An atlas of the binding specificities of transcription factors in Pseudomonas aeruginosa directs prediction of novel regulators in virulence.
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ABSTRACT:
SUBMITTER: Wang T
PROVIDER: S-EPMC8041468 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We performed a high-throughput systematic evolution of ligands by exponential enrichment (HT-SELEX) approach on all 371 annotated TFs in P. aeruginosa. This study provides a valuable resource for TF binding specificities in P. aeruginosa and demonstrates a novel and an integrative analysis for seeking the virulence-associated TFs and its target genes.
2021-03-26 | GSE151518 | GEO
Project description:An Atlas of the Binding Specificities of Transcription Factors in Pseudomonas aeruginosa Directs Prediction of Novel Regulators in Virulence
| PRJNA636188 | ENA
Project description:Gene regulation network in Pseudomonas aeruginosa is complex. With a relatively large genome (6.2 Mb), there is a significant portion of genes that are proven or predicted to be transcriptional regulators. Many of these regulators have been shown to play important roles in biofilm formation and maintenance. In this study, we present a novel transcriptional regulator, PA1226, which modulates biofilm formation and virulence in P. aeruginosa. Mutation in the gene encoding this regulator abolished the ability of P. aeruginosa to produce biofilms in vitro, without any effect on the planktonic growth. This regulator is also essential for the in vivo fitness and pathogenesis in both Drosophila melanogaster and BALB/c mouse lung infection models. Transcriptome analysis revealed that PA1226 regulates many essential virulence genes/pathways, including those involved in alginate, pili, and LPS biosynthesis. Genes/operons directly regulated by PA1226 and potential binding sequences were identified via ChIP-seq. Attempts to confirm the binding sequences by electrophoretic mobility shift assay led to the discovery of a co-regulator, PA1413, via co-immunoprecipitation assay. PA1226 and PA1413 were shown to bind collaboratively to the promoter regions of their regulons. A model is proposed, summarizing our finding on this novel dual-regulation system.
| S-EPMC6158065 | biostudies-literature
Project description:The opportunistic pathogen Pseudomonas aeruginosa explores surfaces using twitching motility powered by retractile extracellular filaments called type IV pili (T4P). Single cells twitch by sequential T4P extension, attachment, and retraction. How single cells coordinate T4P to efficiently navigate surfaces remains unclear. We demonstrate that P. aeruginosa actively directs twitching in the direction of mechanical input from T4P in a process called mechanotaxis. The Chp chemotaxis-like system controls the balance of forward and reverse twitching migration of single cells in response to the mechanical signal. Collisions between twitching cells stimulate reversals, but Chp mutants either always or never reverse. As a result, while wild-type cells colonize surfaces uniformly, collision-blind Chp mutants jam, demonstrating a function for mechanosensing in regulating group behavior. On surfaces, Chp senses T4P attachment at one pole, thereby sensing a spatially resolved signal. As a result, the Chp response regulators PilG and PilH control the polarization of the extension motor PilB. PilG stimulates polarization favoring forward migration, while PilH inhibits polarization, inducing reversal. Subcellular segregation of PilG and PilH efficiently orchestrates their antagonistic functions, ultimately enabling rapid reversals upon perturbations. The distinct localization of response regulators establishes a signaling landscape known as local excitation-global inhibition in higher-order organisms, identifying a conserved strategy to transduce spatially resolved signals.
| S-EPMC8325320 | biostudies-literature
Project description:Pseudomonas syringae is a Gram-negative and model pathogenic bacterium that causes plant diseases worldwide. Here, we set out to identify binding motifs for all 301 annotated transcription factors (TFs) of P. syringae using HT-SELEX. We successfully identify binding motifs for 100 TFs. We map functional interactions between the TFs and their targets in virulence-associated pathways, and validate many of these interactions and functions using additional methods such as ChIP-seq, electrophoretic mobility shift assay (EMSA), RT-qPCR, and reporter assays. Our work identifies 25 virulence-associated master regulators, 14 of which had not been characterized as TFs before.
| S-EPMC7532196 | biostudies-literature
Project description:AmrZ, a member of the Ribbon-Helix-Helix family of DNA binding proteins, functions as both a transcriptional activator and repressor of multiple genes encoding Pseudomonas aeruginosa virulence factors. The expression of these virulence factors leads to chronic and sustained infections associated with worsening prognosis. In this study, we present the X-ray crystal structure of AmrZ in complex with DNA containing the repressor site, amrZ1. Binding of AmrZ to this site leads to auto-repression. AmrZ binds this DNA sequence as a dimer-of-dimers, and makes specific base contacts to two half sites, separated by a five base pair linker region. Analysis of the linker region shows a narrowing of the minor groove, causing significant distortions. AmrZ binding assays utilizing sequences containing variations in this linker region reveals that secondary structure of the DNA, conferred by the sequence of this region, is an important determinant in binding affinity. The results from these experiments allow for the creation of a model where both intrinsic structure of the DNA and specific nucleotide recognition are absolutely necessary for binding of the protein. We also examined AmrZ binding to the algD promoter, which results in activation of the alginate exopolysaccharide biosynthetic operon, and found the protein utilizes different interactions with this site. Finally, we tested the in vivo effects of this differential binding by switching the AmrZ binding site at algD, where it acts as an activator, for a repressor binding sequence and show that differences in binding alone do not affect transcriptional regulation.
| S-EPMC3325190 | biostudies-literature
Project description:The chemotaxis network, one of the most prominent prokaryotic sensory systems, is present in most motile bacteria and archaea. Although the conserved signaling core of this network is well characterized, ligand specificities of a large majority of diverse chemoreceptors encoded in bacterial genomes remain unknown. Here, we performed a systematic identification and characterization of new chemoeffectors for the opportunistic pathogen Pseudomonas aeruginosa, which has 26 chemoreceptors possessing most of the common types of ligand binding domains. By performing capillary chemotaxis assays for a library of growth-promoting compounds, we first identified a number of novel chemoattractants of varying strength. We subsequently mapped specificities of these ligands by performing Förster resonance energy transfer and microfluidic measurements for 16 hybrid chemoreceptors that combine the periplasmic ligand binding domains of P. aeruginosa receptors and the cytoplasmic signaling domain of the Escherichia coli Tar receptor. Direct binding of putative ligands to chemoreceptors was further confirmed using thermal shift assay and microcalorimetry. Altogether, the combination of methods enabled us to assign several new attractants, including methyl 4-aminobutyrate, 5-aminovalerate, L-ornithine, 2-phenylethylamine, and tyramine, to previously characterized chemoreceptors and to annotate a novel purine-specific receptor PctP. Responses of hybrid receptors to changes in pH further revealed a complex bidirectional pH sensing mechanism in P. aeruginosa, which involves at least four chemoreceptors PctA, PctC, TlpQ, and PctP. Our screening strategy could be applied for the systematic characterization of unknown sensory domains in a wide range of bacterial species. IMPORTANCE Chemotaxis of motile bacteria has multiple physiological functions. It enables bacteria to locate optimal ecological niches, mediates collective behaviors, and can play an important role in infection. These multiple functions largely depend on ligand specificities of chemoreceptors, and the number and identities of chemoreceptors show high diversity between organisms. Similar diversity is observed for the spectra of chemoeffectors, which include not only chemicals of high metabolic value but also bacterial, plant, and animal signaling molecules. However, the systematic identification of chemoeffectors and their mapping to specific chemoreceptors remains a challenge. Here, we combined several in vivo and in vitro approaches to establish a systematic screening strategy for the identification of receptor ligands and we applied it to identify a number of new physiologically relevant chemoeffectors for the important opportunistic human pathogen P. aeruginosa. This strategy can be equally applicable to map specificities of sensory domains from a wide variety of receptor types and bacteria.
| S-EPMC10653921 | biostudies-literature
Project description:Bacterial antibiotic resistance is a worldwide health problem that deserves important research attention in order to develop new therapeutic strategies. Recently, the World Health Organization (WHO) classified Pseudomonas aeruginosa as one of the priority bacteria for which new antibiotics are urgently needed. In this opportunistic pathogen, antibiotics efflux is one of the most prevalent mechanisms where the drug is efficiently expulsed through the cell-wall. This resistance mechanism is highly correlated to the expression level of efflux pumps of the resistance-nodulation-cell division (RND) family, which is finely tuned by gene regulators. Thus, it is worthwhile considering the efflux pump regulators of P. aeruginosa as promising therapeutical targets alternative. Several families of regulators have been identified, including activators and repressors that control the genetic expression of the pumps in response to an extracellular signal, such as the presence of the antibiotic or other environmental modifications. In this review, based on different crystallographic structures solved from archetypal bacteria, we will first focus on the molecular mechanism of the regulator families involved in the RND efflux pump expression in P. aeruginosa, which are TetR, LysR, MarR, AraC, and the two-components system (TCS). Finally, the regulators of known structure from P. aeruginosa will be presented.
| S-EPMC6018408 | biostudies-literature
Project description:Pseudomonas aeruginosa infects every type of host that has been examined by deploying multiple virulence factors. Previous studies of virulence regulation have largely focused on chemical cues, but P. aeruginosa may also respond to mechanical cues. Using a rapid imaging-based virulence assay, we demonstrate that P. aeruginosa activates virulence in response to attachment to a range of chemically distinct surfaces, suggesting that this bacterial species responds to mechanical properties of its substrates. Surface-activated virulence requires quorum sensing, but activating quorum sensing does not induce virulence without surface attachment. The activation of virulence by surfaces also requires the surface-exposed protein PilY1, which has a domain homologous to a eukaryotic mechanosensor. Specific mutation of the putative PilY1 mechanosensory domain is sufficient to induce virulence in non-surface-attached cells, suggesting that PilY1 mediates surface mechanotransduction. Triggering virulence only when cells are both at high density and attached to a surface—two host-nonspecific cues—explains how P. aeruginosa precisely regulates virulence while maintaining broad host specificity.
| S-EPMC4250119 | biostudies-literature
Project description:Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that undergoes swarming motility in response to semisolid conditions with amino acids as a nitrogen source. With a genome encoding hundreds of potential intergenic small RNAs (sRNAs), P. aeruginosa can easily adapt to different conditions and stresses. We previously identified 20 sRNAs that were differentially expressed (DE) under swarming conditions. Here, these sRNAs were overexpressed in strain PAO1 and were subjected to an array of phenotypic screens. Overexpression of the PrrH sRNA resulted in decreased swimming motility, whereas a ΔprrH mutant had decreased cytotoxicity and increased pyoverdine production. Overexpression of the previously uncharacterized PA2952.1 sRNA resulted in decreased swarming and swimming motilities, increased gentamicin and tobramycin resistance under swarming conditions, and increased trimethoprim susceptibility. Transcriptome sequencing (RNA-Seq) and proteomic analysis were performed on the wild type (WT) overexpressing PA2952.1 compared to the empty vector control under swarming conditions, and these revealed the differential expression (absolute fold change [FC] ≥ 1.5) of 784 genes and the differential abundance (absolute FC ≥ 1.25) of 59 proteins. Among these were found 73 transcriptional regulators, two-component systems, and sigma and anti-sigma factors. Downstream effectors included downregulated pilus and flagellar genes, the upregulated efflux pump MexGHI-OpmD, and the upregulated arn operon. Genes involved in iron and zinc uptake were generally upregulated, and certain pyoverdine genes were upregulated. Overall, the sRNAs PA2952.1 and PrrH appeared to be involved in regulating virulence-related programs in P. aeruginosa, including iron acquisition and motility.IMPORTANCE Due to the rising incidence of multidrug-resistant (MDR) strains and the difficulty of eliminating P. aeruginosa infections, it is important to understand the regulatory mechanisms that allow this bacterium to adapt to and thrive under a variety of conditions. Small RNAs (sRNAs) are one regulatory mechanism that allows bacteria to change the amount of protein synthesized. In this study, we overexpressed 20 different sRNAs in order to investigate how this might affect different bacterial behaviors. We found that one of the sRNAs, PrrH, played a role in swimming motility and virulence phenotypes, indicating a potentially important role in clinical infections. Another sRNA, PA2952.1, affected other clinically relevant phenotypes, including motility and antibiotic resistance. RNA-Seq and proteomics of the strain overexpressing PA2952.1 revealed the differential expression of 784 genes and 59 proteins, with a total of 73 regulatory factors. This substantial dysregulation indicates an important role for the sRNA PA2952.1.
| S-EPMC7848907 | biostudies-literature