The fluoroquinolone levofloxacin triggers the transcriptional activation of iron transport genes that contribute to cell death in Streptococcus pneumoniae

We studied the transcriptomic response of Streptococcus pneumoniae to levofloxacin (LVX) under conditions inhibiting topoisomerase IV but not gyrase. Although a complex transcriptomic response was observed, the most outstanding result was the upregulation of the genes of the fatDCEB operon, involved in iron (Fe(2+) and Fe(3+)) uptake, which were the only genes varying under every condition tested. Although the inhibition of topoisomerase IV by levofloxacin did not have a detectable effect in the level of global supercoiling, increases in general supercoiling and fatD transcription were observed after topoisomerase I inhibition, while the opposite was observed after gyrase inhibition with novobiocin. Since fatDCEB is located in a topological chromosomal domain downregulated by DNA relaxation, we studied the transcription of a copy of the 422-bp (including the Pfat promoter) region located upstream of fatDCEB fused to the cat reporter inserted into the chromosome 106 kb away from its native position: PfatfatD was upregulated in the presence of LVX in its native location, whereas no change was observed in the Pfatcat construction. Results suggest that topological changes are indeed involved in PfatfatDCE transcription. Upregulation of fatDCEB would lead to an increase of intracellular iron and, in turn, to the activation of the Fenton reaction and the increase of reactive oxygen species. In accordance, we observed an attenuation of levofloxacin lethality in iron-deficient media and in a strain lacking the gene coding for SpxB, the main source of hydrogen peroxide. In addition, we observed an increase of reactive oxygen species that contributed to levofloxacin lethality.This study was supported by grants BIO2011-25343 from Plan Nacional de I+D+i of the Ministerio de Ciencia e Innovación. CIBER de Enfermedades Respiratorias (CIBERES) is an initiative from Instituto de Salud Carlos III.S

Bridging Chromosomal Architecture and Pathophysiology of Streptococcus pneumoniae

The chromosome of Streptococcus pneumoniae is organized into topological domains based on its transcriptional response to DNA relaxation: Up-regulated (UP), down-regulated (DOWN), nonregulated (NR), and AT-rich. In the present work, NR genes found to have highly conserved chromosomal locations (17% of the genome) were categorized as members of position-conserved nonregulated (pcNR) domains, while NR genes with a variable position (36% of the genome) were classified as members of position-variable nonregulated (pvNR) domains. On average, pcNR domains showed high transcription rates, optimized codon usage, and were found to contain only a small number of RUP/BOX/SPLICE repeats. They were also poor in exogenous genes but enriched in leading strand genes that code for proteins involved in primary metabolism with central roles within the interactome. In contrast, pvNR genes coding for cell wall proteins, paralogs, virulence factors and immunogenic candidates for protein-based vaccines were found to be overrepresented. DOWN domains were enriched in genes essential for infection. Many UP and DOWN domain genes were seen to be activated during different stages of competence, whereas pcNR genes tended to be repressed until the competence was switched off. Pneumococcal genes appear to be subject to a topology-driven selection pressure that defines the chromosomal location of genes involved in metabolism, virulence and competence. The pcNR domains are interleaved between UP and DOWN domains according to a pattern that suggests the existence of macrodomain entities. The term "topogenomics" is here proposed to describe the study of the topological rules of genomes and their relationship with physiology.This work was supportedby Ministry of Economy and Competitiveness [BIO2014-55462-R]S

The balance between gyrase and topoisomerase I activities determines levels of supercoiling, nucleoid compaction, and viability in bacteria

Two enzymes are responsible for maintaining supercoiling in the human pathogen Streptococcus pneumoniae, gyrase (GyrA2GyrB2) and topoisomerase I. To attain diverse levels of topoisomerase I (TopoI, encoded by topA), two isogenic strains derived from wild-type strain R6 were constructed: PZn topA, carrying an ectopic topA copy under the control of the ZnSO4-regulated PZn promoter and its derivative ΔtopAPZn topA, which carries a topA deletion at its native chromosomal location. We estimated the number of TopoI and GyrA molecules per cell by using Western-blot and CFUs counting, and correlated these values with supercoiling levels. Supercoiling was estimated in two ways. We used classical 2D-agarose gel electrophoresis of plasmid topoisomers to determine supercoiling density (σ) and we measured compaction of nucleoids using for the first time super-resolution confocal microscopy. Notably, we observed a good correlation between both supercoiling calculations. In R6, with σ = -0.057, the average number of GyrA molecules per cell (2,184) was higher than that of TopoI (1,432), being the GyrA:TopoI proportion of 1:0.65. In ΔtopAPZn topA, the number of TopoI molecules depended, as expected, on ZnSO4 concentration in the culture media, being the proportions of GyrA:TopoI molecules in 75, 150, and 300 μM ZnSO4 of 1:0.43, 1:0.47, and 1:0.63, respectively, which allowed normal supercoiling and growth. However, in the absence of ZnSO4, a higher GyrA:TopoI ratio (1:0.09) caused hyper-supercoiling (σ = -0.086) and lethality. Likewise, growth of ΔtopAPZn topA in the absence of ZnSO4 was restored when gyrase was inhibited with novobiocin, coincidentally with the resolution of hyper-supercoiling (σ change from -0.080 to -0.068). Given that TopoI is a monomer and two molecules of GyrA are present in the gyrase heterotetramer, the gyrase:TopoI enzymes proportion would be 1:1.30 (wild type R6) or of 1:1.26-0.86 (ΔtopAPZn topA under viable conditions). Higher proportions, such as 1:0.18 observed in ΔtopAPZn topA in the absence of ZnSO4 yielded to hyper-supercoiling and lethality. These results support a role of the equilibrium between gyrase and TopoI activities in supercoiling maintenance, nucleoid compaction, and viability. Our results shed new light on the mechanism of action of topoisomerase-targeting antibiotics, paving the way for the use of combination therapies.This work was supported by project PID2021-124738OB-100 to AGC, financed by MCIN/AEI/10.13039/501100011033/FEDER, UE.S

Nonoptimal DNA topoisomerases allow maintenance of supercoiling levels and improve fitness of Streptococcus pneumoniae

Fluoroquinolones, which target gyrase and topoisomerase IV, are used for treating Streptococcus pneumoniae infections. Fluoroquinolone resistance in this bacterium can arise via point mutation or interspecific recombination with genetically related streptococci. Our previous study on the fitness cost of resistance mutations and recombinant topoisomerases identified GyrAE85K as a high-cost change. However, this cost was compensated for by the presence of a recombinant topoisomerase IV (parC and parE recombinant genes) in strain T14. In this study, we purified wild-type and mutant topoisomerases and compared their enzymatic activities. In strain T14, both gyrase carrying GyrAE85K and recombinant topoisomerase IV showed lower activities (from 2.0- to 3.7-fold) than the wild-type enzymes. These variations of in vitro activity corresponded to changes of in vivo supercoiling levels that were analyzed by two-dimensional electrophoresis of an internal plasmid. Strains carrying GyrAE85K and nonrecombinant topoisomerases had lower (11.1% to 14.3%) supercoiling density (σ) values than the wild type. Those carrying GyrAE85K and recombinant topoisomerases showed either partial or total supercoiling level restoration, with σ values being 7.9% (recombinant ParC) and 1.6% (recombinant ParC and recombinant ParE) lower than those for the wild type. These data suggested that changes acquired by interspecific recombination might be selected because they reduce the fitness cost associated with fluoroquinolone resistance mutations. An increase in the incidence of fluoroquinolone resistance, even in the absence of further antibiotic exposure, is envisaged.This study was supported by grants BIO2008-02154 from Plan Nacional de I+D+I of the Ministerio de Ciencia e Innovación and COMBACT-S-BIO-0260/2006 from the Comunidad de Madrid. Ciber Enfermedades Respiratorias is an initiative from the Instituto de Salud Carlos III. A. G. de la Campa is an Investigador Científico from the CSI

Physiologic and Transcriptomic Effects Triggered by Overexpression of Wild Type and Mutant DNA Topoisomerase I in Streptococcus pneumoniae

Topoisomerase I (TopoI) in Streptococcus pneumoniae, encoded by topA, is a suitable target for drug development. Seconeolitsine (SCN) is a new antibiotic that specifically blocks this enzyme. We obtained the topARA mutant, which encodes an enzyme less active than the wild type (topAWT) and more resistant to SCN inhibition. Likely due to the essentiality of TopoI, we were unable to replace the topAWT allele by the mutant topARA version. We compared the in vivo activity of TopoIRA and TopoIWT using regulated overexpression strains, whose genes were either under the control of a moderately (PZn) or a highly active promoter (PMal). Overproduction of TopoIRA impaired growth, increased SCN resistance and, in the presence of the gyrase inhibitor novobiocin (NOV), caused lower relaxation than TopoIWT. Differential transcriptomes were observed when the topAWT and topARA expression levels were increased about 5-fold. However, higher increases (10-15 times), produced a similar transcriptome, affecting about 52% of the genome, and correlating with a high DNA relaxation level with most responsive genes locating in topological domains. These results confirmed that TopoI is indeed the target of SCN in S. pneumoniae and show the important role of TopoI in global transcription, supporting its suitability as an antibiotic target.This research and the APC were funded by project PID2021-124738OB-100 to AGC, financed by MCIN/AEI/10.13039/501100011033/FEDER, UE. M.G.-L. is the beholder of a PhD Contract from Instituto de Salud Carlos III.S

Upregulation of the PatAB Transporter Confers Fluoroquinolone Resistance to Streptococcus pseudopneumoniae

We characterized the mechanism of fluoroquinolone-resistance in two isolates of Streptococcus pseudopneumoniae having fluoroquinolone-efflux as unique mechanism of resistance. Whole genome sequencing and genetic transformation experiments were performed together with phenotypic determinations of the efflux mechanism. The PatAB pump was identified as responsible for efflux of ciprofloxacin (MIC of 4 μg/ml), ethidium bromide (MICs of 8-16 μg/ml) and acriflavine (MICs of 4-8 μg/ml) in both isolates. These MICs were at least 8-fold lower in the presence of the efflux inhibitor reserpine. Complete genome sequencing indicated that the sequence located between the promoter of the patAB operon and the initiation codon of patA, which putatively forms an RNA stem-loop structure, may be responsible for the efflux phenotype. RT-qPCR determinations performed on RNAs of cultures treated or not treated with subinhibitory ciprofloxacin concentrations were performed. While no significant changes were observed in wild-type Streptococcus pneumoniae R6 strain, increases in transcription were detected in the ciprofloxacin-efflux transformants obtained with DNA from efflux-positive isolates, in the ranges of 1.4 to 3.4-fold (patA) and 2.1 to 2.9-fold (patB). Ciprofloxacin-induction was related with a lower predicted free energy for the stem-loop structure in the RNA of S. pseudopneumoniae isolates (-13.81 and -8.58) than for R6 (-15.32 kcal/mol), which may ease transcription. The presence of these regulatory variations in commensal S. pseudopneumoniae isolates, and the possibility of its transfer to Streptococcus pneumoniae by genetic transformation, could increase fluoroquinolone resistance in this important pathogen.This study was supported by grant BIO2014-55462-R from Plan Nacional de I+D+I of the Ministry of Economy and Competitiveness. AM is the recipient of a Miguel Servet contract from the Spanish Ministry of Economy and Competitiveness.S

Reactive Oxygen Species Contribute to the Bactericidal Effects of the Fluoroquinolone Moxifloxacin in Streptococcus pneumoniae

We studied the transcriptomic response of Streptococcus pneumoniae to the fluoroquinolone moxifloxacin at a concentration that inhibits DNA gyrase. Treatment of the wild-type strain R6, at a concentration of 10× the MIC, triggered a response involving 132 genes after 30 min of treatment. Genes from several metabolic pathways involved in the production of pyruvate were upregulated. These included 3 glycolytic enzymes, which ultimately convert fructose 6-phosphate to pyruvate, and 2 enzymes that funnel phosphate sugars into the glycolytic pathway. In addition, acetyl coenzyme A (acetyl-CoA) carboxylase was downregulated, likely leading to an increase in acetyl-CoA. When coupled with an upregulation in formate acetyltransferase, an increase in acetyl-CoA would raise the production of pyruvate. Since pyruvate is converted by pyruvate oxidase (SpxB) into hydrogen peroxide (H2O2), an increase in pyruvate would augment intracellular H2O2. Here, we confirm a 21-fold increase in the production of H2O2 and a 55-fold increase in the amount of hydroxyl radical in cultures treated during 4 h with moxifloxacin. This increase in hydroxyl radical through the Fenton reaction would damage DNA, lipids, and proteins. These reactive oxygen species contributed to the lethality of the drug, a conclusion supported by the observed protective effects of an SpxB deletion. These results support the model whereby fluoroquinolones cause redox alterations. The transcriptional response of S. pneumoniae to moxifloxacin is compared with the response to levofloxacin, an inhibitor of topoisomerase IV. Levofloxacin triggers the transcriptional activation of iron transport genes and also enhances the Fenton reaction.This study was supported by grants BIO2011-25343 and BIO2014-55462-R from Plan Nacional de I+D+I of the Ministry of Economy and Competitiveness. A.J.M.-G. is the recipient of a Miguel Servet contract from the Spanish Ministry of Economy and Competitiveness.S

New alkaloid antibiotics that target the DNA topoisomerase I of Streptococcus pneumoniae

Streptococcus pneumoniae has two type II DNA-topoisomerases (DNA-gyrase and DNA topoisomerase IV) and a single type I enzyme (DNA-topoisomerase I, TopA), as demonstrated here. Although fluoroquinolones target type II enzymes, antibiotics efficiently targeting TopA have not yet been reported. Eighteen alkaloids (seven aporphine and 11 phenanthrenes) were semisynthesized from boldine and used to test inhibition both of TopA activity and of cell growth. Two phenanthrenes (seconeolitsine and N-methyl-seconeolitsine) effectively inhibited both TopA activity and cell growth at equivalent concentrations (∼17 μM). Evidence for in vivo TopA targeting by seconeolitsine was provided by the protection of growth inhibition in a S. pneumoniae culture in which the enzyme was overproduced. Additionally, hypernegative supercoiling was observed in an internal plasmid after drug treatment. Furthermore, a model of pneumococcal TopA was made based on the crystal structure of Escherichia coli TopA. Docking calculations indicated strong interactions of the alkaloids with the nucleotide-binding site in the closed protein conformation, which correlated with their inhibitory effect. Finally, although seconeolitsine and N-methyl-seconeolitsine inhibited TopA and bacterial growth, they did not affect human cell viability. Therefore, these new alkaloids can be envisaged as new therapeutic candidates for the treatment of S. pneumoniae infections resistant to other antibiotics.The work was supported by Comunidad de Madrid Grant CM-BIO0260-2006, COMBACT (to J. H. and A. G. C.); Spanish Ministry of Science and Innovation Grants BIO2008-02154 (to A. G. C.), BFU2008-01711 (to J. A. H.), and SAF2008-03477 (to M. J. S.); and Spanish Ministry of Health, Carlos III Health Institute Grants RIER, RD08/0075/0016 (to M. J. S.).S

StaR Is a Positive Regulator of Topoisomerase I Activity Involved in Supercoiling Maintenance in Streptococcus pneumoniae

The DNA topoisomerases gyrase and topoisomerase I as well as the nucleoid-associated protein HU maintain supercoiling levels in Streptococcus pneumoniae, a main human pathogen. Here, we characterized, for the first time, a topoisomerase I regulator protein (StaR). In the presence of sub-inhibitory novobiocin concentrations, which inhibit gyrase activity, higher doubling times were observed in a strain lacking staR, and in two strains in which StaR was over-expressed either under the control of the ZnSO4-inducible PZn promoter (strain ΔstaRPZnstaR) or of the maltose-inducible PMal promoter (strain ΔstaRpLS1ROMstaR). These results suggest that StaR has a direct role in novobiocin susceptibility and that the StaR level needs to be maintained within a narrow range. Treatment of ΔstaRPZnstaR with inhibitory novobiocin concentrations resulted in a change of the negative DNA supercoiling density (σ) in vivo, which was higher in the absence of StaR (σ = -0.049) than when StaR was overproduced (σ = -0.045). We have located this protein in the nucleoid by using super-resolution confocal microscopy. Through in vitro activity assays, we demonstrated that StaR stimulates TopoI relaxation activity, while it has no effect on gyrase activity. Interaction between TopoI and StaR was detected both in vitro and in vivo by co-immunoprecipitation. No alteration of the transcriptome was associated with StaR amount variation. The results suggest that StaR is a new streptococcal nucleoid-associated protein that activates topoisomerase I activity by direct protein-protein interaction.This research and the APC were funded by project PID2021-124738OB-100 to A.G.d.l.C., financed by MCIN/AEI/10.13039/501100011033/FEDER, UE.S

Reactive Oxygen Species Production Is a Major Factor Directing the Postantibiotic Effect of Fluoroquinolones in Streptococcus pneumoniae.

We studied the molecular mechanisms involved in the postantibiotic effect of the fluoroquinolones levofloxacin and moxifloxacin in Streptococcus pneumoniae Wild-type strain R6 had postantibiotic effects of 2.05 ± 0.10 h (mean ± standard deviation [SD]) and 3.23 ± 0.45 h at 2.5× and 10× MIC of levofloxacin, respectively. Moxifloxacin exhibited lower effects of 0.87 ± 0.1 and 2.41 ± 0.29 h at 2.5× and 10× MIC, respectively. Fluoroquinolone-induced chromosome fragmentation was measured at equivalent postantibiotic effects for levofloxacin (2.5× MIC) and moxifloxacin (10× MIC). After 2 h of drug removal, reductions were approximately 7-fold for levofloxacin and 3-fold for moxifloxacin, without further decreases at later times. Variations in reactive oxygen species production were detected after 4 to 6 h of drug withdrawals, with decreases ≥400-fold for levofloxacin and ≥800-fold for moxifloxacin at 6 h. In accordance, after 4 to 6 h of drug withdrawal, the levofloxacin-induced upregulation of the fatCDEB operon, introducing iron in the bacteria, decreased up to 2- to 3-fold, and the moxifloxacin-induced upregulation of several genes involved in the production of pyruvate was reduced 3- to 7-fold. In accordance, lower postantibiotic effects (up to 1 h) were observed in strain R6 ΔspxB, lacking the main enzyme involved in oxygen peroxide production, than in R6. Although no change in the recovery of chromosome fragmentation was observed between R6 and R6 ΔspxB, 3.5 × 103-fold lower reactive oxygen species production was observed in R6 ΔspxB, without changes after drug removal. These results show that reactive oxygen species are the main factors directing the postantibiotic effect of levofloxacin and moxifloxacin in S. pneumoniae.This study was supported by grant BIO2017-82951-R from Plan Nacional de I+D+I of the Ministry of Economy and Competitiveness.S