Figure 5

Neuraminidase protein production and activity on whole cells. A cytofluorimetric assay with an anti-NanA serum was performed on pneumococci grown on different carbohydrates (panel A). The presence of NanA at the bacterial surface was tested in samples cultivated in glucose (open bar), glucose + ManNAc, ManNAc alone (grey bar), and NeuNAc alone (black bar) (all carbohydrates were at 1 g/L). Data are represented as mean values ± SD of percent bacterial population positive for NanA production and derived from quadruplicates experiments performed independently. Asterisks (*, p < 0.05; **, p < 0.001) indicated statistical significance. Panel B shows the hydrolysis of 2’-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid (4MU-Neu5Ac) in the presence of 40 μl S. pneumoniae FP65 cell samples grown in CAT medium with either glucose (white circles) or N-acetylmannosamine (black circles). The neuraminidase activity was computed as the variation of fluorescence vs time using a linear regression of the data (dashed lines). Inlet. Hydrolysis of 4MU-Neu5Ac by purified NanA neuraminidase, showing the proportionality between enzyme concentration and rate of fluorescence variation. Enzyme concentrations were 10 nM (black circles), 20 nM (triangles), 30 nM (diamonds) and 40 nM (squares). The empty circles show the variation of fluorescence vs time for the substrate alone.