Statistical analysis The Student’s t test was used to calculate the statistical differences between the mean levels of polysaccharide expression of experimental samples (biofilm grown cells) and control samples (planktonic cells). A P value < 0.05 was considered significant. All statistical analyses were done using InStat software (InStat, San Diego, CA). Results Identification of a novel H. somni surface component produced during PI3K inhibitor anaerobic growth To determine if there was variation in expression of membrane components under different environmental conditions, H. somni 738 was grown on CBA plates in 3-5% CO2 or
under anaerobic conditions for 48 h at 37°C. The bacteria were harvested from the plates as described in methods, and Cetavlon was added to the supernatant (0.005 M, final concentration); LOS and protein-enriched outer membranes were prepared
BAY 11-7082 clinical trial from the cell pellets [46, 47]. No substantial qualitative differences were detected in the electrophoretic profiles of the LOS or membrane proteins of bacteria grown OTX015 on CBA under CO2 or anaerobic conditions (data not shown), although growth of H. somni under anaerobic conditions was poor. Nonetheless, when Cetavlon was added to the supernatant of cells washed off CBA plates incubated under anaerobic conditions, a large precipitate formed, whereas little or no precipitate formed from the supernatant of cells grown on CBA in CO2 (data not shown). The Cetavlon precipitate was solubilized in distilled Farnesyltransferase water, and greater than 90% of the precipitate was determined to be carbohydrate. However, it was not LOS, as determined by polyacrylamide gel electrophoresis and silver staining for LOS (data not shown). Electrophoresis of the Cetavlon precipitate followed by staining with alcian blue and ammoniacal silver demonstrated a heterogeneous profile, typical of high molecular size polysaccharide (Figure 1). Figure 1 Electrophoretic profiles of semi-purified Cetavlon precipitates and biofilm. Bacteria were grown anaerobically on plates or to late stationary phase, Cetavlon added, and precipitates
extracted, as described in Methods. Each extract was loaded onto 25% polyacrylamide gels, followed by electrophoresis and staining with Alcian blue and silver. Lanes: 1 and 2, 20 μg and 30 μg of EPS extracted under growth conditions favorable to biofilm formation; 3 and 4, 20 μg and 30 μg of EPS extracted from cells grown to late stationary phase in broth, respectively; 5, buffer alone; 6 and 7, 20 μg and 30 μg of EPS extracted from cells grown anaerobically on plates, respectively. Immuno-transmission electron microscopy of H. somni grown under anaerobic conditions or CO2 The polysaccharide from Cetavlon precipitates obtained from scaled up anaerobic cultures was further purified, as described in methods, and used to immunize a rabbit.