Timeline of vaccination experiments.

<p>Timeline of vaccination experiments.</p

Recovery of WT <i>M. catarrhalis</i> O35E from the nasopharynx of immunized chinchillas three days post infection.

<p>Results are expressed as the mean (± std error) CFU/gr of nasopharyngeal tissues (note the log scale). The asterisk indicates that the reduction in the number of bacteria is statistically significant (Wilcoxon signed rank test, <i>P</i> value is shown in parentheses). Control and His-tagged MhaB groups were tested in parallel on three separate occasions. Each column represents 12 animals (groups of n = 4 animals/experiment).</p

Recovery of <i>M. catarrhalis</i> from the nasopharynx of chinchillas three days post-infection.

<p>Animals were inoculated with ∼1×10⁹ CFU. Results are expressed as the mean (± standard error) CFU/gr of nasopharyngeal tissues. Strains were tested in parallel on two separate occasions. Each column represents 12 animals. The asterisk indicates that the reduction in the number of bacteria is statistically significant (Wilcoxon signed rank test).</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Western blot analysis of serum from chinchillas inoculated with the WT <i>M. catarrhalis</i> strain O35E.

<p>Equivalent amounts of whole cell lysates (WT <i>M. catarrhalis</i> O35E, <i>uspA2</i> KO strain O35E.2, <i>hag</i> transposon mutant strain O35E.TN2, and <i>ompCD</i> KO strain O35E.CD1) were resolved by SDS-PAGE, transferred to PVDF and probed with the indicated primary Abs. <u>Panels A and B:</u> Pre- and post-infection serum samples were pooled and used as primary Abs at a dilution of 1∶250. Goat α-rat IgG conjugated to HRP were used as secondary Abs. <u>Controls:</u> The murine monoclonal Abs 10F3 (Panel C, α-CopB), 5D2 (Panel D, α-Hag), 17H4 (Panel E, α-UspA2) and 1D3 (Panel F, α-OMPCD) were used as primary Abs in combination with goat α-mouse HRP-(IgG+IgA+IgM) secondary Abs. These controls were included to verify the identity of proteins recognized by post-infection chinchilla serum in panel B. MW markers are shown to the left of in kDa.</p

Inhibition of adherence with samples from chinchillas immunized with His-tagged MhaB protein.

<p>The WT <i>M. catarrhalis</i> strains O35E, O12E, and McGHS1 were preincubated for 30 min at 37°C with pooled samples from chinchillas sham-vaccinated with PBS (black bars) or with pooled samples from chinchillas immunized with His-tagged MhaB at dilutions of 1∶50, 1∶100, 1∶200 and/or 1∶2000. These bacteria were then used to perform adherence assays. The adherence of <i>M. catarrhalis</i> preincubated with samples from chinchillas vaccinated with PBS was set at 100%. The adherence of <i>M. catarrhalis</i> preincubated with samples from chinchillas immunized with His-tagged MhaB is expressed as the percentage (±standard error) of that of <i>M. catarrhalis</i> preincubated with samples from chinchillas vaccinated with PBS. Assays were performed in triplicate on three separate occasions. The asterisks indicate that the reduction in adherence is statistically significant (<i>P</i> values <0.05, paired <i>t</i> test). Post-boost samples taken on Day 44 of vaccination experiments (see Fig. 3) were pooled and used in these assays.</p

Western blot and ELISA analyses of samples from chinchillas immunized with the His-tagged MhaB protein.

<p><u>Western blot (panels A, B, C)</u>: Equivalent protein amounts were resolved by SDS-PAGE, transferred to PVDF and probed with the indicated primary and secondary Abs. Post-boost serum and lavage samples taken on Day 44 of the vaccination experiments (see Fig. 3) were pooled and used as primary Abs at the dilution shown in parentheses. Goat α-rat Abs conjugated to HRP were used as secondary Abs. Panel A: western blot of outer membrane protein preparations from the WT <i>M. catarrhalis</i> strain O35E and the <i>mhaB1mhaB2</i> mutant O35E.B1B2. Panels B and C: western blot of the purified recombinant proteins GST-tagged MhaB and GST-tagged McaP (used as negative control). Arrows indicate proteins specifically reacting with chinchilla Abs α-MhaB1/MhaB2. MW markers are shown to the left in kDa. <u>ELISA (panel D)</u>: Individual serum samples were serially diluted and placed in duplicate wells of plates coated with GST-tagged MhaB. Goat α-rat Abs conjugated to HRP were used as secondary Abs. The results are expressed as the mean (± std deviation) end-point titer of samples from n = 12 animals. Individual titers were determined using pre-immune samples as background. Open bars correspond to pre-boost samples taken on Day 19 of the vaccination experiments while black bars represent post-boost samples collected on Day 44 (see Fig. 3).</p

Recovery of WT <i>M. catarrhalis</i> O35E from the nasopharynx of chinchillas.

<p>Animals were inoculated with ∼1×10⁹ CFU. Results are expressed as the mean (± standard error) CFU/mL (lavage fluids, black bars) or CFU/gr (nasopharyngeal tissues, open bars). Each column represents at least 4 animals, and each experimental condition was tested on at least two separate occasions.</p

Detection of aneuploidies.

<p>For each sequenced sample, coverage across each chromosome was compared to genome-wide coverage. Based on DNA content staining, baseline ploidy was assumed to be 1N for haploids and 2N for autodiploids. Euploidy is indicated by empty circles: haploid—green, autodiploids—blue. Aneuploidies are shown as filled circles and labeled by clone.</p