Samples were spun down and pellets were resuspended in anti-NanA rabbit serum diluted 1:100 in PBS/BSA

and incubated at 4°C for 1 h (negative controls were incubated without antibody). After two washes with 1 ml of PBS, 100 μl of fluorescein isothiocyanate (FITC)-conjugated anti-rabbit (1:64; Sigma-Aldrich) was added to bacterial pellets. The resuspensions were incubated at 37°C for 30 min and then washed twice in PBS. Samples were finally resuspended in 300 μl of paraformaldehyde 1% in PBS and subjected to flow cytometry (FACScan, Becton Dickinson, San Diego, CA). Statistical analysis was carried out by using two-tailed Student t test. Neuraminidase activity The neuraminidase activity was measured using the fluorogenic substrate 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4MU-Neu5Ac) (M8639, Sigma-Aldrich, St. Louis, MLN2238 datasheet Miss.). The time dependence of the variation of fluorescence (λexcitation, 335 nm; λemission, 400 nm) in the presence of cell or enzyme samples was recorded with a EnVision multilabel plate reader (Perkin Elmer, Waltham, Mass.) using 50 μM 4MU-Neu5Ac in 10 mM MES buffer at pH 6.0, in a final reaction volume of 200 μl. S. pneumoniae FP65 was grown in CAT medium, containing alternatively glucose or N-acetylmannosamine as the carbon

source, respectively, for 18 hours at 37°C. The sample was prepared as follows; the culture was centrifuged at 10,000 × g (4°C) and the cell pellet washed once in an equal volume of 10 MES buffer pH GS-4997 supplier 6.0, centrifuged and resuspended at a final A600 = 0.4 in 10 mM MES pH 6.0. The method was initially optimized and calibrated using purified NanA neuraminidase of S. pneumoniae D39 produced

in E. coli (0.88 mg/ml) (data not shown). The activity was computed as the variation of fluorescence vs time using a linear regression eltoprazine of the data. In our conditions, 1 μg of purified NanA yielded a activity of 10,690 ΔF/min. Acknowledgements The work was in part funded by the European Commission grant PNEUMOPATH FP7-HEALTH-222983 and by Ricerca Regionale Toscana in Materia di Salute 2009–201. References 1. Kadioglu A, Weiser JN, Paton JC, Andrew PW: The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nat Rev Microbiol 2008, 6:288–301.Pexidartinib PubMedCrossRef 2. King SJ: Pneumococcal modification of host sugars: a major contributor to colonization of the human airway? Mol Oral Microbiol 2010, 25:15–24.PubMedCrossRef 3. Camara M, Boulnois GJ, Andrew PW, Mitchell TJ: A neuraminidase from Streptococcus pneumoniae has the feature of a surface protein. Infect Immun 1994, 62:3688–3695.PubMed 4. Berry AM, Paton JC: Sequence heterogenicity of PsaA, a 37-kilodalton putative adhesin essential for virulence of Streptococcus pneumoniae. Infect Immun 1996, 64:5255–5262.PubMed 5. McCullers JA, Bartmess KC: Role of neuraminidase in lethal synergism between influenza virus and Streptococcus pneumoniae. J Infect Dis 2003, 187:1000–1009.