Construction and test of an efficient biophotonic imaging (BPI) reporter system to study pneumococcal biology in vitro and in vivo

Streptococcus pneumoniae is a common nasopharyngeal resident in healthy persons, but remains a major cause of pneumonia, bacteremia 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 and its shift from commensal to pathogen. 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 interaction and pneumococcal virulence non-invasively. A click beetle luciferase (CBR-luc) containing vector pPP3 under the control of putative highly expressed pneumococcal promoters was constructed. The CBRluc providing red-shifted light production was integrated into known sites in the S. pneumoniae genome. The constructs were compared to a lux-based exist system expressing bacterial luciferase using in vitro growth experiments. The results revealed that CBRluc tagged bacteria, PphrA::luc-wt, showed robust activity of bioluminescence in exponential phase that is 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. Initial 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 system.