Expression data from Pseudomonas aeruginosa PAO1 treated with phenylacetic acid
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ABSTRACT: P. aeruginosa is known to cause acute cytotoxicity against various human and animal cells and tissues. We identified bacterial metabolite - phenylacetic acid (PAA) which acts as an inhibitory molecule counteracting its pathogenic infection. Microarray and genetic analyses were conducted to investigate the inhibitory mechanism of the identified inhibitor PAA on bacterial virulence. Microarray analysis revealed that treatment of P. aeruginosa with PAA down-regulated the transcriptional expression of type 3 secretion systems (T3SS) genes and related regulatory genes including rsmA and vfr, which were confirmed by transcriptional and translational analysis. Our findings present a new insight to the puzzle of high-cell-density-modulated virulence attenuation in P. aeruginosa and the regulatory mechanisms of T3SS which is associated with bacterial acute infection. Overnight PAO1 culture were diluted 1:200 to fresh LB medium supplemented with nitriloacetic acid (NTA) with or without addition of 1 mM of phenylacetic acid (PAA). The growth was continued with shaking at 37M-BM-0C for 4 h to allow OD600 reaching about 1.5 and the cells were used for RNA extraction and hybridization on Affymetrix microarrays.
Project description:P. aeruginosa is known to cause acute cytotoxicity against various human and animal cells and tissues. We identified bacterial metabolite - phenylacetic acid (PAA) which acts as an inhibitory molecule counteracting its pathogenic infection. Microarray and genetic analyses were conducted to investigate the inhibitory mechanism of the identified inhibitor PAA on bacterial virulence. Microarray analysis revealed that treatment of P. aeruginosa with PAA down-regulated the transcriptional expression of type 3 secretion systems (T3SS) genes and related regulatory genes including rsmA and vfr, which were confirmed by transcriptional and translational analysis. Our findings present a new insight to the puzzle of high-cell-density-modulated virulence attenuation in P. aeruginosa and the regulatory mechanisms of T3SS which is associated with bacterial acute infection.
Project description:Colistin is an important cationic antimicrobial peptide (CAMP) in the fight against Pseudomonas aeruginosa infection within the cystic fibrosis (CF) lungs. The effects of sub-inhibitory colistin on gene expression in P. aeruginosa were investigated by transcriptome microarray and functional analysis. Analysis revealed an alteration in the expression of 60 genes in total from a variety of pathways. Genes associated with bacterial chronic colonisation and virulence such as response to osmotic stress, motility, and biofilm formation, as well as those associated with LPS modification and quorum sensing are the most highly represented. Most striking among these is the upregulation of the PQS biosynthesis operon including pqsH, pqsE, and the anthranilate biosynthetic genes phnAB. Early activation of this central component of the QS-network may represent a switch to a more robust population, with increased fitness in the competitive environment of the CF-lung. Experiment Overall Design: Three independent cultures of the P. aeruginosa strain PAO1 were exposed to 0.15 µg colistin mlâ1. The untreated and treated samples were grown from OD600 0.05 to 0.8 and subsequently total RNA was extracted using the Ambion RiboPureTm- Bacteria kit according to the manufacturerâs instructions.
Project description:Regulatory networks including virulence-related transcriptional factors (TFs) determine bacterial pathogenicity in response to different environmental cues. Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen of humans, recruits numerous TFs in quorum sensing (QS) system, type III secretion system (T3SS) and Type VI secretion system (T6SS) to mediate the pathogenicity. Although many virulence-related TFs have been illustrated individually, very little is known about their crosstalks and regulatory network. Here, based on chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) and transcriptome profiling (RNA-seq), we primarily focused on understanding the crosstalks of 20 virulence-related TFs, which led to construction of a virulence regulatory network named PAGnet (Pseudomonas aeruginosa Genomic integrated regulatory network), including 82 crosstalk targets. The PAGnet uncovered the intricate mechanism of virulence regulation and revealed master regulators in QS, T3SS and T6SS pathways. In particular, GacA and ExsA showed novel functions in QS and nitrogen metabolism. In addition, an online PAGnet platform was provided to analyze these TFs and more virulence factors. Taken together, the present study revealed the function-specific crosstalks of virulence regulatory network, which might provide new strategies for treating infections in P. aeruginosa in the future.
Project description:The aryl hydrocarbon receptor (AhR) is a highly conserved ligand-dependent transcription factor that senses environmental toxins and endogenous ligands, thereby inducing detoxifying enzymes and modulating immune cell differentiation and responses. We hypothesized that AhR not only evolved to sense pollutants from the environment, but also insults of microbial origin. We demonstrate that bacterial pigmented virulence factors, namely the phenazines pyocyanin and 1-hydroxyphenazine from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis, are ligands of AhR. AhR activation leads to degradation of these virulence factors and regulated cytokine and chemokine production. The relevance of AhR to host defense is underlined by heightened susceptibility of AhR-deficient mice, both to P. aeruginosa and M. tuberculosis. Our data demonstrate that AhR senses distinct bacterial virulence factors and controls anti-bacterial responses. We provide evidence for a previously unidentified role for AhR as an intracellular pattern recognition receptor, and identify bacterial pigmented virulence factors as a new class of pathogen-associated molecular patterns. Microarray experiments were performed as single-color hybridizations. Quality control and quantification of total RNA amount was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies) and a NanoDrop 1000 spectrophotometer (Kisker).
Project description:Characterization of bacterial behavior in the microgravity environment of spaceflight is of importance towards risk assessment and prevention of infectious disease during long-term missions. Further, this research field unveils new insights into connections between low fluid-shear regions encountered by pathogens during their natural infection process in vivo, and bacterial virulence. This study is the first to characterize the global transcriptomic and proteomic response of an opportunistic pathogen that is actually found in the space habitat, Pseudomonas aeruginosa. Overall, P. aeruginosa responded to spaceflight conditions through differential regulation of 167 genes and 28 proteins, with Hfq identified as a global transcriptional regulator in the response to this environment. Since Hfq was also induced in spaceflight-grown Salmonella typhimurium, Hfq represents the first spaceflight-induced regulator across the bacterial species border. The major P. aeruginosa virulence-related genes induced in spaceflight conditions were the lecA and lecB lectins and the rhamnosyltransferase (rhlA), involved in the production of rhamnolipids. The transcriptional response of spaceflight-grown P. aeruginosa was compared with our previous data of this organism grown in microgravity-analogue conditions using the rotating wall vessel (RWV) bioreactor technology. Interesting similarities were observed, among others with regard to Hfq regulation and oxygen utilization. While LSMMG-grown P. aeruginosa mainly induced genes involved in microaerophilic metabolism, P. aeruginosa cultured in spaceflight adopted an anaerobic mode of growth, in which denitrification was presumably most prominent. Differences in hardware between spaceflight and LSMMG experiments, in combination with more pronounced low fluid shear and mixing in spaceflight when compared to LSMMG conditions, were hypothesized to be at the origin of these observations. Collectively, our data suggest that spaceflight conditions could induce the transition of P. aeruginosa from an opportunistic organism to potential pathogen, results that are of importance for infectious disease risk assessment and prevention, both during spaceflight missions and in the clinic. This study describes the transcriptional response of P. aeruginosa PAO1 to low-Earth orbit environmental conditions. Our aim was to assess whether the microgravity environment of spaceflight could induce virulence traits in P. aeruginosa. To this end, P. aeruginosa cultures were grown in space, and the expression profile was compared with ground control samples (both in biological triplicate). Two RWV samples also examined (did not re-analyze them, only compared the outputs).
Project description:Bacterial interspecies interactions shape the function and structural dynamics of microbial communities and affect disease progression of polymicrobial infections. Here, we present data suggesting that the FemA-FemR-FemI (Fem) cell surface signaling system in P. aeruginosa serves as an interspecies signaling pathway between P. aeruginosa and Mycobacterium species. The Fem system is regulated by the type three secretion system (T3SS) regulator ExsA. Fem system significantly influenced virulence factors in P. aeruginosa, including the quorum sensing systems, pyocyanin production, biofilm formation and the type six secretion systems (T6SSs). Our study using a Galleria mellonella infection model indicates femA deletion significantly increased the host survival rate while femI over-expression decreased it, demonstrating that the Fem system’s role in bacterial pathogenicity in vivo. We propose that the Fem system functions as an interspecies signaling pathway enabling P. aeruginosa to alter its behaviours in response to the presence of mycobacteria.
Project description:Pseudomonas aeruginosa bacterial and (outer membrane vesicles) extracellular vesicles proteome were analysed. Different types of PAO1, dellys and pJNlys were lysed in 1% (v/v) sodium dodecyl sulphate and virulence factors analysed
Project description:Addressing the functionality of predicted genes remains an enormous challenge in the post-genomic era. A prime example of genes lacking functional assignments are the poorly conserved, early expressed genes of lytic bacteriophages, whose products are involved in the subversion of the host metabolism. In this study, we focused on the composition of important macromolecular complexes of Pseudomonas aeruginosa involved in transcription, DNA replication, fatty acid biosynthesis, RNA regulation, energy metabolism and cell division, during infection with members of seven distinct clades of lytic phages. Using affinity purifications of these host protein complexes coupled to mass spectrometric analyses, 37 host complex-associated phage proteins could be identified. Importantly, eight of these show an inhibitory effect on bacterial growth upon episomal expression, suggesting that these phage proteins are potentially involved in hijacking the host complexes. Using complementary protein-protein interaction assays, we further mapped the inhibitory interaction of gp12 of phage 14-1 to the α subunit of the RNA polymerase. Together, our data demonstrate the powerful use of interactomics to unravel the biological role of hypothetical phage proteins, which constitute an enormous untapped source of novel antibacterial proteins.
Project description:To see the effect of sub-inhibitory concentrations of tobramycin on Pseudomonas aeruginosa grown under aerobic conditions. RNA was isolated form 4 biological repeats of P.aeruginosa grown to mid-log in cationic adjusted mueller hinton broth and 4 biological repeats of P.aeruginosa with the addition of 0.25ug/ml tobramycin in cationic adjusted mueller hinton broth. null
Project description:Pseudomonas aeruginosa is an important human pathogen which uses the type III secretion system 21 (T3SS) as a primary virulence factor to establish infections in humans. The results presented in this 22 report revealed that the ATP-binding protein PA4595 (named ArtR, a Regulator that is an ATP-activated 23 Repressor of T3SS) represses T3SS expression in P. aeruginosa. The expression of T3SS genes, 24 including exoS, exoY, exoT, exsCEBA and exsD-pscB-L, increased significantly when artR was 25 knockout. The effect of ArtR on ExsA is at the transcriptional level, not at the translational level. The 26 regulatory role and cytoplasm localization of ArtR suggest it belongs to the REG sub-family of 27 ATP-binding cassette (ABC) family. Purified GST-tagged ArtR showed ATPase activity in vitro. The 28 conserved aspartate residues in the dual Walker B motifs prove to be essential for the regulatory 29 function of ArtR. The regulation of T3SS by ArtR is unique, which does not involve the known 30 GacS/A-RsmY/Z-RsmA-ExsA pathway or Vfr. This is the first REG subfamily of ATP-binding 31 cassette that is reported to regulate T3SS genes in bacteria. The results specify a novel player in the 32 regulatory networks of T3SS in P. aeruginosa.