RESUMO
Streptococcus pneumoniae is a major cause of disease and death that develops resistance to multiple antibiotics. DNA topoisomerase I (TopoI) is a novel pneumococcal drug target. TopoI is the sole type-I pneumococcal topoisomerase that regulates supercoiling homeostasis in this bacterium. In this study, a direct in vitro interaction between TopoI and RNA polymerase (RNAP) was detected by surface plasmon resonance. To understand the interplay between transcription and supercoiling regulation in vivo, genome-wide association of RNAP and TopoI was studied by ChIP-Seq. RNAP and TopoI were enriched at the promoters of 435 and 356 genes, respectively. Higher levels of expression were consistently measured in those genes whose promoters recruit both RNAP and TopoI, in contrast with those enriched in only one of them. Both enzymes occupied a narrow region close to the ATG codon. In addition, RNAP displayed a regular distribution throughout the coding regions. Likewise, the summits of peaks called with MACS tool, mapped around the ATG codon in both cases. However, RNAP showed a broader distribution towards ATG-downstream positions. Remarkably, inhibition of RNAP with rifampicin prevented the localization of TopoI at promoters and, vice versa, inhibition of TopoI with seconeolitsine prevented the binding of RNAP to promoters. This indicates a functional interplay between RNAP and TopoI. To determine the molecular factors responsible for RNAP and TopoI co-recruitment, we looked for DNA sequence motifs. We identified a motif corresponding to a -10-extended promoter for TopoI and for RNAP. Furthermore, RNAP was preferentially recruited to genes co-directionally oriented with replication, while TopoI was more abundant in head-on genes. TopoI was located in the intergenic regions of divergent genes pairs, near the promoter of the head-on gene of the pair. These results suggest a role for TopoI in the formation/stability of the RNAP-DNA complex at the promoter and during transcript elongation.
Assuntos
DNA Topoisomerases Tipo I/genética , RNA Polimerases Dirigidas por DNA/genética , Infecções Pneumocócicas/genética , Streptococcus pneumoniae/genética , Transcrição Gênica/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genoma Bacteriano/genética , Motivos de Nucleotídeos/efeitos dos fármacos , Infecções Pneumocócicas/tratamento farmacológico , Infecções Pneumocócicas/microbiologia , Regiões Promotoras Genéticas/genética , Rifampina/farmacologia , Streptococcus pneumoniae/patogenicidade , Ressonância de Plasmônio de SuperfícieRESUMO
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.
Assuntos
DNA Topoisomerases Tipo I , Transcriptoma , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Streptococcus pneumoniae/genética , DNA Girase/genética , DNA Girase/metabolismo , Antibacterianos/farmacologiaRESUMO
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.
Assuntos
DNA Topoisomerases Tipo I , Streptococcus pneumoniae , Humanos , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Novobiocina/farmacologia , DNA Bacteriano/genética , DNA Girase/genética , DNA Girase/metabolismoRESUMO
We studied the transcriptional response to an increase in DNA supercoiling in Streptococcus pneumoniae by using seconeolitsine, a new topoisomerase I inhibitor. A homeostatic response allowing recovery of supercoiling was observed in cells treated with subinhibitory seconeolitsine concentrations. Supercoiling increases of 40.7% (6 µM) and 72.9% (8 µM) were lowered to 8.5% and 44.1%, respectively. Likewise, drug removal facilitated the recovery of cell viability and DNA-supercoiling. Transcription of topoisomerase I depended on the supercoiling level. Also specific binding of topoisomerase I to the gyrase A gene promoter was detected by chromatin-immunoprecipitation. The transcriptomic response to 8 µM seconeolitsine had two stages. An early stage, associated to an increase in supercoiling, affected 10% of the genome. A late stage, manifested by supercoiling recovery, affected 2% of the genome. Nearly 25% of the early responsive genes formed 12 clusters with a coordinated transcription. Clusters were 6.7-31.4 kb in length and included 9-22 responsive genes. These clusters partially overlapped with those observed under DNA relaxation, suggesting that bacteria manage supercoiling stress using pathways with common components. This is the first report of a coordinated global transcriptomic response that is triggered by an increase in DNA supercoiling in bacteria.
Assuntos
DNA Topoisomerases Tipo I/genética , DNA Super-Helicoidal/genética , Homeostase/genética , Família Multigênica , Streptococcus pneumoniae/genética , Benzodioxóis/farmacologia , DNA Girase/genética , DNA Topoisomerases Tipo I/biossíntese , DNA Topoisomerases Tipo I/metabolismo , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/genética , DNA Super-Helicoidal/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos/genética , Homeostase/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/genética , Fenantrenos/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Fluoroquinolonas/farmacologia , Ferro/metabolismo , Levofloxacino/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/metabolismo , Ativação Transcricional/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Testes de Sensibilidade MicrobianaRESUMO
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 (â¼17 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.
Assuntos
Alcaloides , Antibacterianos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Fenantrenos , Streptococcus pneumoniae/enzimologia , Inibidores da Topoisomerase I , Alcaloides/química , Alcaloides/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Linhagem Celular , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Fenantrenos/química , Fenantrenos/farmacologia , Estrutura Terciária de Proteína , Streptococcus pneumoniae/crescimento & desenvolvimento , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/farmacologiaRESUMO
The transcriptional response of Streptococcus pneumoniae was examined after exposure to the GyrB-inhibitor novobiocin. Topoisomer distributions of an internal plasmid confirmed DNA relaxation and recovery of the native level of supercoiling at low novobiocin concentrations. This was due to the up-regulation of DNA gyrase and the down-regulation of topoisomerases I and IV. In addition, >13% of the genome exhibited relaxation-dependent transcription. The majority of the responsive genes (>68%) fell into 15 physical clusters (14.6-85.6 kb) that underwent coordinated regulation, independently of operon organization. These genomic clusters correlated with AT content and codon composition, showing the chromosome to be organized into topology-reacting gene clusters that respond to DNA supercoiling. In particular, down-regulated clusters were flanked by 11-40 kb AT-rich zones that might have a putative structural function. This is the first case where genes responding to changes in the level of supercoiling in a coordinated manner were found organized as functional clusters. Such an organization revealed DNA supercoiling as a general feature that controls gene expression superimposed on other kinds of more specific regulatory mechanisms.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Streptococcus pneumoniae/genética , Códon , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo II/genética , DNA Bacteriano/química , DNA Super-Helicoidal/metabolismo , Inibidores Enzimáticos/farmacologia , Novobiocina/farmacologia , RNA Mensageiro/metabolismo , Streptococcus pneumoniae/enzimologia , Inibidores da Topoisomerase II , Transcrição GênicaRESUMO
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.
RESUMO
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.
Assuntos
DNA Topoisomerases/metabolismo , Streptococcus pneumoniae/enzimologia , Antibacterianos/farmacologia , DNA Girase/genética , DNA Girase/metabolismo , DNA Topoisomerases/genética , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Eletroforese em Gel Bidimensional , Fluoroquinolonas/farmacologia , Streptococcus pneumoniae/efeitos dos fármacosRESUMO
Eight rifampin-resistant streptococci of the mitis group were identified at the species level by using a concatenated 16S rRNA gene-sodA-rpoB-hlpA sequence. Characterization of their rpoB alleles showed single amino acid changes involved in rifampin resistance. Comparison of RpoB sequences from pneumococcal recombinant isolates, viridans isolates, and type strains revealed a species-specific amino acid signature, which allowed it to be ascertained that recombinant RpoBs were originated in genetic interchanges with Streptococcus mitis and Streptococcus oralis.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Rifampina/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Filogenia , RNA Ribossômico 16S/genética , Streptococcus mitis/efeitos dos fármacos , Streptococcus mitis/genética , Streptococcus oralis/efeitos dos fármacos , Streptococcus oralis/genética , Streptococcus pneumoniae/classificaçãoRESUMO
Streptococcus suis is an emerging zoonotic pathogen. With the lack of an effective vaccine, antibiotics remain the main tool to fight infections caused by this pathogen. We have previously observed a reserpine-sensitive fluoroquinolone (FQ) efflux phenotype in this species. Here, SatAB and SmrA, two pumps belonging to the ATP binding cassette (ABC) and the major facilitator superfamily (MFS), respectively, have been analyzed in the fluoroquinolone-resistant clinical isolate BB1013. Genes encoding these pumps were overexpressed either constitutively or in the presence of ciprofloxacin in this strain. These genes could not be cloned in plasmids in Escherichia coli despite strong expression repression. Finally, site-directed insertion of smrA and satAB in the amy locus of the Bacillus subtilis chromosome using ligated PCR amplicons allowed for the functional expression and study of both pumps. Results showed that SatAB is a narrow-spectrum fluoroquinolone exporter (norfloxacin and ciprofloxacin), susceptible to reserpine, whereas SmrA was not involved in fluoroquinolone resistance. Chromosomal integration in Bacillus is a novel method for studying efflux pumps from Gram-positive bacteria, which enabled us to demonstrate the possible role of SatAB, and not SmrA, in fluoroquinolone efflux in S. suis.
Assuntos
Anti-Infecciosos/metabolismo , Farmacorresistência Bacteriana , Fluoroquinolonas/metabolismo , Streptococcus suis/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Animais , Anti-Infecciosos/farmacologia , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Fluoroquinolonas/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Streptococcus suis/metabolismoRESUMO
Streptococcus pneumoniae is the main cause of bacterial pneumonia, a condition that currently produces significant global morbidity and mortality. The initial immune response to this bacterium occurs when the innate system recognizes common motifs expressed by many pathogens, events driven by pattern recognition receptors like the Toll-like family receptors (TLRs). In this study, lung myeloid-cell populations responsible for the innate immune response (IIR) against S. pneumoniae, and their dependence on the TLR4-signaling axis, were analyzed in TLR4-/- and Myeloid-Differentiation factor-88 deficient (MyD88-/-) mice. Neutrophils and monocyte-derived cells were recruited in infected mice 3-days post-infection. Compared to wild-type mice, there was an increased bacterial load in both these deficient mouse strains and an altered IIR, although TLR4-/- mice were more susceptible to bacterial infection. These mice also developed fewer alveolar macrophages, weaker neutrophil infiltration, less Ly6Chigh monocyte differentiation and a disrupted classical and non-classical monocyte profile. The pro-inflammatory cytokine profile (CXCL1, TNF-α, IL-6, and IL-1ß) was also severely affected by the lack of TLR4 and no induction of Th1 was observed in these mice. The respiratory burst (ROS production) after infection was profoundly dampened in TLR4-/- and MyD88-/- mice. These data demonstrate the complex dynamics of myeloid populations and a key role of the TLR4-signaling axis in the IIR to S. pneumoniae, which involves both the MyD88 and TRIF (Toll/IL-1R domain-containing adaptor-inducing IFN-ß) dependent pathways.
Assuntos
Pulmão/imunologia , Monócitos/imunologia , Fator 88 de Diferenciação Mieloide/fisiologia , Mielopoese/fisiologia , Pneumonia Pneumocócica/imunologia , Pneumonia Pneumocócica/patologia , Transdução de Sinais/fisiologia , Streptococcus pneumoniae/imunologia , Receptor 4 Toll-Like/fisiologia , Administração Intranasal , Animais , Carga Bacteriana , Citocinas/biossíntese , Imunidade Inata , Pulmão/patologia , Macrófagos Alveolares/imunologia , Camundongos , Monócitos/patologia , Fator 88 de Diferenciação Mieloide/deficiência , Infiltração de Neutrófilos , Espécies Reativas de Oxigênio/metabolismo , Células Th1/imunologia , Receptor 4 Toll-Like/deficiênciaRESUMO
The histone-like protein HU is a conserved nucleoid-associated protein that is involved in the maintenance of the bacterial chromosome architecture. It is the only known nucleoid-associated protein in Streptococcus pneumoniae, but it has not been studied. The pneumococcal gene encoding this protein, hlp, is shown herein to be essential for cell viability. Its disruption was only possible either when it was duplicated in the chromosome and its expression induced from the P Zn promoter, or when hlp was cloned into a plasmid under the control of the inducible P mal promoter. In vitro assays indicated that pneumococcal HU shows a preference for binding to supercoiled DNA rather than to linear or nicked DNA. In vivo experiments in which the amount of HU was manipulated showed a relationship between the amount of HU and the level of DNA supercoiling. A twofold reduction in the amount of HU triggered a 21% increase in DNA relaxation in untreated cells. However, in cells treated with novobiocin, a drug that relaxes DNA by inhibiting DNA gyrase, a 35% increase in DNA relaxation was observed, instead of the expected 20% in cells with a constitutive HU amount. Conversely, a fourfold HU increase caused only 14% of DNA relaxation in the presence of novobiocin. Taken together, these results support an essential role for HU in the maintenance of DNA supercoiling in S. pneumoniae.
RESUMO
Genetic studies aimed at eliminating expression of the atp operon (F(0)F(1) H(+)-ATPase) of Streptococcus pneumoniae by genetic disruption of atpC, the first gene of the operon, with a chloramphenicol-resistance cassette were performed. Resistant transformants were obtained only when the recipient strain had a duplication of atpC, recombination occurring in such a way that transcription of the operon from its own promoter was allowed. These results imply that the atp operon is essential for the viability of the cells.
Assuntos
Membrana Celular/enzimologia , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Streptococcus pneumoniae/crescimento & desenvolvimento , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Deleção de Genes , Genes Essenciais , ATPases Mitocondriais Próton-Translocadoras/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética , Streptococcus pneumoniae/enzimologia , Transformação BacterianaRESUMO
The most basic level of transcription regulation in Streptococcus pneumoniae is the organization of its chromosome in topological domains. In response to drugs that caused DNA-relaxation, a global transcriptional response was observed. Several chromosomal domains were identified based on the transcriptional response of their genes: up-regulated (U), down-regulated (D), non-regulated (N), and flanking (F). We show that these distinct domains have different expression and conservation characteristics. Microarray fluorescence units under non-relaxation conditions were used as a measure of gene transcriptional level. Fluorescence units were significantly lower in F genes than in the other domains with a similar AT content. The transcriptional level of the domains categorized them was D>U>F. In addition, a comparison of 12 S. pneumoniae genome sequences showed a conservation of gene composition within U and D domains, and an extensive gene interchange in F domains. We tested the organization of chromosomal domains by measuring the relaxation-mediated transcription of eight insertions of a heterologous Ptccat cassette, two in each type of domain, showing that transcription depended on their chromosomal location. Moreover, transcription from the four promoters directing the five genes involved in supercoiling homeostasis, located either in U (gyrB), D (topA), or N (gyrA and parEC) domains was analyzed both in their chromosomal locations and in a replicating plasmid. Although expression from the chromosomal PgyrB and PtopA showed the expected domain regulation, their expression was down-regulated in the plasmid, which behaved as a D domain. However, both PparE and PgyrA carried their own regulatory signals, their topology-dependent expression being equivalent in the plasmid or in the chromosome. In PgyrA a DNA bend acted as a DNA supercoiling sensor. These results revealed that DNA topology functions as a general transcriptional regulator, superimposed upon other more specific regulatory mechanisms.
Assuntos
Proteínas de Bactérias/genética , DNA Super-Helicoidal/genética , Regulação Bacteriana da Expressão Gênica , Streptococcus pneumoniae/genética , DNA Bacteriano/genética , DNA Super-Helicoidal/fisiologia , Regiões Promotoras Genéticas , TranscriptomaRESUMO
Antibiotic resistance in Streptococcus pneumoniae has increased worldwide by the spread of a few clones. Fluoroquinolone resistance occurs mainly by alteration of their intracellular targets, the type II DNA topoisomerases, which is acquired either by point mutation or by recombination. Increase in fluoroquinolone-resistance may depend on the balance between antibiotic consumption and the cost that resistance imposes to bacterial fitness. In addition, pneumococcal prophages could play an important role. Prophage induction by fluoroquinolones was confirmed in 4 clinical isolates by using Southern blot hybridization. Clinical isolates (105 fluoroquinolone-resistant and 160 fluoroquinolone-susceptible) were tested for lysogeny by using a PCR assay and functional prophage carriage was studied by mitomycin C induction. Fluoroquinolone-resistant strains harbored fewer inducible prophages (17/43) than fluoroquinolone-susceptible strains (49/70) (Pâ=â0.0018). In addition, isolates of clones associated with fluoroquinolone resistance [CC156 (3/25); CC63 (2/20), and CC81 (1/19)], had lower frequency of functional prophages than isolates of clones with low incidence of fluoroquinolone resistance [CC30 (4/21), CC230 (5/20), CC62 (9/21), and CC180 (21/30)]. Likewise, persistent strains from patients with chronic respiratory diseases subjected to fluoroquinolone treatment had a low frequency of inducible prophages (1/11). Development of ciprofloxacin resistance was tested with two isogenic strains, one lysogenic and the other non-lysogenic: emergence of resistance was only observed in the non-lysogenic strain. These results are compatible with the lysis of lysogenic isolates receiving fluoroquinolones before the development of resistance and explain the inverse relation between presence of inducible prophages and fluoroquinolone-resistance.
Assuntos
Farmacorresistência Bacteriana/genética , Fluoroquinolonas/farmacologia , Prófagos/fisiologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/virologia , Ativação Viral/efeitos dos fármacos , Southern Blotting , Doença Crônica , Ciprofloxacina/farmacologia , Progressão da Doença , Farmacorresistência Bacteriana/efeitos dos fármacos , Humanos , Lisogenia/efeitos dos fármacos , Mitomicina/farmacologia , Infecções Pneumocócicas/microbiologia , Infecções Pneumocócicas/patologia , Reação em Cadeia da Polimerase , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/isolamento & purificaçãoRESUMO
In enteropathogenic and enterohemorraghic Escherichia coli (EPEC and EHEC), two members of the SlyA family of transcriptional regulators have been identified as SlyA. Western blot analysis of the wild type and the corresponding hosA and slyA deletion mutants indicated that SlyA and HosA are distinct proteins whose expression is not interdependent. Of 27 different E. coli strains (EPEC, EHEC, enteroinvasive, enteroaggregative, uropathogenic, and commensal) examined, 14 were positive for both genes and proteins. To investigate hosA expression, a hosA::luxCDABE reporter gene fusion was constructed. hosA expression was significantly reduced in the hosA but not the slyA mutant and was influenced by temperature, salt, and pH. In contrast to SlyA, HosA did not activate the cryptic E. coli K-12 hemolysin ClyA. Mutation of hosA did not influence type III secretion, the regulation of the LEE1 and LEE4 operons, or the ability of E2348/69 to form attaching-and-effacing lesions on intestinal epithelial cells. HosA is, however, involved in the temperature-dependent positive control of motility on swim plates and regulates fliC expression and FliC protein levels. In electrophoretic mobility shift assays, purified HosA protein bound specifically to the fliC promoter, indicating that HosA directly modulates flagellin expression. While direct examination of flagellar structure and the motile behavior of individual hosA cells grown in broth culture at 30 degrees C did not reveal any obvious differences, hosA mutants, unlike the wild type, clumped together, forming nonmotile aggregates which could account for the markedly reduced motility of the hosA mutant on swim plates at 30 degrees C. We conclude that SlyA and HosA are independent transcriptional regulators that respond to different physicochemical cues to facilitate the environmental adaptation of E. coli.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Aderência Bacteriana , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Humanos , Intestinos/microbiologia , Dados de Sequência Molecular , Movimento , Mutação , Análise de Sequência de DNA , Temperatura , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
A total of 103 (0.7%) of 14,236 Streptococcus pneumoniae isolates collected in four Spanish hospitals from 1989 to 2003 were resistant to rifampin (MICs, 4 to 512 microg/ml). Only sixty-one (59.2%) of these isolates were available for molecular characterization. Resistance was mostly related to human immunodeficiency virus (HIV) infection in adult patients and to conjunctivitis in children. Thirty-six different pulsed-field gel electrophoresis patterns were identified among resistant isolates, five of which were related to international clones (Spain23F-1, Spain6B-2, Spain9V-3, Spain14-5, and clone C of serotype 19F), and accounted for 49.2% of resistant isolates. Single sense mutations at cluster N or I of the rpoB gene were found in 39 isolates, while double mutations, either at cluster I, at clusters I and II, or at clusters N and III, were found in 14 isolates. The involvement of the mutations in rifampin resistance was confirmed by genetic transformation. Single mutations at clusters N and I conferred MICs of 2 microg/ml and 4 to 32 microg/ml, respectively. Eight isolates showed high degrees of nucleotide sequence variations (2.3 to 10.8%) in rpoB, suggesting a recombinational origin for these isolates, for which viridans group streptococci are their potential gene donors. Although the majority of rifampin-resistant isolates were isolated from individual patients without temporal or geographical relationships, the clonal dissemination of rifampin-resistant isolates was observed among 12 HIV-infected patients in the two hospitals with higher rates of resistance.
Assuntos
Antibacterianos/farmacologia , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana/genética , Transferência Genética Horizontal , Mutação , Rifampina/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Sequência de Aminoácidos , Sequência de Bases , RNA Polimerases Dirigidas por DNA/química , Hospitais , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Filogenia , Infecções Pneumocócicas/epidemiologia , Infecções Pneumocócicas/microbiologia , Análise de Sequência de DNA , Espanha/epidemiologia , Streptococcus pneumoniae/genéticaRESUMO
A total of 46 ciprofloxacin-resistant (Cip(r)) Streptococcus pneumoniae strains were isolated from 1991 to 2001 at the Hospital of Bellvitge. Five of these strains showed unexpectedly high rates of nucleotide variations in the quinolone resistance-determining regions (QRDRs) of their parC, parE, and gyrA genes. The nucleotide sequence of the full-length parC, parE, and gyrA genes of one of these isolates revealed a mosaic structure compatible with an interspecific recombination origin. Southern blot analysis and nucleotide sequence determinations showed the presence of an ant-like gene in the intergenic parE-parC regions of the S. pneumoniae Cip(r) isolates with high rates of variations in their parE and parC QRDRs. The ant-like gene was absent from typical S. pneumoniae strains, whereas it was present in the intergenic parE-parC regions of the viridans group streptococci (Streptococcus mitis and Streptococcus oralis). These results suggest that the viridans group streptococci are acting as donors in the horizontal transfer of fluoroquinolone resistance genes to S. pneumoniae.
Assuntos
DNA Topoisomerase IV/genética , Streptococcus pneumoniae/genética , Estreptococos Viridans/genética , Sequência de Bases , DNA Girase/genética , DNA Intergênico , Transferência Genética Horizontal , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Recombinação Genética , Mapeamento por Restrição , Streptococcus pneumoniae/enzimologia , Estreptococos Viridans/enzimologiaRESUMO
Five Spain(9V-3) Streptococcus pneumoniae strains were isolated from a patient with bronchiectasis who had received long-term ciprofloxacin therapy. One ciprofloxacin-susceptible strain was isolated before treatment, and four ciprofloxacin-resistant strains were isolated during treatment. The resistant strains were derived from the susceptible strain either by a parC mutation (low-level resistance) or by parC and gyrA mutations (high-level resistance). This study shows that ciprofloxacin therapy in a patient colonized by susceptible S. pneumoniae may select fluoroquinolone-resistant mutants.