Comment[ArrayExpressAccession] E-GEOD-26976 MAGE-TAB Version 1.1 Public Release Date 2013-04-01 Investigation Title Streptococcus pneumoniae in a biofilm are hyper-adhesive, avirulent, and when used as a killed whole-cell vaccine confers protection against lethal challenge Comment[Submitted Name] Streptococcus pneumoniae in a biofilm are hyper-adhesive, avirulent, and when used as a killed whole-cell vaccine confers protection against lethal challenge Experiment Description For Streptococcus pneumoniae, biofilms have been suggested to promote long-term colonization of the nasopharynx and contribute to the pathology of recurrent middle ear infections. To date numerous studies have investigated the contribution of specific genetic determinants for the development of pneumococcal biofilms, however, studies examining the global changes that occur during biofilm development and how they contribute to disease are lacking. Using Scanning and Transmission electron microscopy we examined development of a mature pneumococcal biofilm in a continuous flow through reactor. We determined that a mature biofilm is formed in discrete stages, is marked by the formation of complex 3-dimensional structures, and is primarily composed of dead pneumococci. Using genomic microarrays we determined that pneumococci in mature biofilms down regulate genes involved in protein synthesis, energy production, metabolism, capsular polysaccharide production, and virulence. We confirmed these changes by testing bacterial resistance to antimicrobials, measuring capsule production by ELSIA, and immunoblotting for pneumolysin production. We determined that biofilm pneumococci are hyper-adhesive, binding to cell lines at levels 9 to 11-fold greater than planktonic counterparts. Using Western blot and ELISA, we determined that biofilm bacteria produce greater amounts of the adhesins PsrP, CbpA, and surface exposed phosphorylcholine. We subsequently determined that the hyper-adhesive phenotype was in part due to selection of the transparent phase variant during biofilm growth. Intranasal, intratracheal and intraperitoneal challenge of mice with biofilm and planktonic pneumococci determined that biofilm bacteria were highly attenuated for invasive disease but not nasopharyngeal colonization. Immunization of mice with ethanol-killed biofilm pneumococci of serotype 4 conferred protection against challenge with same isolate but not a serotype 3. ELISA for reactive IgG levels subsequently determined that biofilm pneumococci do not provide high levels of cross-reactive protein antigens. Together these studies suggest that biofilms do not directly contribute to disease but instead confer a protected mode of growth for the pneumococcus. Pneumococcal biofilms compared to planktonic control at 4, 12, 24, 48 hours. 3 biological replicates each of 4 and 12 hour time points, and 2 biological replicates each of 24 and 48 hour time points. Flip dye (technical replicates) performed for 4, 12, and 24 hour time points; no technical replicate performed for 48 hour time point due to limiting material. Ratios were determined by averaging across technical and biological replicates. The following hybridizations made up each biological replicate: 14090167.tav.annot and 14090190.tav.annot (4hr biol rep 1); 14090169.tav.annot and 14090176.tav.annot (4hr biol rep 2); 14090192.tav.annot and 14090188.tav.annot (4hr biol rep 3); 14087688.tav.annot and 14090180.tav.annot (12hr biol rep 1); 14090185.tav.annot and 14090168.tav.annot (12hr biol rep 2); 14090191.tav.annot and 14090174.tav.annot (12hr biol rep 3); 14090170.tav.annot and 14090175.tav.annot (24hr biol rep 2); 14090193.tav.annot and 14087687.tav.annot (24hr biol rep 3); 14090181.tav.annot (48hr biol rep 1); 14090187.tav.annot (48hr biol rep 2) Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name KUMAR Sanchez Kumar Shivshankar Lizcano Hotopp Jorgensen Tettelin Orihuela Person First Name NIKHIL Carlos Nikhil Pooja Anel Julie James Hervé Carlos Person Mid Initials J C H J Person Email nkumar@som.umaryland.edu Person Affiliation University of Maryland, Baltimore Person Address Institute for Genome Sciences, University of Maryland, Baltimore, 801 W. Baltimore Street, Baltimore, MD, USA Person Roles submitter Protocol Name P-GSE26976-4 P-GSE26976-3 P-GSE26976-1 P-GSE26976-2 P-GSE26976-9 P-GSE26976-6 P-GSE26976-11 P-GSE26976-10 P-GSE26976-5 P-GSE26976-7 P-GSE26976-8 Protocol Description ID_REF = VALUE = normalized average log2 ratio test/control (G54 probes) 48hr_avg = 48hr_pvalue = ID_REF = VALUE = normalized average log2 ratio test/control (G54 probes) 24hr_avg = 24hr_pvalue = ID_REF = VALUE = normalized average log2 ratio test/control (G54 probes) 4hr_avg = 4hr_pvalue = ID_REF = VALUE = normalized average log2 ratio test/control (G54 probes) 12hr_avg = 12hr_pvalue = Slides were prehybridized in a 50 ml solution of 5X SSC, 0.1% SDS and 1% BSA for at least one hour at 42C, washed 10X in water and once in isopropanol, then dried by brief centrifugation. Labeled probes were re-suspended in hybridization buffer (50% formamide, 5X SSC, 0.1% SDS, 1 uL 0.1M DTT, 0.6 ug/uL salmon sperm DNA) and hybridized to the microarray slides in a 42C water bath for 16-20 h. Slides were washed twice in a low stringency buffer (2X SSC, 0.1% SDS) at 55C for 5 min, twice in a medium stringency buffer (0.1X SSC, 0.1% SDS) at room temperature for 5 min, twice in a high stringency buffer (0.1X SSC) at room temperature for 5 min, and finally in water for 2 min, and then dried by brief centrifugation. Bacterial strains were grown on tryptic soy blood agar plates at 37C in 5% CO2. For planktonic growth, Todd Hewitt Broth (THB) was inoculated with overnight plate cultures and grown to mid-logarithmic phase (OD620 = 0.5; ~1.0 X 108 CFU/mL) using normal culture conditions. Mature S. pneumoniae biofilms were grown under once-through flow conditions using a once-through biofilm line reactor, as previously described. Iterative log-mean normalization and filtering using MIDAS. Scanned on GenePix 4000B. S. pneumoniae grown under biofilm conditions for 4, 12, 24, and 48 hours were collected, immediately suspended in RNAprotect and stored at -20C. Total RNA extracted using Rneasy Mini Kit, following manufacturer's instructions, with exception to bacterial lysis. Aliquots of 2 ug of the total RNAs were reverse transcribed into single-stranded cDNA using Superscript II reverse transcriptase (Invitrogen), random hexamers (Invitrogen), 1X first strand buffer, 10 mM dithiothreitol (DTT), 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.3 mM dTTP and 0.2 mM of aminoallyl-modified nucleotide (Invitrogen). The mixture was incubated overnight at 42C. Amine-modified cDNA was purified using QIAquick PCR purification kit (Qiagen), followed by chemical labeling with Cy3- or Cy5-NHS-ester fluorescent dyes (GE Healthcare). Protocol Type bioassay_data_transformation bioassay_data_transformation bioassay_data_transformation bioassay_data_transformation hybridization grow feature_extraction image_aquisition specified_biomaterial_action nucleic_acid_extraction labeling Experimental Factor Name SAMPLE TYPE TIME Experimental Factor Type sample type time Publication Title Streptococcus pneumoniae in biofilms are unable to cause invasive disease due to altered virulence determinant production. Publication Author List Sanchez CJ, Kumar N, Lizcano A, Shivshankar P, Dunning Hotopp JC, Jorgensen JH, Tettelin H, Orihuela CJ PubMed ID 22174882 Publication DOI 10.1371/journal.pone.0028738 Comment[SecondaryAccession] GSE26976 Comment[GEOReleaseDate] 2013-04-01 Comment[ArrayExpressSubmissionDate] 2011-01-31 Comment[GEOLastUpdateDate] 2013-04-03 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-26976.sdrf.txt