Real time PCR for expression of <i>S. pneumoniae</i> housekeeping and virulence genes show differential gene regulation in co-culture with NTHi.

<p><i>S. pneumoniae</i> were grown in mono- and co-culture biofilms and expression of <i>spxB, ply,</i> and <i>pavA</i> are recorded and normalized to expression of the 16s rRNA gene using the ΔΔCt method. Up and down-regulation are displayed as ΔΔCt in panel A. Fold change from the reference gene are displayed in panel B. Changes are considered significant when p≤0.05. Significance is indicated by *. Here, we show significant up-regulation of <i>spxB</i> in co-culture with NTHi, and down-regulation of <i>ply,</i> and <i>pavA.</i></p

List of primer sequences and target gene function.

<p>Genes for NTHi and <i>S. pneumoniae</i> are listed along with primers sequences (5′-3′), gene target, function, and GenBank accession number, if applicable.</p

Chemical factors secreted in co-culture conditions regulate the expression of key virulence genes.

<p>Conditioned media from <i>S. pneumoniae/</i>NTHi 24h co-cultures was used to treat 24h single species biofilms (Conditioned Media column). Cells were grown in 100% media, and treated with 100%, or 10% media to distinguish the effects of nutrient depletion. The ΔΔCt is presented in Panel A. Fold change from 16s gene expression is displayed in Panel B. Conditioned media did not induce <i>pilA</i> expression. <i>S. pneumoniae spxB</i> and the 16s rRNA genes were down-regulated in the presence of CM compared to the media controls. Student's t-test determined significant down-regulation of <i>spxB</i> only when conditioned media was used.</p

Real time PCR analysis of expression of NTHi type IV pili.

<p>NTHi were grown in mono- and co-culture biofilms and expression of 16s rRNA reference gene and <i>pilA</i> are recorded and the ΔΔCt calculated. Fold change, or 2^ΔΔCt, from the reference gene are displayed in panel B. Standard deviation is presented, and changes are considered significant when p≤0.05, determined by Students t-test. Significance is indicated by *.</p