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:The surge of antimicrobial resistance in recent decades threatens efficacy of current antibiotics, particularly against Pseudomonas aeruginosa, a highly resistant gram-negative pathogen. The asymmetric outer membrane of P. aeruginosa combined with its array of efflux pumps provide a barrier to xenobiotic intracellular accumulation, thus making the discovery of novel drugs with whole cell antibacterial activity very challenging. We adapted PROSPECT, a genome-wide, target-based, whole cell screening strategy, to take a focused approach to discover small molecule probes with specific activity against engineered P. aeruginosa mutants depleted for essential proteins localized at the outer membrane. We identified BRD1401, a small molecule that has specific activity against a P. aeruginosa mutant depleted for the essential lipoprotein, OprL. Genetic studies identified a novel link between OprL and the non-essential, outer membrane β barrel protein, OprH, to modulate BRD1401 activity. BRD1401 directly bound to OprH to disrupt the known interaction between OprH and lipopolysaccharide (LPS) in vitro and in whole bacteria. OprH also biochemically interacted with OprL, thus providing a link between outer membrane and peptidoglycan in P. aeruginosa. Thus, a whole cell, multiplexed screen against P. aeruginosa identified a species-specific inhibitor and probe molecule that revealed novel pathogen biology.
Project description:The surge of antimicrobial resistance in recent decades threatens efficacy of current antibiotics, particularly against Pseudomonas aeruginosa, a highly resistant gram-negative pathogen. The asymmetric outer membrane of P. aeruginosa combined with its array of efflux pumps provide a barrier to xenobiotic intracellular accumulation, thus making the discovery of novel drugs with whole cell antibacterial activity very challenging. We adapted PROSPECT, a genome-wide, target-based, whole cell screening strategy, to take a focused approach to discover small molecule probes with specific activity against engineered P. aeruginosa mutants depleted for essential proteins localized at the outer membrane. We identified BRD1401, a small molecule that has specific activity against a P. aeruginosa mutant depleted for the essential lipoprotein, OprL. Genetic studies identified a novel link between OprL and the non-essential, outer membrane β barrel protein, OprH, to modulate BRD1401 activity. BRD1401 directly bound to OprH to disrupt the known interaction between OprH and lipopolysaccharide (LPS) in vitro and in whole bacteria. OprH also biochemically interacted with OprL, thus providing a link between outer membrane and peptidoglycan in P. aeruginosa. Thus, a whole cell, multiplexed screen against P. aeruginosa identified a species-specific inhibitor and probe molecule that revealed novel pathogen biology.
Project description:Analysis of a SigX knockout mutant of Pseudomonas aeruginosa H103 strain in minimal medium with glucose as carbon source (M9G). SigX, one of the 19 extra-cytoplasmic function sigma factors of P. aeruginosa, was only known to be involved in transcription of the gene encoding the major outer membrane protein OprF in Pseudomonas aeruginosa. Deletion of the ECF sigma factor sigX gene provide insights into the SigX role in several virulence and biofilm- related phenotypes in Pseudomonas aeruginosa.
Project description:To understand molecular pathways responding to loss of essential proteins in the inner- or outer-membrane, we knocked down essential genes in the inner- and outer membrane using a CRISPRi approach in Pseudomonas aeruginosa. CRISPRi is an inducible system with which even essential genes can be targeted by a specifig sg-RNA. In this study, we investigated the transcriptomic changes upon loss of BamA, LptD and FtsH. Loss of BamA and LptD, both outer membrane proteins, resulted in strong upregulation of genes involved in LipidA modification and upregulation of several two component systems. Depletion of FtsH resulted in strong upregulation in genes responsible for amino acid biosynthesis and regulatory genes. Surprisngly, knockdown of bamA and lptD resulted in (mild) upregulation of H1-T6SS genes.
Project description:Pseudomonas aeruginosa, an opportunistic human bacterial pat hogen, constitutively secretes outer membrane vesicles (OMVs ) into the extracellular milieu. In this study, we identifi ed vesicular proteins with high confidence by five separate LC-MS/MS analyses.
Project description:The previously uncharacterized proteins HigB (unannotated) and HigA (PA4674) of Pseudomonas aeruginosa PA14 were found to form a type II TA system in which antitoxin HigA masks the RNase activity of toxin HigB through direct binding. To determine the physiological role of HigB/HigA in P. aeruginosa, a whole-transcriptome experiment was performed for the higA antitoxin deletion mutant of the PA14 strain compared to the wild-type PA14 strain. The rationale was that for the strain that lacks the antitoxin, the effect of the toxin could be discerned due to enhanced activity of the toxin. Furthermore, toxin HigB reduces production of the virulence factors pyochelin, pyocyanin, swarming, and biofilm formation.
Project description:The Pseudomonas aeruginosa MvfR-dependent QS regulatory pathway controls the expression of key virulence genes; and is activated via the extracellular signals 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS). Our findings reveal a multi-layered homeostatic regulation of PQS and HHQ and the MvfR regulon component PqsE which is a key mediator in orchestrating this homeostatic regulation.
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.
