Table 1.
List of primer sequences and target gene function.
Figure 1.
Growth curve and colony forming units of planktonic mono and co-cultures of NTHi and S. pneumoniae.
Planktonic NTHi and S. pneumoniae were grown in sBHI for 10h. Optical density (600nm) and CFU were recorded. Growth inhibition was not detected in co-culture by this method.
Figure 2.
Scanning electron microscopy of NTHi mono-culture and co-culture with S. pneumoniae show increase in pili-like structures in co-culture.
NTHi was grown for 24h in mono- and co-culture with S. pneumoniae and SEM was used to identify pili-like structures. While no pili could be identified at 10,000X magnification in mono-culture, at least 7 structures were identified in co-culture with S. pneumoniae.
Table 2.
Real time PCR analysis of expression of NTHi type IV pili.
Figure 3.
Real time PCR for expression of S. pneumoniae housekeeping and virulence genes show differential gene regulation in co-culture with NTHi.
S. pneumoniae were grown in mono- and co-culture biofilms and expression of spxB, ply, and pavA 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 spxB in co-culture with NTHi, and down-regulation of ply, and pavA.
Figure 4.
Real time PCR for S. pneumoniae and NTHi virulence gene transcripts from CRS and non-CRS excised tissue.
Transcripts for virulence factors of S. pneumoniae and NTHi were observed in human tissue from 7 CRS and 3 non-CRS patients (x-axis). A positive control of the appropriate DNA spike was included. Panels A and B show the average Ct for spxB (A) and pilA (B) genes, and the average among all positive samples in CRS and non-CRS (red bar). Panel C displays the fold change (2ΔΔCt) of spxB and pilA relative to the appropriate 16s rRNA gene expression in CRS vs. non-CRS tissue. This figure shows the presence of S. pneumoniae spxB in 7/7 CRS and 3/3 non-CRS; however, expression was significantly greater in CRS patients (P = 0.002). NTHi pilA was demonstrated in 4/7 CRS samples and 1/3 non-CRS samples. Expression of pilA was significantly greater in CRS than non-CRS patients. Standard deviation is presented and significance (P<0.05) is indicated by *, determined by students t-test.
Figure 5.
Physical contact regulates the expression of NTHi pilA, not S. pneumoniae spxB.
Virulence factors pilA and spxB were assayed for when S. pneumoniae and NTHi were in mono-culture, co-culture with physical contact (dark gray bars), and in co-culture without physical contact (light gray bars). The ΔΔCt is presented in Panel A. Fold change from 16s gene expression is displayed in Panel B. Expression of pilA was induced only when physical contact was allowed. S. pneumoniae spxB was only significantly up-regulated when physical contact was allowed (p = 0.002). Standard deviation is presented and significance (P<0.05) is indicated by *, determined by students t-test.
Figure 6.
Chemical factors secreted in co-culture conditions regulate the expression of key virulence genes.
Conditioned media from S. pneumoniae/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 pilA expression. S. pneumoniae spxB 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 spxB only when conditioned media was used.