruginosa.ImportanceA variety of bacterial pathogens, such as Pseudomonas aeruginosa, cause acute and chronic infections in humans. During infections, pathogens produce different sets of virulence genes for colonization, tissue damage, and dissemination and for countering host immune responses. Complex regulatory networks control the delicate tuning of gene expression in response to host environments to enable the survival and growth of invading pathogens. Here we identified suhB as a critical gene for the regulation of virulence factors in P. aeruginosa. The expression of suhB was upregulated during acute infection in an animal model, and mutation of suhB rendered P. aeruginosa avirulent. Moreover, we demonstrate that SuhB is required for the activation of virulence factors associated with acute infections while suppressing virulence factors associated with chronic infections. Our report provides new insights into the multilayered regulatory network of virulence genes in P. aeruginosa.
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Project description:Pyocyanin is a toxin produced by Pseudomonas aeruginosa. Here we describe a novel paper-based electrochemical sensor for pyocyanin detection, manufactured with a simple and inexpensive approach based on electrode printing on paper. The resulting sensors constitute an effective electrochemical method to quantify pyocyanin in bacterial cultures without the conventional time consuming pretreatment of the samples. The electrochemical properties of the paper-based sensors were evaluated by ferri/ferrocyanide as a redox mediator, and showed reliable sensing performance. The paper-based sensors readily allow for the determination of pyocyanin in bacterial cultures with high reproducibility, achieving a limit of detection of 95 nM and a sensitivity of 4.30 μA/μM in standard culture media. Compared to the similar commercial ceramic based sensors, it is a 2.3-fold enhanced performance. The simple in-house fabrication of sensors for pyocyanin quantification allows researchers to understand in vitro adaptation of P. aeruginosa infections via rapid screenings of bacterial cultures that otherwise are expensive and time-consuming.
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Project description:Pseudomonas aeruginosa PAO1 contains gshA and gshB genes, which encode enzymes involved in glutathione (GSH) biosynthesis. Challenging P. aeruginosa with hydrogen peroxide, cumene hydroperoxide, and t-butyl hydroperoxide increased the expression of gshA and gshB. The physiological roles of these genes in P. aeruginosa oxidative stress, bacterial virulence, and biofilm formation were examined using P. aeruginosa ?gshA, ?gshB, and double ?gshA?gshB mutant strains. These mutants exhibited significantly increased susceptibility to methyl viologen, thiol-depleting agent, and methylglyoxal compared to PAO1. Expression of functional gshA, gshB or exogenous supplementation with GSH complemented these phenotypes, which indicates that the observed mutant phenotypes arose from their inability to produce GSH. Virulence assays using a Drosophila melanogaster model revealed that the ?gshA, ?gshB and double ?gshA?gshB mutants exhibited attenuated virulence phenotypes. An analysis of virulence factors, including pyocyanin, pyoverdine, and cell motility (swimming and twitching), showed that these levels were reduced in these gsh mutants compared to PAO1. In contrast, biofilm formation increased in mutants. These data indicate that the GSH product and the genes responsible for GSH synthesis play multiple crucial roles in oxidative stress protection, bacterial virulence and biofilm formation in P. aeruginosa.
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Project description:AmrZ is a putative ribbon-helix-helix (RHH) transcriptional regulator. RHH proteins utilize residues within the β-sheet for DNA binding, while the α-helices promote oligomerization. AmrZ is of interest due to its dual roles as a transcriptional activator and as a repressor, regulating genes encoding virulence factors associated with both chronic and acute Pseudomonas aeruginosa infection. In this study, cross-linking revealed that AmrZ forms oligomers in solution but that the amino terminus, containing an unordered region and a β-sheet, were not required for oligomerization. The first 12 unordered residues (extended amino terminus) contributed minimally to DNA binding. Mutagenesis of the AmrZ β-sheet demonstrated that residues 18, 20, and 22 were essential for DNA binding at both activation and repressor sites, suggesting that AmrZ utilizes a similar mechanism for binding to these sites. Mice infected with amrZ mutants exhibited reduced bacterial burden, morbidity, and mortality. Direct in vivo competition assays showed a 5-fold competitive advantage for the wild type over an isogenic amrZ mutant. Finally, the reduced infection phenotype of the amrZ-null strain was similar to that of a strain expressing a DNA-binding-deficient AmrZ variant, indicating that DNA binding and transcriptional regulation by AmrZ is responsible for the in vivo virulence defect. These recent infection data, along with previously identified AmrZ-regulated virulence factors, suggest the necessity of AmrZ transcriptional regulation for optimal virulence during acute infection.
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Project description:In a number of bacterial pathogens, the production of virulence factors is induced at 37 °C; this effect is often regulated by mRNA structures formed in the 5' untranslated region (UTR) that block translation initiation of genes at environmental temperatures. At 37 °C, the RNA structures become unstable and ribosomes gain access to their binding sites in the mRNAs. Pseudomonas aeruginosa is an important opportunistic pathogen and the expression of many of its virulence-associated traits is regulated by the quorum-sensing (QS) response, but the effect of temperature on virulence-factor expression is not well-understood. The aim of this work is the characterization of the molecular mechanism involved in thermoregulation of QS-dependent virulence-factor production. We demonstrate that traits that are dependent on the QS transcriptional regulator RhlR have a higher expression at 37 °C, correlating with a higher RhlR concentration as measured by Western blot. We also determined, using gene fusions and point mutations, that RhlR thermoregulation is a posttranscriptional effect dependent on an RNA thermometer of the ROSE (Repression Of heat-Shock gene Expression) family. This RNA element regulates the expression of the rhlAB operon, involved in rhamnolipid production, and of the downstream rhlR gene. We also identified a second functional thermometer in the 5' UTR of the lasI gene. We confirmed that these RNA thermometers are the main mechanism of thermoregulation of QS-dependent gene expression in P. aeruginosa using quantitative real-time PCR. This is the first description, to our knowledge, of a ROSE element regulating the expression of virulence traits and of an RNA thermometer controlling multiple genes in an operon through a polar effect.
| S-EPMC4217398 | biostudies-literature
Project description:The emergence of multidrug-resistant Pseudomonas aeruginosa in healthcare settings poses a tremendous challenge to traditional antibiotic therapy. Pseudomonas aeruginosa utilizes quorum sensing (QS) to coordinate the production of virulence factors and the formation of drug-resistant biofilms. QS is mediated by signal compounds produced by P. aeruginosa as well as signal molecules produced by other non-pseudomonad bacteria. A potential strategy to prevent bacterial pathogenicity is utilizing enzymes to interfere with QS. Here, we used AidC, a quorum-quenching (QQ) enzyme from Chryseobacterium sp. strain StRB126 that can effectively hydrolyze N-(3-oxododecanoyl) homoserine lactone (3OC12-HSL) and N-butanoyl-homoserine lactone (C4-HSL), the major signal molecules synthesized by P. aeruginosa. The exogenous addition of AidC to P. aeruginosa wild-type strain PAO1 cultures significantly reduced the total protease and elastase activities and the production of pyocyanin. In addition, the application of AidC resulted in thin and sparse biofilm formation. Later, we used a metagenomic-derived QQ enzyme, QQ-2, in combination with AidC to attenuate PAO1 virulence when the presence of a non-pseudomonad signal compound, autoinducer-2, aggravated it. These findings suggest that using a combined antimicrobial approach may lead to a more efficacious therapeutic intervention against P. aeruginosa PAO1 infection, as its behavior is modulated in the presence of intraspecies and interspecies signal compounds.One-sentence summaryIn this work, the potential of dual enzymes was investigated to interfere with quorum sensing as a novel concept for reducing the virulence of P. aeruginosa, which is influenced by both intra species and interspecies communication.
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Project description:The virulence of Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is regulated by many transcriptional factors (TFs) that control the expression of quorum sensing and protein secretion systems. Here, we report a genome-wide, network-based approach to dissect the crosstalk between 20 key virulence-related TFs. Using chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), as well as RNA-seq, we identify 1200 TF-bound genes and 4775 differentially expressed genes. We experimentally validate 347 of these genes as functional target genes, and describe the regulatory relationships of the 20 TFs with their targets in a network that we call 'Pseudomonas aeruginosa genomic regulatory network' (PAGnet). Analysis of the network led to the identification of novel functions for two TFs (ExsA and GacA) in quorum sensing and nitrogen metabolism. Furthermore, we present an online platform and R package based on PAGnet to facilitate updating and user-customised analyses.
| S-EPMC6610081 | biostudies-literature
Project description:To assess the role of core metabolism genes in bacterial virulence - independently of their effect on growth - we correlated the genome, the transcriptome and the pathogenicity in flies and mice of 30 fully sequenced Pseudomonas strains. Gene presence correlates robustly with pathogenicity differences among all Pseudomonas species, but not among the P. aeruginosa strains. However, gene expression differences are evident between highly and lowly pathogenic P. aeruginosa strains in multiple virulence factors and a few metabolism genes. Moreover, 16.5%, a noticeable fraction of the core metabolism genes of P. aeruginosa strain PA14 (compared to 8.5% of the non-metabolic genes tested), appear necessary for full virulence when mutated. Most of these virulence-defective core metabolism mutants are compromised in at least one key virulence mechanism independently of auxotrophy. A pathway level analysis of PA14 core metabolism, uncovers beta-oxidation and the biosynthesis of amino-acids, succinate, citramalate, and chorismate to be important for full virulence. Strikingly, the relative expression among P. aeruginosa strains of genes belonging in these metabolic pathways is indicative of their pathogenicity. Thus, P. aeruginosa strain-to-strain virulence variation, remains largely obscure at the genome level, but can be dissected at the pathway level via functional transcriptomics of core metabolism.
| S-EPMC7289854 | biostudies-literature
Project description:Posttranscriptional regulation plays an essential role in the quick adaptation of pathogenic bacteria to host environments, and RNases play key roles in this process by modifying small RNAs and mRNAs. We find that the Pseudomonas aeruginosa endonuclease YbeY is required for rRNA processing and the bacterial virulence in a murine acute pneumonia model. Transcriptomic analyses reveal that knocking out the ybeY gene results in downregulation of oxidative stress response genes, including the catalase genes katA and katB Consistently, the ybeY mutant is more susceptible to H2O2 and neutrophil-mediated killing. Overexpression of katA restores the bacterial tolerance to H2O2 and neutrophil killing as well as virulence. We further find that the downregulation of the oxidative stress response genes is due to defective expression of the stationary-phase sigma factor RpoS. We demonstrate an autoregulatory mechanism of RpoS and find that ybeY mutation increases the level of a small RNA, ReaL, which directly represses the translation of rpoS through the 5' UTR of its mRNA and subsequently reduces the expression of the oxidative stress response genes. In vitro assays demonstrate direct degradation of ReaL by YbeY. Deletion of reaL or overexpression of rpoS in the ybeY mutant restores the bacterial tolerance to oxidative stress and the virulence. We also demonstrate that YbeZ binds to YbeY and is involved in the 16S rRNA processing and regulation of reaL and rpoS as well as the bacterial virulence. Overall, our results reveal pleiotropic roles of YbeY and the YbeY-mediated regulation of rpoS through ReaL.IMPORTANCE The increasing bacterial antibiotic resistance imposes a severe threat to human health. For the development of effective treatment and prevention strategies, it is critical to understand the mechanisms employed by bacteria to grow in the human body. Posttranscriptional regulation plays an important role in bacterial adaptation to environmental changes. RNases and small RNAs are key players in this regulation. In this study, we demonstrate critical roles of the RNase YbeY in the virulence of the pathogenic bacterium Pseudomonas aeruginosa We further identify the small RNA ReaL as the direct target of YbeY and elucidate the YbeY-regulated pathway on the expression of bacterial virulence factors. Our results shed light on the complex regulatory network of P. aeruginosa and indicate that inference with the YbeY-mediated regulatory pathway might be a valid strategy for the development of a novel treatment strategy.
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