Figure 4

Increase in the binding of lactoferrin on the surface of M. catarrhalis as a result of cold shock. A, solid-phase lactoferrin binding assay. B, strain O35E exposed to 26°C or to 37°C for 3 h was preincubated with saliva samples from healthy adults or human milk lactoferrin, followed by a mouse anti-human lactoferrin antibody. Alexa 488-conjugated anti-mouse antibody was added, followed by flow-cytometric analysis. Representative flow-cytometric profiles of M. catarrhalis strain O35E after exposure at 26°C (gray) or at 37°C (black) show cold shock-dependent binding to salivary lactoferrin (sLf) and lactoferrin isolated from human milk (Lf). The dotted line represents the negative control (bacteria incubated with secondary antibodies only). C, binding of human lactoferrin to OMPs isolated from M. catarrhalis strain O35E exposed to 26°C or 37°C was analyzed by SDS-PAGE Coomassie blue staining (left panel) and Western blot (right panel). Proteins were probed with human lactoferrin. Molecular weight markers in kDa are indicated to the left. D, increase in CopB surface expression due to cold shock. Strain O35E exposed to 26°C or to 37°C for 3 h was incubated with the copB-specific 10F3 mouse monoclonal antibodies, followed by Alexa 488-conjugated anti-mouse antibody. Representative flow-cytometric profiles of M. catarrhalis strain O35E after exposure at 26°C (gray) or at 37°C (black) show cold shock-dependent CopB upregulation. The mean fluorescence intensity ± 1 standard deviation for 2 experiments performed is shown (E). *, p < 0.05 for 26°C versus 37°C (one-way analysis of variance).