Project description:Untargeted metabolomics analysis of in vitro headspace volatiles from 81 Pseudomonas aeruginosa bacterial isolates from individuals with cystic fibrosis. Headspace volatiles were collected using solid-phase microextraction (SPME) (in triplicate) and comprehensive two-dimensional gas chromatography and time-of-flight mass spectrometry (GCxGC-TOFMS). 15 replicates of un-inoculated media were prepared and analyzed in parallel, for a total of 258 samples.
Project description:In the Pseudomonas aeruginosa type strain PA14, 40 genes are known to encode for diguanylate cyclases (DGCs) and/or phosphodiesterases (PDEs), which modulate the intracellular pool of the nucleotide second messenger c-di-GMP. While in general, high levels of c-di-GMP drive the switch from highly motile phenotypes towards a sessile lifestyle, the different c-di-GMP modulating enzymes are responsible for smaller and in parts non-overlapping phenotypes. In this study, we sought to utilize previously recorded P. aeruginosa gene expression datasets on 414 clinical isolates to uncover transcriptional changes as a result of a high expression of genes encoding diguanylate cyclases. We demonstrate that the selection of sub-groups of clinical isolates with high versus low expression of regulatory genes served the identification of their downstream regulons. Thus, analyzing transcriptional profiles of clinical isolates with variant expression of diguanylate cyclase genes might provide a unique opportunity to bypass the problem that for many c-di-GMP modulating enzymes it is not known under which conditions their expression is activated. However, because the high c-di-GMP associated phenotypes were rapidly lost in the clinical isolates, we were unable to confirm diguanylate specific gene regulation. It is possible that the elevated c-di-GMP concentrations observed in the clinical P. aeruginosa isolates were due to a memory response, in which the bacteria maintain a certain level of c-di-GMP even after being removed from the conditions that induced the production of c-di-GMP. Further studies would be needed to determine the specific mechanisms underlying this memory response in P. aeruginosa.
Project description:Comparative genomics has greatly facilitated the identification of shared as well as unique features among individual cells or tissues, and thus offers the potential to find disease markers. While proteomics is recognized for its potential to generate quantitative maps of protein expression, comparative proteomics in bacteria has been largely restricted to the comparison of single cell lines or mutant strains. In this study, we used a data independent acquisition (DIA) technique, which enables global protein quantification of large sample cohorts, to record the proteome profiles of overall 27 whole genome sequenced and transcriptionally profiled clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa. Analysis of the proteome profiles across the 27 clinical isolates grown under planktonic and biofilm growth conditions led to the identification of a core biofilm-associated protein profile. Furthermore, we found that protein-to-mRNA ratios between different P. aeruginosa strains are well correlated, indicating conserved patterns of post-transcriptional regulation. Uncovering core regulatory pathways, which drive biofilm formation and associated antibiotic tolerance in bacterial pathogens, promise to give clues to interactions between bacterial species and their environment and could provide useful targets for new clinical interventions to combat biofilm-associated infections.
Project description:Purpose : The goal of this study was to use RNA Seq to explore the success of lasR mutants in the opportunistic pathogen Pseudomonas aeruginosa in a clinical context by profiling the functional consequences of patho-adaptive mutations in clinical isolates. Methods : mRNA profiles were generated for Pseudomonas aerugionsa samples by deep sequencing.Ribosomal RNA was removed by using the RiboZero Bacteria kit (Illumina). cDNA libraries were synthesized using the SMARTScribe Reverse Transcriptase (Takara) followed by a PCR enrichment using the AccuPrime HiFi Taq polymerase (Invitrogen). Enzymatic reactions were carried out in the presence of SUPERase·In™ RNase Inhibitor (Invitrogen); RNACleanXP beads (Agencourt) were used for all RNA purification steps. Quality checks were performed before, during and after cDNA library preparation with the RNA Nano Kit and an Agilent Bioanalyzer 2100 (Agilent Technologies). Libraries were sequenced on an Illumina NovaSeq 6000 (paired-end mode; 2 x 50 bp) and mRNA reads were trimmed using the tool ‘cutadapt’ (version 3.5) with customized settings and mapped to the NC_008463.1 (PA14) reference genome from NCBI using ‘bowtie2’ (version 2.3.5.1) with the settings “--very-sensitive-local; --no-mixed; --fr; --no-unal”.
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:Pseudomonas aeruginosa is known as opportunistic pathogen frequently isolated from different infection sites such as burned wounds, lung and urinary tract. To shed light on the expression rates of cytoplasmic P. aeruginosa proteins, commonly expressed by eleven different clinical isolates, absolute protein quantities were determined using P. aeruginosa PAO1 as a reference strain and employing a highly precise gel-free and data-independent LC-IMSE approach. Moreover, the metabolic diversity of these isolates has been investigated by 13C-metabolic flux analyses. 903 proteins were reproducibly identified and absolutely quantified for P. aeruginosa PAO1, 363 of which were also identified and relatively quantified in all of the tested clinical isolates. The vast majority of these proteins is expressed in constant amounts in all strains and exhibits a relatively low relative standard deviation. In contrast, the expression rates of 42 proteins were highly variable between the isolates. Notably, the outer membrane protein OprH and the response regulator PhoP were strongly expressed in isolates from burned wounds when compared to isolates from lung or urinary tract. Moreover, proteins involved in the uptake of iron and amino acids (i.e. HitA, BfrB, PA5217, BraC, PA5153) were found to be more abundant in urinary tract isolates compared to lung isolates. The fluxome data revealed a conserved glycolysis, and a niche-specific divergence in fluxes through the glyoxylate shunt and the TCA cycle among the isolates. The integrated analysis of proteome and fluxome did not indicate straightforward correlation between the amount of proteins and their flux, but rather points to additional layers of regulation that mediate metabolic adaption of P. aeruginosa to different host environments.
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-1), 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:Gene expression profiles of two Pseudomonas aeruginosa taxonomic outlier clinical isolates, CLJ1 and CLJ3 [CLJ3] Pseudomonas aeruginosa taxonomic outliers emerged recently as infectious for humans, provoking hemorrhagic pneumonia. Those bacteria lack classical type III secretion system, and utilize the pore-forming toxin for infection. Two clones CLJ1 and CLJ3 belonging to these taxonomic outliers have been isolated from the same patient at two different times during hospitalization. P. aeruginosa CLJ3 displays antibiotic resistance phenotype, while CLJ1 is more cytotoxic on epithelial and endothelial cells.
Project description:Gene expression profiles of two Pseudomonas aeruginosa taxonomic outlier clinical isolates, CLJ1 and CLJ3 [CLJ1] Pseudomonas aeruginosa taxonomic outliers emerged recently as infectious for humans, provoking hemorrhagic pneumonia. Those bacteria lack classical type III secretion system, and utilize the pore-forming toxin for infection. Two clones CLJ1 and CLJ3 belonging to these taxonomic outliers have been isolated from the same patient at two different times during hospitalization. P. aeruginosa CLJ3 displays antibiotic resistance phenotype, while CLJ1 is more cytotoxic on epithelial and endothelial cells.