Major Proteomic Changes Associated with Amyloid Induced Biofilm Formation in Pseudomonas aeruginosa PAO1
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Pseudomonas Aeruginosa Pao1 (ncbitaxon:208964)
SUBMITTER: 
Per Halkj��r Nielsen
PROVIDER: MSV000082917 | MassIVE |
SECONDARY ACCESSION(S): PXD001373
REPOSITORIES: MassIVE
Publications
Major proteomic changes associated with amyloid-induced biofilm formation in Pseudomonas aeruginosa PAO1.
Herbst Florian-Alexander FA Søndergaard Mads T MT Kjeldal Henrik H Stensballe Allan A Nielsen Per H PH Dueholm Morten S MS
Journal of proteome research 20141031 1
The newly identified functional amyloids in Pseudomonas (Fap) are associated with increased aggregation and biofilm formation in the opportunistic pathogen P. aeruginosa; however, whether this phenomenon can be simply ascribed to the mechanical properties of the amyloid fibrils remains undetermined. To gain a deeper understanding of the Fap-mediated biofilm formation, the physiological consequences of Fap expression were investigated using label-free protein quantification. The functional amyloi ...[more]
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Project description:The newly identified functional amyloids in <i>Pseudomonas</i> (Fap) are associated with increased aggregation and biofilm formation in the opportunistic pathogen <i>P. aeruginosa</i>. However, whether this phenomenon can be simply ascribed to the mechanical properties of the amyloid fibrils remains undetermined. To gain a deeper understanding of the Fap mediated biofilm formation, the physiological consequences of Fap expression were investigated using label-free protein quantification. The functional amyloids were found to not solely act as inert structural biofilm components. Their presence induced major changes in the global proteome of the bacterium. These included the lowered abundance of classical virulence factors such as elastase B and the secretion system of alkaline protease A. Amyloid mediated biofilm formation furthermore increased abundance of the alginate and pyoverdine synthesis machinery, turning <i>P. aeruginosa</i> PAO1 into an unexpected mucoid phenotype. The results imply a significant impact of functional amyloids in <i>P. aeruginosa</i> for biofilm formation and chronic infections.
2014-10-17 | PXD001373 | Pride
Project description:Biofilms are surface-adhered bacterial communities encased in an extracellular matrix composed of polysaccharides, proteins, and extracelluar (e)DNA, with eDNA being required for the formation and integrity of biofilms. Here we demonstrate that the spatial and temporal release of eDNA is regulated by BfmR, a regulator essential for Pseudomonas aeruginosa biofilm development. The expression of bfmR coincided with localized cell death and DNA release, with high eDNA concentrations localized to the outer part of microcolonies in the form of a ring and as a cap on small clusters. Additionally, eDNA release and cell lysis increased significantly following bfmR inactivation. Genome-wide transcriptional profiling indicated that bfmR was required for repression of genes associated with bacteriophage assembly and bacteriophage-mediated lysis. In order to determine which of these genes were directly regulated by BfmR, we utilized chromatin immunoprecipitation (ChIP) analysis to identify the promoter of PA0691, termed here phdA, encoding a previously undescribed homologue of the prevent-host-death (Phd) family of proteins. Lack of phdA expression coincided with impaired biofilm development, increased cell death and bacteriophage release, a phenotype comparable to ΔbfmR. Expression of phdA in ΔbfmR biofilms restored eDNA release, cell lysis, release of bacteriophages, and biofilm formation to wild type levels. Moreover, overexpression of phdA rendered P. aeruginosa resistant to lysis mediated by superinfective bacteriophage Pf4 which was only detected in biofilms. The expression of bfmR was stimulated by conditions resulting in membrane perturbation and cell lysis. Thus, we propose that BfmR regulates biofilm development by controlling bacteriophage-mediated lysis and thus, cell death and eDNA release, via PhdA. For biofilm growth experiments, three independent replicates of P. aeruginosa strains PAO1 and ΔbfmR were grown as biofilms in a flow-through system using a once-through continuous flow tube reactor system for biofilm sample collection and in flow cells (BioSurface Technologies) for the analysis of biofilm architecture as previously described (Sauer et al., 2002, Sauer et al., 2004, Petrova & Sauer, 2009). Cells were treated with RNAprotect (Qiagen), and total RNA was extracted using an RNeasy mini purification kit (Qiagen) per the manufacturer’s instructions. RNA quality and the presence of residual DNA were checked on an Agilent Bioanalyzer 2100 electrophoretic system pre- and post-DNase treatment. Ten micrograms of total RNA was used for cDNA synthesis, fragmentation, and labeling according to the Affymetrix GeneChip P. aeruginosa genome array expression analysis protocol. Sauer, K., A. K. Camper, G. D. Ehrlich, J. W. Costerton & D. G. Davies, (2002) Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J. Bacteriol. 184: 1140-1154. Sauer, K., M. C. Cullen, A. H. Rickard, L. A. H. Zeef, D. G. Davies & P. Gilbert, (2004) Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm. J. Bacteriol. 186: 7312-7326. Petrova, O. E. & K. Sauer, (2009) A novel signaling network essential for regulating Pseudomonas aeruginosa biofilm development. PLoS Pathogens 5: e1000668.
2011-06-01 | E-GEOD-28953 | biostudies-arrayexpress
Project description:An antivirulence approach targets bacterial virulence rather than cell viability in the antibiotic approach that can readily lead to drug resistance. Opportunistic human pathogen Pseudomonas aeruginosa produces a variety of virulence factors, and biofilm cells of this bacterium are much more resistant to antibiotics than planktonic cells. To identify novel inorganic antivirulence compounds, the dual screenings of thirty-six metal ions were performed to identify that zinc ions and ZnO nanoparticle inhibited the pyocyanin production and biofilm formation in P. aeruginosa without affecting the growth of planktonic cells. Moreover, zinc ion and ZnO nanoparticle markedly reduced the production of 2-heptyl-3-hydroxy-4(1H)-quinolone and siderophore pyochelin, while increased the production of another sideropore pyoverdine and swarming motility. Further, zinc ion and ZnO nanoparticle clearly suppressed hemolytic activity in P. aeruginosa. Transcriptome analyses showed that ZnO nanoparticle induced zinc cation efflux pump czc operon, porin genes (oprD and opdT), and Pseudomonas type III repressor A ptrA, while repressed pyocyanin-related phz operon, which partially explains the phenotypic changes. Overall, ZnO nanoparticle is a potential candidate for use in an antivirulence approach against persistent P. aeruginosa infection.
2015-05-25 | GSE46947 | GEO
Project description:A hallmark of the biofilm architecture is the presence of microcolonies. However, little is known about the underlying mechanisms governing microcolony formation. In the human pathogen Pseudomonas aeruginosa, microcolony formation is dependent on the two-component regulator MifR, with mifR mutant biofilms exhibiting an overall thin structure lacking microcolonies, and overexpression of mifR resulting in hyper-microcolony formation. Here, we made use of the distinct MifR-dependent phenotypes to elucidate mechanisms associated with microcolony formation. Using global transcriptomic and proteomic approaches, we demonstrate that cells located within microcolonies experience stressful, oxygen limited, and energy starving conditions, as indicated by the activation of stress response mechanisms and anaerobic and fermentative processes, in particular pyruvate fermentation. Inactivation of genes involved in pyruvate utilization including uspK, acnA and ldhA abrogated microcolony formation in a manner similar to mifR inactivation. Moreover, depletion of pyruvate from the growth medium impaired biofilm and microcolony formation, while addition of pyruvate significantly increased microcolony formation. Addition of pyruvate partly restored microcolony formation in M-bM-^HM-^FmifR biofilms. Moreover, addition of pyruvate to or expression of mifR in lactate dehydrogenase (ldhA) mutant biofilms did not restore microcolony formation. Consistent with the finding of denitrification genes not demonstrating distinct expression patterns in biofilms forming or lacking microcolonies, addition of nitrate did not alter microcolony formation. Our findings indicate the fermentative utilization of pyruvate to be a microcolony-specific adaptation to the oxygen limitation and energy starvation of the P. aeruginosa biofilm environment. For biofilm growth experiments, three independent replicates of P. aeruginosa strains PAO1 and M-NM-^TmifR were grown as biofilms in a flow-through system using a once-through continuous flow tube reactor system for biofilm sample collection and in flow cells (BioSurface Technologies) for the analysis of biofilm architecture as previously described (Sauer et al., 2002, Sauer et al., 2004, Petrova & Sauer, 2009). Cells were treated with RNAprotect (Qiagen) and total RNA was extracted using an RNeasy mini purification kit (Qiagen) per the manufacturerM-bM-^@M-^Ys instructions. RNA quality and the presence of residual DNA were checked on an Agilent Bioanalyzer 2100 electrophoretic system pre- and post-DNase treatment. Ten micrograms of total RNA was used for cDNA synthesis, fragmentation, and labeling according to the Affymetrix GeneChip P. aeruginosa genome array expression analysis protocol. Sauer, K., A. K. Camper, G. D. Ehrlich, J. W. Costerton & D. G. Davies, (2002) Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J. Bacteriol. 184: 1140-1154. Sauer, K., M. C. Cullen, A. H. Rickard, L. A. H. Zeef, D. G. Davies & P. Gilbert, (2004) Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm. J. Bacteriol. 186: 7312-7326. Petrova, O. E. & K. Sauer, (2009) A novel signaling network essential for regulating Pseudomonas aeruginosa biofilm development. PLoS Pathogens 5: e1000668.
2012-04-30 | E-GEOD-35286 | biostudies-arrayexpress
Project description:. The transition of the opportunistic pathogen Pseudomonas aeruginosa from free-living bacteria into surface-associated biofilm communities represents a viable target for the prevention and treatment of chronic infectious disease. We have established a proteomics platform that identified 2443 and 1142 high-confidence proteins in P. aeruginosa whole cells and outer membrane vesicles (OMVs), respectively, at three time points during biofilm development. Analysis of cellular systems, specifically the phenazine biosynthetic pathway, demonstrates that whole cell protein abundance correlates to end product (i.e., pyocyanin) concentrations in biofilm but not planktonic cultures. Furthermore, increased cellular protein abundance in this pathway results in quantifiable pyocyanin in early biofilm OMVs, and OMVs from both growth modes isolated at later time points. Overall, our data indicate that the OMVs being released from the surface of the biofilm whole cells have unique proteomes in comparison to their planktonic counterparts. The relative abundance of OMV proteins from various subcellular sources showed considerable differences between the two growth modes over time, supporting the existence and preferential activation of multiple OMV biogenesis mechanisms under different conditions. The consistent detection of cytoplasmic proteins in the OMV subproteome suggests that these proteins may contribute a small but functionally relevant component to biofilm OMVs. Direct comparisons of outer membrane protein abundance levels between OMVs and whole cells shows ratios that vary greatly from 1:1, and supports previous studies that advocate specific inclusion, or “packaging”, of proteins into OMVs. The detailed analysis of packaged protein groups indicates biogenesis mechanisms that involve untethered, rather than absent, peptidoglycan-binding proteins. Collectively, individual protein and biological system analyses of biofilm OMVs show that drug-binding cytoplasmic proteins and porins are shuttled from the whole cell into the OMVs, potentially contributing to the antibiotic resistance of P. aeruginosa whole cells within biofilms.
2015-09-21 | PXD002605 | Pride
Project description:Intercellular signal indole and its derivative hydroxyindoles inhibit Escherichia coli biofilm and diminish Pseudomonas aeruginosa virulence. However, indole and bacterial indole derivatives were unstable in microbial community due to the widespread of diverse oxygenases that could quickly degrade them. Hence, we sought to identify novel non-toxic, stable, and potent indole derivatives from plant sources for inhibiting biofilm formation of E. coli O157:H7 and P. aeruginosa PAO1. Here, plant auxin 3-indolylacetonitrile (IAN) was found to inhibit biofilm formation of both E. coli O157:H7 and P. aeruginosa without affecting its growth. IAN inhibited biofilms more effectively than indole for both E. coli and P. aeruginosa. Additionally, IAN decreased the production of virulence factor pyocyanin in P. aeruginosa. DNA microarray analysis indicated that IAN repressed genes involved in curli formation and glycerol metabolism, while IAN induced indole-related genes and prophage genes in E. coli. It appears that IAN inhibits biofilm formation of E. coli by reducing curli formation and inducing indole production. Furthermore, unlike bacterial indole derivatives, plant-originated IAN was stable in the presence of either E. coli or P. aeruginosa.
2011-02-20 | GSE21508 | GEO
Project description:Intercellular signal indole and its derivative hydroxyindoles inhibit Escherichia coli biofilm and diminish Pseudomonas aeruginosa virulence. However, indole and bacterial indole derivatives were unstable in microbial community due to the widespread of diverse oxygenases that could quickly degrade them. Hence, we sought to identify novel non-toxic, stable, and potent indole derivatives from plant sources for inhibiting biofilm formation of E. coli O157:H7 and P. aeruginosa PAO1. Here, plant auxin 3-indolylacetonitrile (IAN) was found to inhibit biofilm formation of both E. coli O157:H7 and P. aeruginosa without affecting its growth. IAN inhibited biofilms more effectively than indole for both E. coli and P. aeruginosa. Additionally, IAN decreased the production of virulence factor pyocyanin in P. aeruginosa. DNA microarray analysis indicated that IAN repressed genes involved in curli formation and glycerol metabolism, while IAN induced indole-related genes and prophage genes in E. coli. It appears that IAN inhibits biofilm formation of E. coli by reducing curli formation and inducing indole production. Furthermore, unlike bacterial indole derivatives, plant-originated IAN was stable in the presence of either E. coli or P. aeruginosa.
2010-12-31 | GSE19842 | GEO
Project description:The bifidogenic effect of the prebiotic inulin and its hydrolysed form (fructoligosaccharide, FOS) is well established since they promote the growth of specific beneficial (probiotic) gut bacteria such as bifidobacteria. Previous studies of the opportunistic nosocomial pathogen Pseudomonas aeruginosa PAO1 have shown that inulin and FOS reduce growth, biofilm formation, through decrease of its ability to motility and exotoxin secretion. However, the transcriptional basis for these phenotypic alterations remains unclear. To address this question we conducted RNA-sequence analysis. In most cases changes in the transcript level induced by inulin and FOS were similar. However, a set of transcripts were increased in expression response to inulin, but reduced in the presence of FOS. In the presence of inulin or FOS 260 and 217 transcript levels, respectively, were altered as compared to the control to which no polysaccharide was added. Importantly, changes in transcript levels of 57 and 83 genes were found to be specific for either inulin or FOS, respectively, indicating that both compounds trigger different changes. Gene pathway analysis of different exposure genes (DEG) revealed a specific FOS-mediated reduction in transcript levels of genes that participate in several canonical pathways involved in metabolism and growth, motility, biofilm formation, β-lactam resistance and in the modulation of type III and VI secretion system which were supported by real time quantitative PCR measurements. These results may provide solid information to suggest that FOS selectively modulates the P. aeruginosa PAO1 pathogenecity through distinct signaling pathways.
2020-01-17 | GSE124468 | GEO
Project description:Analysis of Pseudomonas aeruginosa PA01 (ATCC 15692) Psl polysaccharide deficient mutant. Psl polysaccharide deficient mutant is evaluated with microarray to understand the genes affected by this polysaccharide. Our results provide new vision on the roles played by Psl polysaccharide in P. aeruginosa. Exopolysaccharide Psl is a critical biofilm matrix component in Pseudomonas aeruginosa, which forms a fiber-like matrix to enmesh bacteria communities. Iron has been shown to serve as a signal in P. aeruginosa biofilm development, yet how iron controls biofilm formation is not clear. Here we perform a transcriptomic analysis to compare Psl negative strain versus its isogenic wild-type strain PAO1. The results indicate that the expression of genes involved in iron homeostasis and oxidative stress response increased drastically at transcriptional level in Psl negative strain, suggesting Psl deficiency induces iron limitation. Subsequent studies confirm that Psl can efficiently bind iron in vitro and Psl fibers functions as an iron storage channel in P. aeruginosa biofilms. We used two wild type (WT) replicates and two mutant (MT) replicates to compare the differential gene expression.
2016-01-26 | E-GEOD-77163 | biostudies-arrayexpress
Project description:Pseudomonas aeruginosa is a versatile opportunistic pathogen requiring iron for its survival and virulence within the host. The ability to switch to heme as an iron source provides an advantage in chronic infection. We have recently shown the extracellular heme metabolites biliverdin IX (BVIX) and/or BVIX positively regulate the heme dependent cell surface signaling cascade. We further investigated the role of BVIX and BVIX in cell signaling utilizing allelic strains lacking a functional HemO (hemOin), or one reengineered to produce BVIX (hemO). Compared to PAO1 both strains show a heme dependent growth defect, decreased swarming and twitching and less robust biofilm formation. Interestingly, the motility and biofilm defects were partially rescued on addition of exogenous BVIX and BVIX. Utilizing LC-MS/MS we performed a comparative proteomics and metabolomics analysis of PAO1 versus the allelic strains in shaking and static conditions. In shaking conditions, the hemO allelic strains showed a significant increase in proteins involved in quorum sensing (QS), phenazine production and chemotaxis.Metabolite profiling further revealed increased levels of PQS and phenazine metabolites. In static conditions we observed a significant repression of chemosensory pathways and Type IV pili (TFP) biogenesis proteins as well as several phosphodiesterases associated with biofilm dispersal. We propose BVIX metabolites function as signaling and chemotactic molecules integrating heme utilization as an iron source into the adaptation of P. aeruginosa from a planktonic to sessile lifestyle.
2024-01-19 | PXD045835 | Pride