Project description:ClpV3 is a cytoplasmic AAA+ ATPase protein and is an essential component of H3-T6SS in Pseudomonas aeruginosa. Here, we report that an H3-T6SS deletion mutant PAO1(ΔclpV3) significantly affected the virulence-related phenotypes including pyocyanin production, biofilm formation, proteolytic activity and motilities. Most interestingly, the expression of T3SS genes was markedly affected, indicating a strong link between H3-T6SS and T3SS. RNA-Sequencing was performed to globally identify the genes differentially expressed when H3-T6SS was inactivated and the results obtained could be well correlated to the observed phenotypes.
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:To further determine the origin of the increased virulence of Pseudomonas aeruginosa PA14 compared to Pseudomonas aeruginosa PAO1, we report a transcriptomic approach through RNA sequencing. Next-generation sequencing (NGS) has revolutioned sistems-based analsis of transcriptomic pathways. The goals of this study are to compare the transcriptomic profile of all 5263 orthologous genes of these nearly two strains of Pseudomonas aeruginosa.
Project description:Pseudomonas aeruginosa is an opportunistic pathogen which causes acute and chronic infections that are difficult to treat. Comparative genomic analysis has showed a great genome diversity among P. aeruginosa clinical strains and revealed important regulatory traits during chronic adaptation. While current investigation of epigenetics of P. aeruginosa is still lacking, understanding the epigenetic regulation may provide biomarkers for diagnosis and reveal important regulatory mechanisms. The present study focused on characterization of DNA methyltransferases (MTases) in a chronically adapted P. aeruginosa clinical strain TBCF10839. Single-molecule real-time sequencing (SMRT-seq) was used to characterize the methylome of TBCF. RCCANNNNNNNTGAR and TRGANNNNNNTGC were identified as target motifs of DNA MTases, M.PaeTBCFI and M.PaeTBCFII, respectively.
Project description:Pseudomonas aeruginosa (P. aeruginosa) lung infection is a significant cause of mortality in patients with cystic fibrosis (CF). Most CF patients acquire unique P. aeruginosa strains from the environment; however clonal strains have been identified in CF communities in several countries. Two clonal strains infect 10% to 40% of patients in three CF clinics in mainland eastern Australia. The expression profiles of four planktonically-grown isolates of one Australian clonal strain (AES-2), and four non–clonal CF P. aeruginosa isolates were compared to each other and to the reference strain PAO1 using the Affymetrix P. aeruginosa PAO1 genome array, to gain insight into properties mediating the enhanced infectivity of AES-1. The isolates were subsequently grown as 3-day old biofilms and similarly extracted for RNA and compared as above. Data analysis was carried out using BIOCONDUCTOR software. Keywords: Comparative strain hybridization
Project description:The Pseudomonas aeruginosa quorum-sensing (QS) systems contribute to bacterial homeostasis and pathogenicity. Although the AraC family transcription factor VqsM has been characterized to control the production of virulence factors and QS signaling molecules, its detailed regulatory mechanisms still remain elusive. Here, we report that VqsM directly binds to the lasI promoter region, and thus regulates its expression. To identify additional targets of VqsM in P. aeruginosa PAO1, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) which detected 48 enriched loci harboring VqsM-binding peaks in P. aeruginosa genome. The direct regulation of these genes by VqsM has been confirmed by Electrophoretic mobility shift assays (EMSAs) and quantitative real-time polymerase chain reactions (qRT-PCR). A VqsM-binding motif is found by using MEME suite and verified by footprint assays in vitro. In addition, VqsM directly binds to the promoter regions of antibiotic resistance regulator NfxB and the master type III system regulator ExsA. Notably, the vqsM mutant displayed more resistance to two types of antibiotics and promoted bacterial survival in a mouse model, compared to the wild type PAO1 strain. Collectively, this work provides new cues to better understand the detailed regulatory networks of QS systems, T3SS, and antibiotic resistance. Pseudomonas aeruginosa MAPO1 containing empty pAK1900 or pAK1900-VqsM-VSV
Project description:We identified PhaF as an RNA binding protein in Pseudomonas aeruginosa. In order to identify the different target transcripts of PhaF, we carried out the CLIP (Crosslinking and immunoprecipitation) and CLAP-Seq (Covalent linkage affinity purification) approaches, which utilizes UV irradiation to crosslink PhaF with the transcripts that are in close vicinity to it. For CLIP-Seq experiments, we used the Pseudomonas aeruginosa PAO1 strain along with a derivative of PAO1 that harbors a C-terminal VSVG tag on PhaF. In order to determine the importance of the C-terminal domain (CTD) of PhaF on RNA binding, we used PAO1ΔphaF strains carrying plasmids that express a C-terminal VSVG tagged version of PhaF-CTD (pPhaF-CTD-V), along with a control strain that carries the same plasmid (pPhaF-CTD) but without the VSVG tag. For CLAP-Seq experiments, we used the Pseudomonas aeruginosa PAO1 strain along with a derivative of PAO1 that harbors a C-terminal Halo tag on PhaF. All the strains were subjected to UV irradiation, lysed, immunoprecipitated using either anti-VSVG-antibody coated beads or Magne Halo tagged beads. Following immunoprecipitation, the RNAs were purified. We also purified Total (Tot) RNA (samples collected before immunoprecipitation) from the PAO1 strains harboring the C-terminal VSVG tag on PhaF. The purified RNA samples were converted to cDNA libraries, which were subsequently sequenced using the Illumina NextSeq. The sequences were mapped to the genome to identify the target transcripts. In a separate series of experiments we also carried out RNA-Seq to identify the genes that are differentially regulated using Pseudomonas aeruginosa PAO1 and PAO1ΔphaF strains. The isolated RNA was sent to SeqCenter (Pittsburgh, PA) and the sequences obtained were mapped to the genome and DESeq analysis was performed.