Construction and Testing of An Efficient Biophotonic Imaging (BPI) Reporter System to Study Pneumococcal Biology In Vitro and In Vivo
thesisposted on 18.08.2021, 11:17 by Nada Alkhorayef
Streptococcus pneumoniae is a common nasopharyngeal resident in healthy persons, but it remains a major cause of pneumonia, bacteraemia and otitis media despite vaccines and effective antibiotics. There is an urgent need for novel therapeutic approaches, but such advances require a detailed knowledge of S. pneumoniae biology. To better understand pneumococcal biology and infections, we need sensitive in vivo imaging technologies. To this end, bioluminescence imaging can be used, for example, to evaluate anti-infectives, intraspecies interactions and pneumococcal virulence non-invasively. Pneumococcal strains containing click beetle luciferase (CBRluc) under the control of putative highly expressed pneumococcal promoters were constructed. The CBRluc constructs were integrated into known sites in the S. pneumoniae genome. The bioluminescent strains were compared to a lux-based system expressing bacterial luciferase using in vitro growth experiments and in vivo mouse model of pneumonia. The results revealed that CBRluc tagged bacteria showed robust activity of bioluminescence in exponential phase that is also maintained during stationary phase, whereas lux-expressing pneumococci emitted a light signal with high background that peaked during exponential phase and was significantly reduced in intensity during stationary phase. The presence of CBRluc did not affect the growth and virulence properties of the bioluminescent pneumococcal strains. In vivo bioluminescence activity obtained with CBRluc labelled bacteria was much higher than the lux containing strain under different promoters. This study also established that CBRluc reporter system can be used to study pneumococcal virulence in G. mellonella model.
The findings demonstrate that the CBRluc reporter system is more efficient than lux, providing a potential platform for utilization in understanding of the mechanisms of pneumococcal pathogenesis in vivo.