This latter suggests that the adaptive immune response developed

This latter suggests that the adaptive immune response developed towards biofilm bacteria during colonization would have restricted utility during invasive disseminated disease. Our studies also identify PsrP as one possible antigen

that may confer protection against both colonization and invasive disease. The other proteins identified as enhanced during biofilm formation and immunogenic during invasive disease may also represent novel targets for intervention. Methods All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee at The University of Texas Health Science Center at San Antonio under protocol number 09022x-34. Strain and bacterial growth conditions Streptococcus pneumoniae strain TIGR4 is a serotype 4 clinical isolate whose genome has been sequenced and annotated

[44]. A66.1 is a serotype PLX4032 3 isolate that has also been previously described [24]. For planktonic growth, Todd Hewitt Broth (THB) was inoculated with overnight plate cultures and grown to mid-logarithmic phase (OD620 = 0.5; ~1.0 × 108 CFU/ml) at 37°C in 5% CO2. Mature biofilms were grown under once-through flow conditions as previously described [14]. Briefly, planktonic seed cultures were used to inoculate 1 meter long silicone tubing (0.89 mm internal diameter, Cole Parmer Inc., Vernon Hills, IL). Bacteria in the line were allowed to attach for 2 hours, after which the flow rate of THB was

adjusted to 0.035 ml/minute. Biofilm derived bacteria were harvested after 3 days by pinching the tube along its entire length, thereby removing the bacterial cells. One and two-dimensional gel electrophoresis and differential protein analysis For one-dimensional (1DGE) comparative analysis of proteins, whole cell lysates (25 μg) from the biofilm and planktonic pneumococci were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver stained using standard methods. Two-dimensional electrophoresis (2DGE) was conducted according to the principles of O’Farrell [45], and done using the optimized conditions for S. pneumoniae as previously described by Allegrucci et al. [24]. Briefly, planktonic and biofilm pneumococci were collected, washed, and suspended in TE buffer (10 mM Tris-HCl, 1 Carnitine palmitoyltransferase II mM EDTA, pH 8.0) supplemented with 300 μg/ml phenylmethyslfonylfluoride (Sigma, St. Louis, MO). Bacteria were disrupted by sonication on ice using 6, 10-second bursts. Samples were prepared for isoelectric focusing (IEF) using a ReadyPrep 2-D cleanup kit (Bio-Rad, Hercules, CA) after which the protein pellet was dissolved in DeStreak rehydration solution (GE Healthcare, Piscataway, NJ). Protein levels were quantified using a Non-Interfering protein assay (G-Biosciences, Maryland Heights, MO). For each sample, 300 μg of protein were applied to 11-cm Immobiline DryStrips (pH 3-5.

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