Dataset Information

A serial sample processing strategy with improved performance for in-depth quantitative analysis of type III secretion events in Pseudomonas aeruginosa

ABSTRACT: Bacterial pathogens have the ability to secrete effector proteins to promote host cell invasion. The efficient analysis of the secretomes in close to wildtype cell systems is important to study the mechanisms of bacterial secretion efficiently at different levels. In particular the secretome analysis of bacteria that rely on rich media for optimal secretion may hamper analysis via modern sample preparation shotgun proteomics workflows due to higher degree of sample impurities. This is may be a reason for the low number of quantitative secretome investigations in such cells. We assessed the efficiency of different workflows and their amenability for secretome analysis including precipitation, SP3 and a combined, serial workflow. Using the model organism Pseudomonas aeruginosa, we found that, the combined TCA-SP3 strategy outperforms the other tested methods on all levels and proofed most efficient in the recovery of proteins related to the type III secretion system (T3SS), including all known effector proteins and secretion machinery components. Moreover, the serial workflows resulted in improved quantification accuracy using MS1 label-free quantification. Finally by monitoring the compositional changes of secretome samples over time we observed strong increase of the fraction made up by T3SS related proteins with parallel decrease of proteins not linked to T3SS. Our study shows how combining sample preparation procedures can lead to orthogonality in depleting impurities that result in better chromatographic peptide separation, and more efficient MS detection with improved quantification parameters.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Pseudomonas Aeruginosa Pao1 Bacteria

TISSUE(S): Cell Suspension Culture

SUBMITTER: Timo Glatter

LAB HEAD: Timo Glatter

PROVIDER: PXD015153 | Pride | 2019-12-11

REPOSITORIES: Pride

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			Action	DRS
	DL_Sec-3-10re.mzid.gz	Mzid
	DL_Sec-3-10re.mzid_DL_Sec-3-10re.MGF	Mzid
	DL_Sec-3-10re.mzid_DL_Sec-3-10re.pride.mgf.gz	Mzid
	DL_Sec-3-10re.raw	Raw
	DL_Sec-3-11re.mzid.gz	Mzid

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A Serial Sample Processing Strategy with Improved Performance for in-Depth Quantitative Analysis of Type III Secretion Events in Pseudomonas aeruginosa.

Lampaki Dimitrios D Diepold Andreas A Glatter Timo T

Journal of proteome research 20191219 1

The efficient analysis of secretomes is important to study the mechanisms of bacterial secretion. However, secretome analysis of bacteria that rely on rich media for optimal secretion via modern quantitative shotgun proteomics workflows is often hampered by the higher degree of sample impurities. This may be a reason for the low number of quantitative secretome investigations in such cases. We assessed the efficiency and amenability for rich media secretome analysis of different workflows includ ...[more]

PMID: 31814412

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Project description:Prenatal exposure to neurotoxicants such as lead (Pb) may cause stable changes in the DNA methylation (5mC) profile of the fetal genome. However few studies have examined its effect on the DNA de-methylation pathway, specifically the dynamic changes of the 5-hydroxymethylcytosine (5hmC) profile. Therefore, in this study, we investigate the relationship between Pb exposure and 5mC and 5hmC modifications during early development. To study the changes in the 5hmC profile, we use a novel modification of the Infinium™ Human methylation 450K assay (Illumina, Inc.), which we named HMeDIP-450K assay, in an in vitro human embryonic stem cell model of Pb-exposure. We model Pb-exposure associated 5hmC changes as clusters of correlated, adjacent CpG sites, which are co-responding to Pb. We further extend our study to look at Pb-dependent changes in high density 5hmC regions in umbilical cord blood DNA from 48 mother-infant pairs from the Early Life Exposure in Mexico to Environmental Toxicants (ELEMENT) cohort. For our study, we randomly selected UCB from 24 male and 24 female children from the 1st and 4th quartiles of Pb levels. Our data show that Pb-associated changes in the 5hmC and 5mC profiles can be divided into sex-dependent and sex-independent categories. Interestingly, differential 5mC sites are better markers of Pb-associated sex-dependent changes compared to differential 5hmC sites. In this study we identified several 5hmC and 5mC genomic loci, which we believe might have some potential as early biomarkers of prenatal Pb-exposure. Human Methylation 450K array coupled with 5-hydroxymethylcytosine (5hmC) IP or HMeDIP-450K array, was used to determine the high density 5hmC profile of 46 Umbilical cord blood samples from the ELLEMENT cohort. HM450K array, without IP was used to determine the 5-methylcytosine(5mC) plus 5hmC profile. The 5hmC region detected from the HMeDIP-450K array was subtracted from the HM450K(without IP) to get putative high density 5mC regions.

Project description:Background Dickeya dadantii is a necrotrophic pathogen causing disease in many plants. Previous studies have demonstrated that the type III secretion system (T3SS) of D. dadantii is required for full virulence. HrpL is an alternative sigma factor that binds to the hrp box promoter sequence of T3SS genes to up-regulate their expression. Methodology/Principal Findings To explore the inventory of HrpL-regulated genes of D. dadantii 3937 (3937), transcriptome profiles of wild-type 3937 and a hrpL mutant grown in a T3SS-inducing medium were examined. Using a cut-off value of 1.5, significant differential expression was observed in sixty-three genes, which are involved in various cellular functions such as type III secretion, chemotaxis, metabolism, regulation, and stress response. A hidden Markov model (HMM) was used to predict candidate hrp box binding sites in the intergenic regions of 3937, including the promoter regions of HrpL-regulated genes identified in the microarray assay. In contrast to biotrophic phytopathgens such as Pseudomonas syringae, among the HrpL up-regulated genes in 3937 only those within the T3SS were found to contain a hrp box sequence. Moreover, direct binding of purified HrpL protein to the hrp box was demonstrated for hrp box-containing DNA fragments of hrpA and hrpN using the electrophoretic mobility shift assay (EMSA). In this study, a putative T3SS effector DspA/E was also identified as a HrpL-upregulated gene, and shown to be translocated into plant cells in a T3SS-dependent manner. Conclusion/Significances We provide the genome-wide study of HrpL-regulated genes in a necrotrophic phytopathogen (D. dadantii 3937) through a combination of transcriptomics and bioinformatics, which led to identification of several effectors. Our study indicates the extent of differences for T3SS effector protein inventory requirements between necrotrophic and biotrophic pathogens, and may allow the development of different strategies for disease control for these different groups of pathogens. Analysis used aerobic steady state RNA from wild-type as control samples for comparison to the experimental samples of hrpL mutant taken 6 h post-inoculation in T3SS inducible minimum media. The microarray results are based on the geomean of ten normalized expression values derived from three biological replicates and two dye-swap experiments with a technology replicate for each array.

Project description:Preparation of nuclear extracts D. melanogaster Schneider cells were prepared as previously described by Andrews and Faller (1991). Briefly, in a typical preparation, 8x106 cells (grown in Schneider medium plus 10% fetal calf serum; GIBCO-BRL) were pelleted and resuspended in 1,5 mL cold PBS1X; the cell suspension is then tranferred to a microfuge tube. Cells are pelleted for 10 seconds and resuspended in 400 M-5L cold Buffer A (10mM HEPES-KOH pH 7.9 at 4M-0C, 1.5mM MgCl2, 10mM KCl, 0.5mM duthiothreitol, 0.2mM PMSF, 0.2U/microL RNasin). The cells are allowed to swell on ice for 10 minutes, and then vortexed for 10 seconds. Samples are centrifuged for 10 seconds, and the supernatant fraction is discarded. The pellet is resuspended in 100 M-5L of cold Buffer C (20mM HEPES-KOH pH 7.9, 25% glycerol, 420mM NaCl, 1.5mM MgCl2, 0.2mM EDTA, 0.5mM dithiothreitol, 0.2mM PMSF) and incubated on ice for 20 min for high-salt extraction. Cellular debris is removed by centrifugation for 2 min at 4M-0C and the supernatant fraction is stored at M-^V70M-0C. All buffers were prepared from double-distilled autoclaved water that had been treated with 0.1% DEPC (Sigma). RNA immunoprecipitation and Reverse Transcription For RNA immunopreciptation, the nuclear extract was incubated whit 50M-5g of monoclonal C1A9 anti HP1 antibody and the mixture was kept at 4M-0C overnight under continuous gentle movement. One-hundred microliters ofprotein G-Sepharose (Sigma) suspension (50% packed Sepharose in Buffer C) was added and the incubation was continued overnight as described.The beads were pelleted by 2 min centrifugation at 240 g at 4M-0C; the pellet was washed briefly three times whit each 1mL of IP Wash Solution (150mM NaCl, 50mM Tris pH 7.5, 0.5% NP40) and the Sepharose was transferred for eluition into a fresh plastic tube , pelleted again and then the supernatant was removed completely. To elute the immunocomplexes for protein analysis, an aliquot of beads were suspended in 50M-5L SDS-PAGE sample buffer and incubated for 10 min at 90M-0C; following centrifugation the supernatant was removed and used for Western blot for testing the presence of HP1 protein. To elute the immunoprecipitated RNAs, the pelleted beads were boiled in 200M-5L of DEPC wather for 5 min, spun, and the supernatant recovered; 1mL of Trizol (Invitrogen) was added to 200 M-5L of supernatant and mixed well, followed by the addition of 200M-5L of chloroform. This mixture was incubated at 4M-0C for 5 min and then centrifuged at 12000 g for 15 min; the RNAs in the aqueous phase was precipitated whit half volume of isopropanol; after precipitation, the RNAs was resuspend in 10M-5L of DEPC wather. Contaminating DNA was digested whit RNase-free DNase I (Sigma). The RNA purified from the previous step is used as a template to synthesize cDNA using oligo dT , random hexamers and SuperScript reverse transcriptase III (Invitrogen) according to the manufacturer's protocol. cDNA amplification and labeling The cDNA was used as template for a two-step random PCR amplification; in Round A, Sequenase is used to extend randomly annealed primers (Primer A) to generate templates for subsequent PCR; during Round B, the specific primer B is used to aplify the templates previously generated and finally round C consist of additional PCR cycles to inorporate the amino allyl dUTP nucleotide. About 25 ng of each cDNA sample was used for two 8 min extension with 2,7 mM Round A primer (5M-^R-GTT TCC CAG TCA CGA TCN NNN NNN NN-3M-^R, N being a mixture of all four nucleotides with 60% A+T and 40% G+C) at 37M-0C with 267U/ml Sequenase version 2.0 (usb). DNA was denatured at 94M-0C for 2 min and cooled to 10M-0C , and Sequenase 2.0 was added between extensions. The resulting products were used as template for 25 cycles of PCR using 1U/100M-5l Taq polymerase (Platinum Taq Invitrogen) and 10mM Round B primer (5M-^R-GTT TCC CAG TCA CGA TC-3M-^R). Finally this DNA was used as template for 25 cycles PCR to inorporate the amino allyl dUTP nucleotides to which the fluorescent dye may then be attached. To remove Tris buffer which interferes with the indirect coupling, the aminoallyl-cDNA samples were desalted by filtering through a Microcon M-^V30 and then mixed with the succinimidyl esters of the Cy3 or Cy5 dyes (Amersham Biosciences) in 0,1M sodium bicarbonate buffer (pH 9); the coupling reaction was icubated overnight in the dark at room temperature. Each dye labeled sample was purified by AutoSeq MicroSpin G-50 columns (Amersham Biosciences) following the manufacterers directions. Microarrays Hybridization and washing The dried labeled cDNAs pellet were resuspended in 80M-5l of DIG Easy Hyb (Roche) hybridization buffer containg 0,5 mg/ml yeast tRNa and 0,5 mg/ml salmon sperm DNA. Finally the DNA probe was denaturated by incubation at 65M-0C for 10 min and, after a brief centrifugation to spin down the drops, the mixture was pipetted onto a microarray; a coverslip was applied and the slide was placed in a microarray hybridation chamber (BioRad) and incubated overnight at 37M-0C. After hybridization, the slide was submerged in 0,01% SDS, 1%SSC until the coverslip slipped off the surface; The slide was transferred to a solution of 1%SSC and shaken at 50M-0C for 10 min, then washed once more by shaking in 0,1%SSC. Slide was dried by centrifugation for 3 min at 550 rpm and scanned with an ScanArray Lite Microarray Scanner (Packard Bioscience) with laser intensities chosen to maximize signals while avoiding pixel saturation. ScanArray express softwere was used to quantify hybridation signals; bad spots were flagged automatically by softwere and subsequently each slide was inspected manually.

Dataset Information

A serial sample processing strategy with improved performance for in-depth quantitative analysis of type III secretion events in Pseudomonas aeruginosa

Dataset's files

Publications

A Serial Sample Processing Strategy with Improved Performance for in-Depth Quantitative Analysis of Type III Secretion Events in <i>Pseudomonas aeruginosa</i>.

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