Genome-wide antibiotic-CRISPRi profiling identifies LiaR activation as a strategy to resensitize fluoroquinolone-resistant Streptococcus pneumoniae

Streptococcus pneumoniae is a human pathogen that has become increasingly resistant to synthetic fluoroquinolone antibiotics that target bacterial topoisomerases. To identify pathways essential under fluoroquinolone stress and potential novel targets to revitalize use of this antibiotic class, we perform genome-wide CRISPRi-seq screens and generate antibiotic-gene essentiality signatures. Expectedly, genes involved in DNA recombination and repair become more important under fluoroquinolone-induced DNA damage, including recA, recJ, recF, recO, rexAB, and ruvAB. Surprisingly, specific downregulation of the gene encoding the histidine kinase LiaS caused fluoroquinolone hypersensitivity. LiaS is part of the LiaFSR (VraTSR) three-component regulatory system involved in cell envelope homeostasis. We show that LiaS keeps the response regulator LiaR inactive, and that liaS deletion causes LiaR hyperphosphorylation and upregulation of the LiaR regulon. We use RNA-seq to refine the LiaR regulon, highlighting the role of heat-shock response and pleiotropic regulator SpxA2 in fluoroquinolone sensitivity. Activating the LiaR-regulon by the cell envelope-targeting antibiotic bacitracin synergized with ciprofloxacin and levofloxacin, restoring sensitivity in fluoroquinolone-resistant strains in vitro. Furthermore, bacitracin/levofloxacin combination therapy is effective in vivo and improved treatment of fluoroquinolone-resistant S. pneumoniae infection in a zebrafish meningitis model. These findings offer a starting point for identification and validation of potent combination therapies to treat antibiotic-resistant pneumococcal infections.