Comment[ArrayExpressAccession]	E-BUGS-117
Investigation Title	Extracellular Matrix Formation Enhances the Ability of Streptococcus pneumoniae to Cause Invasive Disease
Comment[Submitted Name]	Extracellular Matrix Formation Enhances the Ability of Streptococcus pneumoniae to Cause Invasive Disease
Experimental Design	growth_condition_design
Experimental Factor Name	GrowthCondition	Phenotype
Experimental Factor Type	GrowthCondition	Phenotype
Person Last Name	Trappetti	BuG@S group
Person First Name	Glaudia
Person Mid Initials
Person Email		awitney@sgul.ac.uk
Person Phone		+44(0)208 725 0698
Person Fax
Person Address		Medical Microbiology
Person Affiliation	University of Adelaide
Person Roles	investigator	submitter
Quality Control Type
Public Release Date	2011-04-20
PubMed ID	21611130
Publication DOI	10.1371/journal.pone.0019844
Publication Author List	Trappetti C, Ogunniyi AD, Oggioni MR, Paton JC.
Publication Title	Extracellular matrix formation enhances the ability of Streptococcus pneumoniae to cause invasive disease.
Publication Status
Experiment Description	 Background. During infection, pneumococci exist mainly in sessile biofilms rather than in planktonic form, except during sepsis. However, relatively little is known about how biofilms contribute to pneumococcal pathogenesis. Methods. We performed a biofilm assay and mouse infection experiments on opaque and transparent variants of a clinical serotype 19F strain.  Results. After 4 days incubation, scanning electron microscopy revealed that opaque biofilm bacteria produced an extracellular matrix, whereas the transparent variant did not. The opaque biofilm-derived bacteria translocated from the nasopharynx to the lungs and brain of mice, and showed 100-fold greater in vitro adherence to A549 cells. Microarray analysis of planktonic and sessile bacteria from transparent and opaque variants showed differential gene expression in two operons: the lic operon, involved in choline uptake, and in the two-component system, ciaRH. Mutants of these genes did not form an extracellular matrix, could not translocate from the nasopharynx to the lungs or the brain, and adhered poorly to A549 cells. Conclusions. We conclude that only the opaque phenotype is able to form extracellular matrix, and that the lic operon and ciaRH contribute to this process. We propose that during infection, extracellular matrix formation enhances the ability of pneumococci to cause invasive disease. Data is also available from http://bugs.sgul.ac.uk/E-BUGS-117
Protocol Name	P-MTAB-20413	P-MTAB-20414	P-MTAB-20415	P-MTAB-20416	P-MTAB-20417	P-MTAB-20418	P-MTAB-20419	BuG@S hybridization	P-MTAB-20420
Protocol Type	image_acquisition	grow	split	nucleic_acid_extraction	labeling	split	labeling	hybridization	bioassay_data_transformation
Protocol Description	Microarray slides were scanned with a GenePix 4000B Microarray Scanner (Axon Instruments) using PMT voltages usually of 800 for cDNA experiments. Basic analysis was performed using the software provided with the scanner, GenePix Pro. Care was taken to ensure that control spots and spots with high local background were removed from the results so that they were unable to interfere with later analysis. 	Bacteria were grown in flat-bottom polystyrene tissue culture plates (96-well plates; Sarstedt). Frozen pneumococcal cultures (about 1  108 cfu ml-1) were diluted 1:100 in 200 µl THY and plates were incubated for 4 days at 37°C in a CO2-enriched atmosphere, and medium was changed every day.	After removal of planktonic bacteria, biofilm cells were detached by subjecting plates to a 3 s sonication in a water bath. Detached cells (100 µl) were then recovered from plates for cfu counts and microarray analysis.	The bacteria were resuspended completely in 300 µl prewarmed (65 °C) acid-phenol and incubated for 5 min at 65 °C. To this, 300 µl prewarmed NAES buffer (50 mM sodium acetate pH 5·1, 10 mM EDTA, 1% SDS) was added, and the mixture was incubated for another 5 min at 65 °C, with intermittent mixing. The mixture was cooled on ice for 1 min and the phases were separated by centrifugation at 15500 g for 1 min. The aqueous phase was re-extracted twice with acid-phenol followed by two further extractions with chloroform. The extract was then precipitated at –80 °C overnight in the presence of 40 ng glycogen µl–1 (Sigma 1767). Subsequently, the preparation was treated with 10 U RNase-free DNase (Roche) at 37 °C for 30 min in the presence of 1 U µl–1 recombinant RNasin ribonuclease inhibitor (Promega N251A), after which RQ1 DNase stop buffer (Promega M198A) was added to inactivate the DNase. The amount of RNA recovered following purification/enrichment was determined by OD260/280 measurements.	 2-10ug of RNA sample was placed in a microfuge tube (0.5mL) with 3ug of random primers (1uL) and made up to a final volume of 11uL with H2O (DNase and RNase free, molecular biology grade). The RNA was then heated to 95C for 5min, snap cooled on ice and centrifuged. 5uL First Strand Buffer (5x), 2.5uL DTT (100mM), 2.3ul dNTP's (5mM dA/G/TTP, 2mM dCTP), 1.7uL of Cy5(1mM) and 2.5uL of SuperScript II (200U/uL) were added to make a final volume of 25uL. The solution was incubated in the dark at 25C (room temperature) for 10min and then at 42C in the dark for 90min. 	Planktonic bacteria were harvested into ice-cold eppendorf and wells were further washed three times each with ice-cold THY, after which 100 µl THY was added to each well.	For RNA samples labelled with Cy3 dCTP. 2-10ug of RNA sample was placed in a microfuge tube (0.5mL) with 3ug of random primers (1uL)and made up to a final volume of 11uL with water (DNase and RNase free, molecular biology grade). The RNA was then heated to 95C for 5min, snap cooled on ice and centrifuged. 5uL First Strand Buffer (5x), 2.5uL DTT (100mM), 2.3ul dNTP's (5mM dA/G/TTP, 2mM dCTP), 1.7uL of Cy3(1mM) and 2.5uL of SuperScript II (200U/uL) were added to make a final volume of 25uL. The solution was incubated in the dark at 25C (room temperature) for 10min and then at 42C in the dark for 90min.	1)Excess Cy3 and Cy5 dCTP was removed from labelled DNA or cDNA samples using the MinElute Reaction Cleanup Kit (Qiagen). Cy3 and Cy5 labelled DNA and/or cDNA samples were combined in a single tube (1.5mL) and 5 volumes of PB buffer added. The solution was applied to a MinElute column in a collection tube and centrifuged at 13,000rpm for 1min. The flow-through was discarded and the MinElute column placed back into the same collection tube. 500uL of Buffer PE was added to MinElute column, centrifuged at 13,000rpm for 1min and the flow-through discarded. The MinElute column was placed back in the same collection tube and the previous step repeated with 250uL of PE. The MinElute column was placed into a fresh 1.5mL tube, 15.9uL of water (22x22mm LifterSlip) or 30.2uL (22x50mm LifterSlip) was added to the centre of the membrane, allowed to stand for 1min and then centrifuged at 13,000rpm for 1min. 2)50mL of prehybridization solution (3.5xSSC, 0.1% SDS, 10mg/mL BSA) was placed in Coplin jar and incubated at 65C to preheat for 1h 30min. The microarray slide was placed in the pre-hybridization solution and incubated at 65C for 20min. The slide was then rinsed in 400mL water for 1min and 400mL of propan-2-ol for 1min. The slide was placed in a 50mL centrifuge tube and centrifuged at 1,500rpm for 5min to dry. Each slide was stored in a dark, dust free box until hybridization (<1h). 3)The prehybridized microarray slide was placed in the hybridization cassette and two 15uL aliquots of water added to the wells of the cassette. A 4xSSC 0.3% SDS hybridization solution containing the Cy3/Cy5 labelled samples was prepared with a final volume of 23ul (22x22mm LifterSlips)or 45uL (22x50mm LifterSlips). The hybridization solution was heated at 95C for 2min, allowed to cool slightly at room temperature and briefly centrifuged. A LifterSlip was placed carefully over the arrayed area of the slide, to avoid scratching its surface. The hybridization solution was pipetted under one corner of the LifterSlip, allowing the solution to be drawn completely across the array by capillary action. Any excess hybridization solution was pipetted under the opposite corner of the LifterSlip. The hybridization cassette was sealed and submerged in a water bath at 65C in the dark for 16-20h. 4)Wash A (1xSSC, 0.05% SDS) was preheated to 65C and placed in a staining trough pre-heated to 65C. The microarray slide was removed from the hybridization cassette and carefully washed in the staining trough of Wash A at 65C to remove LifterSlip. The slide was then placed in a slide rack and agitated in Wash A for a further 2min. Slides were transferred to a clean rack and agitated in a trough of 400mL of Wash B (0.06xSSC) for 2min at room temperature. Slides were transferred to a second trough of 400mL of Wash B (0.06xSSC) and agitated for a further 2min at room temperature. The slide was then dried by centrifugation at 1,500rpm for 5min in a 50mL centrifuge tube. 	mRNAs were ranked from those most likely to be differentially expressed to the least likely using false-discovery rate values of p< 0.05. A fold change of ≥ 2.0 in gene expression with P value of ≤ 0.01 was considered to be significant.
Protocol Parameters					RNA quantity;fluorescence label;labelled extract yield		RNA quantity;fluorescence label;labelled extract yield	hybridization volume;hybridization temperature;hybridization time;Wash A temperature;Wash A time;Wash B temperature;Wash B time 1;Wash B time 2
Comment[AEExperimentType]	transcription profiling by array
SDRF File	E-BUGS-117.sdrf.txt