Figure 3. Surface localized sialidase is required for growth.
(A) Sialidase activity of intact bacteria, measured with the substrate MUAN as the mean of triplicate measurements and s.d. of a representative experiment. (B) Viable counts after challenge with 2×106 Cc5 (black), siaC (light grey) or siaC complemented with plasmids containing siaC FL, siaC Δ1–21 and siaC Y488C after 24 h in presence of J774.1 with the grey dotted line indicating the bacterial number inoculated. Sialidase was detected by immunoblotting with a polyclonal antibody against SiaC in total cells (TC). (C) Outer membrane protein fractions (OMP), cell free supernatants (SN) of the J774.1 cultures shown in (B) including as control TC of Cc5 were analyzed by immunoblotting for the presence of SiaC. (D) Surface localization of SiaC was tested by immunofluorescence on paraformaldehyde fixed but not permeabilized bacteria using anti-SiaC followed by anti- rabbit IgG conjugated to FITC.