A pooled CRISPRi screen to identify new cell cycle proteins in the opportunistic human pathogen Streptococcus pneumoniae

Objetivo

There is much truth in the ancient Chinese saying; “if you want to win the battle, you have to know your enemy”. When it comes to bacterial infections, we often don’t know our enemy very well and we are losing. Despite our best efforts to combat Streptococcus pneumoniae infections, this opportunistic pathogen remains a serious threat to human health, killing over 826 000 children each year and causing severe illness in 14 million more. Mankind is therefore in desperate need of novel therapies that can eradicate S. pneumoniae infections. The search for these therapies, however, is impeded by the lack of insight into the life cycle of this important pathogen. In this proposal, I therefore aim to generate unprecedented insight into the S. pneumoniae cell cycle. I will do so by performing an innovative pooled CRISPRi (Clustered Regularly Interspaced Short Palindromic Repeats Interference) screen combined with FACS (Fluorescence Activated Cell Sorting) to assess the effect of downregulation of all S. pneumoniae genes on important cell cycle parameters such as cell morphology, DNA content and the formation of the Z-ring that is required for cell division. This genome-wide screen will reveal several gene products that are important for the correct progression of the S. pneumoniae cell cycle. I will further characterize the most promising targets to unravel their cell cycle-related function at the molecular level. Moreover, newly-identified gene products that are important for cell cycle progression could be interesting novel drug targets. The efficacy of these prospective targets will be validated in an in vivo infection model in Galleria mellonella (wax moth) larvae. In the future, these candidates can be used as a starting point for the development of novel and effective therapies. Besides generating valuable fundamental insight into the S. pneumoniae cell cycle, the results of this project will thus also assist in our fight against this notorious pathogen.