Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Expression data from Pseudomonas aeruginosa (wild type and lasRrhlR mutant strains) exposed to human neutrophils

ABSTRACT: In the present in vitro study, interactions between P. aeruginosa (sessile biofilms as well as planktonic cells) and PMNs were analyzed by means of DNA microarray based transcriptomics. We found that the P. aeruginosa wild type biofilms, in contrast to planktonic cultures and quorum sensing (QS) deficient strains, respond to PMN exposure in a rather aggressive manner. The response does not involve protective mechanisms such as those involved in oxidative stress. Rather it is dominated by QS controlled virulence determinants such as those encoded by pqs, phz, rhlAB, all of which are designed to cripple Eukaryotic cells including PMNs and macrophages. Our comparative analysis supports the view that QS plays a major role in mechanisms by which P. aeruginosa evades host defense systems. Keywords: Stress response The biofilms were allowed to grow and develop in the biofilm flow chambers for 3 days before challenge with PMNs. Fresh PMNs from human volunteers were isolated and resuspended in RPMI-1640 medium (BIOCHROM AG) as previously described by Bjarnsholt et al. 2005 (Bjarnsholt et al., 2005). Prior to PMN injection the flow was halted. Ten million PMNs were resuspended in 6 ml of RPMI media and injected into the flow chamber. The concentration of PMNs was found by microscopy to be approximately 1 PMN per 1000 bacterial cells. PMNs and biofilm were incubated for 2 hours. The flow chambers were rolled every 15 minutes to ensure that the entire biofilm was exposed to the PMNs. After incubation the fluid inside the chamber was discarded and the attached biofilm cells were loosened by rolling. The cells were then mechanically removed and collected in 6 ml of RNAlater®

ORGANISM(S): Pseudomonas aeruginosa

SUBMITTER: Morten Alhede

PROVIDER: E-GEOD-6769 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Pseudomonas aeruginosa recognizes and responds aggressively to the presence of polymorphonuclear leukocytes.

Alhede Morten M Bjarnsholt Thomas T Jensen Peter Ø PØ Phipps Richard Kerry RK Moser Claus C Christophersen Lars L Christensen Louise Dahl LD van Gennip Maria M Parsek Matt M Høiby Niels N Rasmussen Thomas Bovbjerg TB Givskov Michael M

Microbiology (Reading, England) 20090730 Pt 11

Polymorphonuclear neutrophilic leukocytes (PMNs) play a central role in innate immunity, where they dominate the response to infections, in particular in the cystic fibrosis lung. PMNs are phagocytic cells that produce a wide range of antimicrobial agents aimed at killing invading bacteria. However, the opportunistic pathogen Pseudomonas aeruginosa can evade destruction by PMNs and thus cause persistent infections. In this study, we show that biofilm cells of P. aeruginosa recognize the presence ...[more]

PMID: 19643762

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Project description:Background: Biofilm formation by Salmonella species enhances the capacity of these pathogenic bacteria to survive stresses that are commonly encountered within food processing and during host infection. The persistence of Salmonella within the food chain has become a major health concern, as biofilms can serve as a reservoir to contaminate food products. While the molecular mechanisms required for the survival of bacteria on surfaces are not fully understood, transcriptional studies of other bacteria have demonstrated that biofilm growth triggers the expression of specific sets of genes, compared with planktonic cells. Until now, most transcriptomic studies of Salmonella have focused on the effect of infection-relevant stressors on virulence and on comparing mutant and wild-type (WT). However little is known about the physiological responses taking place inside a Salmonella biofilm. Results: We have determined the transcriptomic and proteomic profiles of biofilms of Salmonella enterica serovar Typhimurium. We discovered that 124 of detectable proteins were differentially expressed in the biofilm compared with planktonic cells, and that 10% (433 genes) of S. Typhimurium genes showed a biofilm-specific pattern of transcription (i.e. a 2-fold or more change in the biofilm compared with planktonic cells). The genes that were significantly up-regulated implicated specific cellular processes in biofilm development including amino acid metabolism, cell motility, global regulation and tolerance to stress. We found that the most highly down-regulated genes in the biofilm were located on Salmonella Pathogenicity Island 2 (SPI2), but that a functional SPI2 secretion system regulator (ssrA) was required for WT biofilm formation. We identified STM0341 as a gene of unknown function that was needed for WT biofilm growth. Genes involved in tryptophan (trp) biosynthesis and transport were up-regulated in the biofilm. Deletion of trpE led to a decrease in bacterial attachment and we show that this biofilm defect is restored by exogenous tryptophan or indole. Conclusion: Biofilm growth of S. Typhimurium causes distinct changes in gene and protein expression. Our results show that aromatic amino acids make an important contribution to biofilm formation, and reveal that SPI2 genes, required for virulence in host cells, show opposing patterns of expression to biofilm-specific genes in S. Typhimurium. Overnight cultures of SL1344 grown in CFA broth were standardized to achieve an initial concentration of 10^6 CFU ml-1 and injected into a stirring influent flask containing 5 L of pre-warmed (25C) sterile CFA medium. The inoculated influent was then pumped through silicon tubing (5 mm internal diameter, Samco Silicon Products Ltd.) at 60 ml h-1 using a peristaltic pump (Minipulse 3, Gilson). The bacteria were allowed to flow through the closed system and either attach to a vertical piece of silicon tubing (1 meter length, 16 mm internal diameter, Samco Silicon Products Ltd.) or flow out into a waste reservoir. The tubing was positioned vertically to collect biofilm cells adherent to the tubing and minimise collecting bacteria that had simply sedimented. Possible backflow of media or bacteria into the influent from the silicon tubing was eliminated using media breaks. Samples taken to determine the pH of the medium or bacterial cell counts were removed via a media port located near the effluent and influent vessel, respectively. Planktonic cells were removed after 72 h of growth from the influent vessel to avoid any contamination with biofilm cells. The entire system was kept at a constant temperature of 25C and biofilm cells were isolated after 72 h of growth. All planktonic and biofilm samples were isolated from the same experimental system and used for both RNA and protein isolation. For this study, four biological replicates were performed at four different dates (Jun25, Jul5, Jul16, Oct11; as indicated in the sample title). For the dual-colour microarray hybridisations, we used Salmonella genomic DNA as the comparator which also acted as the control for spot quality. Individual labelled cDNA samples (RNA) were hybridised up to 4 times (technical replicates).

Project description:The physiological and transcriptional response of Nitrosomonas europaea biofilms to phenol and toluene was examined and compared to suspended cells. Biofilms were grown in Drip Flow Biofilm Reactors under continuous flow conditions of growth medium containing ammonia as growth substrate. The responses of N. europaea biofilms to the aromatic hydrocarbons phenol and toluene were determined during short-term (3 h) additions of each compound to the biofilms. Ammonia oxidation in the biofilms was inhibited 50% by 60 uM phenol and 100 uM toluene. These concentrations were chosen for microarray analysis of phenol- and toluene-exposed N. europaea biofilms. Liquid batch cultures of exponentially growing N. europaea cells were harvested alongside the biofilms to determine differential gene expression between attached and suspended growth of N. europaea. Four sample groups of N. europaea cells were used in this study, with biological triplicates of each group. Groups were: Control (untreated) biofilms, phenol-exposed biofilms, toluene-exposed biofilms, and exponentially growing suspended cells. Biofilms were grown in Drip Flow Biofilm Reactors containing 4 independent growth channels and subject to 2 hour inhibition tests. During each experiment, 2 biofilm channels served as control with no inhibitor present and the other 2 biofilm channels were exposed to either 60 uM phenol or 100 uM toluene. Nitrite production was monitored throughout the experiment, and the given concentrations of phenol and toluene resulted in 50% inhibition of ammonia oxidation by the biofilms. Suspended cells were grown in batch reactors. Three 4-plex NimbleGen microarray chips were used, and each chip contained one sample from each experimental group. QC of samples was determined by spectrophotometric methods and using Agilent bioanalyzer traces to determine purity and integrity of RNA and cDNA. A sample tracking report was used to verify the correct hybridization of each sample to the intended array.

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.

Dataset Information

Expression data from Pseudomonas aeruginosa (wild type and lasRrhlR mutant strains) exposed to human neutrophils

Publications

Pseudomonas aeruginosa recognizes and responds aggressively to the presence of polymorphonuclear leukocytes.

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