CRISPRi-seq identifies pneumococcal genes influencing fluoroquinolone susceptibility at a genome-wide level.

A Schematic overview of the chemogenomic screen workflow. The S. pneumoniae CRISPRi library was treated with a sub-lethal dose of each antibiotic (AB) in the absence or presence of IPTG to induce dCas9. The results of the pooled CRISPRi-seq screen were then validated by testing individual mutants. B Heatmap depicting all the sgRNAs (representing operons that have been transcriptionally repressed) that showed a significant differential fitness effect upon antibiotic treatment shared between all three fluoroquinolones as compared to no antibiotic treatment (|Δlog₂FC| >  1, P_adj < 0.05; DESeq2³² differential enrichment analysis using a negative binomial generalized linear model with two-tailed P-values adjusted for false discovery rate (FDR)). Source data available in Supplementary Data 1.

The ssDNA repair complex RecJFOR is crucial for pneumococcal survival during fluoroquinolone treatment.

A At DNA gaps, the ssDNA-specific exonuclease RecJ enlarges the ssDNA region. RecF is a recombination mediator protein and works in complex with RecOR to facilitate the binding of RecA onto the ssDNA. The recombinase RecA, then, promotes homology search, homologous pairing, and strand exchange. Figure adapted from ref. ³⁸. Depletion strains were constructed for (B) recJ and CrecF, and the mutants were treated with the sub-lethal concentration of LEV used in the screens. Deletion of recF or recJ resulted in increased sensitivity to LEV, and the phenotype was rescued upon complementation. D Deletion of efp resulted in a fitness advantage in the presence of a lethal concentration of LEV. Growth curve data represent the mean ± SEM of three biological replicates. E Blocking translation elongation using chloramphenicol (CHL) results in an antagonistic interaction with LEV. Source data are provided as a Source Data file.

Deleting liaS of the LiaFSR three-component sensor system in S. pneumoniae increases sensitivity to fluoroquinolone treatment through the upregulation of the LiaR regulon.

A The LiaFSR operon consists of three genes, liaF a proposed signal sensor, liaS (HK) and liaR (RR). B Growth curves of deletion mutants ΔliaF, ΔliaR and ΔliaFSR treated with sub-lethal dose of CIP showed sensitivity equal to wild-type (WT). Only the ∆liaS mutant showed increased sensitivity to CIP. C Complementation of liaS restored CIP sensitivity back to WT levels. D RNA-seq revealed a set of genes upregulated in a ∆liaS mutant, including the entire known LiaR regulon, highlighted by a black outline. Statistical significance was defined as |log₂FC|> 1, and P_adj < 0.05 (DESeq2³² differential enrichment analysis using a negative binomial generalized linear model with two-tailed P values adjusted for false discovery rate (FDR)). Source data available in Supplementary Data 2. E At 40 °C, ∆liaS mutants are more susceptible to CIP than WT and the ∆liaR mutant. F Overexpression of spxA2 from an IPTG-inducible promoter by addition of IPTG (dashed line) caused increased sensitivity to a sub-lethal concentration of CIP. Growth curve data represent the mean ± SEM of three biological replicates. Source data are provided as a Source Data file.

LiaS acts primarily as a phosphatase or inhibitor of LiaR phosphorylation.

A The luc reporter gene was placed under the control of the promoter of spv_0803, a gene that is strongly induced by LiaR-P. Upon bacitracin (BAC) treatment, luciferase was highly expressed, indicating induction of the LiaR regulon. Deletion of liaS results in overexpression of luciferase. A ∆liaF mutant displays slight induction of the LiaR regulon, even in the absence of BAC. B A phosphomimetic version of LiaR (LiaR*, D53E) was constructed under the control of the IPTG-inducible P_lac promoter. The strain P_{spv_0803}-luc, P_lac-liaR* phenocopied a ∆liaS mutant in both growth phenotype and induction of the regulon. C Activation of the LiaR regulon by expression of LiaR* results in increased sensitivity to ciprofloxacin (CIP). Growth curve data represent the mean ± SEM of three biological replicates. Source data are provided as a Source Data file.

Schematic representation of the role of the LiaFSR three-component regulatory system in fluoroquinolone susceptibility. Hypothetical model of the LiaFSR system modeled with AlphaFold.

A In the absence of a stimulus, LiaFSR remains an intact complex. LiaS exhibits phosphatase activity, ensuring LiaR remains unphosphorylated and the regulon remains inactive. LiaF senses cell envelope stress, resulting in conformational changes in LiaS. B Upon cell membrane disruption, for example by bacitracin, the complex dissociates. LiaS no longer dephosphorylates LiaR, allowing LiaR to become phosphorylated, dimerize and bind to DNA to activate transcription of the genes within the LiaR regulon. C In the absence of LiaS (∆liaS), there is constitutive phosphorylation of LiaR, likely due to the metabolite acetyl phosphate present in the cell. The LiaR regulon is highly upregulated, and the overexpression of these genes, specifically heat-shock genes and spxA2, result in increased sensitivity to ciprofloxacin and levofloxacin.

A combination of bacitracin together with ciprofloxacin or levofloxacin increases the potency of these fluoroquinolones.

A Ciprofloxacin (5 mg/ml) disc diffusion assays show an increased zone-of-inhibition of strain D39V with the addition of 0.5 μg/ml bacitracin (BAC). Checkerboard assays with levofloxacin (LEV) and BAC indicate a synergistic interaction (FICI = 0.5) for (B) D39V and the fluoroquinolone-resistant strains (C) FQR_25191 and D FQR_40109 (see Methods).

Levofloxacin and bacitracin act synergistically in vivo in a zebrafish embryo meningitis model.

A Experimental set up. S. pneumoniae was injected into the hindbrain ventricle of zebrafish larvae at 2 days post-fertilization. Zebrafish were treated at 1 hour post injection with: DMSO vehicle controls (group 1), BAC injected into the bloodstream and DMSO vehicle control added to the water (group 2), LEV added into the water and DMSO vehicle control injection (group 3), or a combination of both LEV added to the water and BAC injection (group 4). B Survival curves of zebrafish embryos injected with ~300 CFUs of S. pneumoniae D39V. Zebrafish embryos were treated with DMSO (control), LEV, BAC or a combination of both BAC and LEV. The data represent the mean ± SEM of three biological replicates with 15 larvae per group (n = 45 in total/group); ns = not significant, *p = 0.0064, ****p < 0.0001; determined by Log-rank test. C Survival curves of zebrafish embryos injected with ~300 CFUs of fluoroquinolone-resistant S. pneumoniae strain FQR_25191. Zebrafish embryos were treated with DMSO (control), LEV, BAC or a combination of both BAC and LEV. The data represent the mean ± SEM of three biological replicates with 15 larvae per set (n = 45 in total/group); ns not significant, *p = 0.0437; determined by Log-rank test.