Streptococcus pneumoniae is a common nasopharyngeal resident in healthy people, but at the same time one of the major causes of infectious diseases such as pneumonia, meningitis and sepsis. The shift from commensal to pathogen and its interaction with host cells is poorly understood. One of the major limitations for research on pneumococcal-host interactions is the lack of suitable tools for live cell imaging. To address this issue, we developed a generally applicable strategy to create genetically stable, highly fluorescent bacteria. Our strategy relies on fusing superfolder green fluorescent protein (GFP) or a far-red fluorescent protein (RFP) to the abundant histone-like protein HlpA. Due to efficient translation and limited cellular diffusion of these fusions, the cells are 25-fold brighter than the currently best available imaging S. pneumoniae strain. These novel bright pneumococcal strains are fully virulent and the GFP-reporter can be used for in situ imaging in mouse tissue. We used our reporter strains to study the effect of the polysaccharide capsule, a major pneumococcal virulence factor, on different stages of infection. By dual-color live cell imaging experiments, we show that unencapsulated pneumococci adhere significantly better to human lung epithelial cells compared to encapsulated strains, in line with previous data obtained by classical approaches. We also confirm with live cell imaging that the capsule protects pneumococci from neutrophil phagocytosis, demonstrating the versatility and usability of our reporters. The described imaging tools will pave the way for live cell imaging of pneumococcal infection and help understand the mechanisms of pneumococcal pathogenesis.
- pneumococcal virulence