RNA was analyzed by semi-quantitative reverse-transcription PCR. PCR products were analyzed on 1.5% agarose Dactolisib gels, stained with ethidium bromide and subsequently visualized. To confirm equal loading, PCR for 16S rRNA was LOXO-101 molecular weight performed in parallel. Ctrl indicates control reactions with no cDNA templates. Because lactoferrin rather than transferrin is the primary carrier of iron on mucosal surfaces and lactoferrin binding proteins are thought to be important virulence factors in some gram-negative bacteria [28], we investigated whether cold shock affects the expression

of these genes. As shown in Figure 2, cold shock increased the mRNA level of lbpB and lbpA genes in strain O35E after 3 h of incubation at 26°C (Figure 2C). Furthermore, cold shock increased the transcriptional level of lbpA and lbpB of other clinical isolates indicating that this effect is a general characteristic of M. catarrhalis (Figure 2D). Enhanced binding of transferrin and lactoferrin on the surface of M. catarrhalis induced by cold shock Because a temperature drop from 37°C to 26°C induces an increase in the copy numbers of genes involved in iron Combretastatin A4 mw acquisition, we investigated whether it also affects the binding

to human transferrin and lactoferrin. Strain O35E and its TbpB-deficient mutant were exposed to 26°C or 37°C and evaluated for their ability to bind transferrin. Binding to transferrin was increased when bacteria were exposed to 26°C (Figure 3A and 3B). The absence of TbpB reduced binding to transferrin, indicating that TbpB is required for maximum binding of transferrin on the surface of cold shock-induced M. catarrhalis. Figure 3 Increase in the binding of transferrin on the surface of M. catarrhalis as a result of cold shock. A, strain O35E and its isogenic mutant O35E.tbpB exposed to 26°C or 37°C for 3 h were incubated with fluorescein isothiocyanate (FITC)-conjugated transferrin

(0.1 μg/mL) and flow cytometry analysis was performed. Shown are representative flow cytometry profiles of strain O35E and O35E.tbpB after exposure Methisazone at 26°C (gray) or at 37°C (black), which demonstrate that TbpB is required for maximum binding of transferrin on the surface of cold shock-induced Moraxella catarrhalis. The dotted line represents the negative control (bacteria only). The mean fluorescence intensity ± 1 standard deviation for three experiments performed is shown in panel B. *, P< 0.05 for 26°C versus 37°C (one-way analysis of variance). Binding to lactoferrin in a whole-cell solid-phase binding assay was significantly increased when bacteria were exposed to 26°C, in comparison with exposure to 37°C (Figure 4A). The surface binding of human salivary and milk lactoferrin (sLf and Lf, respectively) was further quantitated using flow cytometry, resulting in a clear shift of fluorescence intensity for M. catarrhalis exposed at 26°C (Figure 4B).