scholarly journals Interactions of the Streptococcus pneumoniae Toxin-Antitoxin RelBE Proteins with Their Target DNA

2021 ◽  
Vol 9 (4) ◽  
pp. 851
Author(s):  
Inmaculada Moreno-Córdoba ◽  
Wai-Ting Chan ◽  
Concha Nieto ◽  
Manuel Espinosa
Keyword(s):  
Streptococcus Pneumoniae ◽  
Self Regulation ◽  
Mrna Translation ◽  
Dna Supercoiling ◽  
Bacterial Toxin ◽  
Type Ii ◽  
Cellular Functions ◽  
Dna Backbone ◽  
Different Strains ◽  
Palindromic Sequences

Type II bacterial toxin-antitoxin (TA) systems are found in most bacteria, archaea, and mobile genetic elements. TAs are usually found as a bi-cistronic operon composed of an unstable antitoxin and a stable toxin that targets crucial cellular functions like DNA supercoiling, cell-wall synthesis or mRNA translation. The type II RelBE system encoded by the pathogen Streptococcus pneumoniae is highly conserved among different strains and participates in biofilm formation and response to oxidative stress. Here, we have analyzed the participation of the RelB antitoxin and the RelB:RelE protein complex in the self-regulation of the pneumococcal relBE operon. RelB acted as a weak repressor, whereas RelE performed the role of a co-repressor. By DNA footprinting experiments, we show that the proteins bind to a region that encompasses two palindromic sequences that are located around the −10 sequences of the single promoter that directs the synthesis of the relBE mRNA. High-resolution footprinting assays showed the distribution of bases whose deoxyriboses are protected by the bound proteins, demonstrating that RelB and RelB:RelE contacted the DNA backbone on one face of the DNA helix and that these interactions extended beyond the palindromic sequences. Our findings suggest that the binding of the RelBE proteins to its DNA target would lead to direct inhibition of the binding of the host RNA polymerase to the relBE promoter.

scholarly journals Cleavable-Complex Formation by Wild-Type and Quinolone-Resistant Streptococcus pneumoniae Type II Topoisomerases Mediated by Gemifloxacin and Other Fluoroquinolones

2002 ◽  
Vol 46 (2) ◽  
pp. 413-419 ◽  
Author(s):  
Genoveva Yague ◽  
Julia E. Morris ◽  
Xiao-Su Pan ◽  
Katherine A. Gould ◽  
L. Mark Fisher
Keyword(s):  
Streptococcus Pneumoniae ◽  
Complex Formation ◽  
Dna Cleavage ◽  
Dna Supercoiling ◽  
Type Ii ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Drug Induced ◽  
Drug Concentrations ◽  
Cleavable Complex

ABSTRACT Gemifloxacin is a recently developed fluoroquinolone with potent activity against Streptococcus pneumoniae. We show that the drug is more active than moxifloxacin, gatifloxacin, levofloxacin, and ciprofloxacin against S. pneumoniae strain 7785 (MICs, 0.03 to 0.06 μg/ml versus 0.25, 0.25, 1, and 1 to 2 μg/ml, respectively) and against isogenic quinolone-resistant gyrA-parC mutants (MICs, 0.5 to 1 μg/ml versus 2 to 4, 2 to 4, 16 to 32, and 64 μg/ml, respectively). Gemifloxacin was also the most potent agent against purified S. pneumoniae DNA gyrase and topoisomerase IV in both catalytic inhibition and DNA cleavage assays. The drug concentrations that inhibited DNA supercoiling or DNA decatenation by 50% (IC50s) were 5 to 10 and 2.5 to 5.0 μM, respectively. Ciprofloxacin and levofloxacin were some four- to eightfold less active against either enzyme; moxifloxacin and gatifloxacin showed intermediate activities. In assays of drug-mediated DNA cleavage by gyrase and topoisomerase IV, the same order of potency was seen: gemifloxacin > moxifloxacin > gatifloxacin > levofloxacin ≈ ciprofloxacin. For gemifloxacin, the drug concentrations that caused 25% linearization of the input DNA by gyrase and topoisomerase IV were 2.5 and 0.1 to 0.3 μM, respectively; these values were 4-fold and 8- to 25-fold lower than those for moxifloxacin, respectively. Each drug induced DNA cleavage by gyrase at the same spectrum of sites but with different patterns of intensity. Finally, for enzymes reconstituted with quinolone-resistant GyrA S81F or ParC S79F subunits, although cleavable-complex formation was reduced by at least 8- to 16-fold for all the quinolones tested, gemifloxacin was the most effective; e.g., it was 4- to 16-fold more active than the other drugs against toposiomerase IV with the ParC S79F mutation. It appears that the greater potency of gemifloxacin against both wild-type and quinolone-resistant S. pneumoniae strains arises from enhanced stabilization of gyrase and topoisomerase IV complexes on DNA.

scholarly journals Differential behaviors of Staphylococcus aureus and Escherichia coli type II DNA topoisomerases.

1996 ◽  
Vol 40 (12) ◽  
pp. 2714-2720 ◽  
Author(s):  
F Blanche ◽  
B Cameron ◽  
F X Bernard ◽  
L Maton ◽  
B Manse ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Strong Support ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Type Ii ◽  
Topoisomerase Iv ◽  
E Coli ◽  
Dna Relaxation

Staphylococcus aureus gyrA and gyrB genes encoding DNA gyrase subunits were cloned and coexpressed in Escherichia coli under the control of the T7 promoter-T7 RNA polymerase system, leading to soluble gyrase which was purified to homogeneity. Purified gyrase was catalytically indistinguishable from the gyrase purified from S. aureus and did not contain detectable amounts of topoisomerases from the E. coli host. Topoisomerase IV subunits GrlA and GrlB from S. aureus were also expressed in E. coli and were separately purified to apparent homogeneity. Topoisomerase IV, which was reconstituted by mixing equimolar amounts of GrlA and GrlB, had both ATP-dependent decatenation and DNA relaxation activities in vitro. This enzyme was more sensitive than gyrase to inhibition by typical fluoroquinolone antimicrobial agents such as ciprofloxacin or sparfloxacin, adding strong support to genetic studies which indicate that topoisomerase IV is the primary target of fluoroquinolones in S. aureus. The results obtained with ofloxacin suggest that this fluoroquinolone could also primarily target gyrase. No cleavable complex could be detected with S. aureus gyrase upon incubation with ciprofloxacin or sparfloxacin at concentrations which fully inhibit DNA supercoiling. This suggests that these drugs do not stabilize the open DNA-gyrase complex, at least under standard in vitro incubation conditions, but are more likely to interfere primarily with the DNA breakage step, contrary to what has been reported with E. coli gyrase. Both S. aureus gyrase-catalyzed DNA supercoiling and S. aureus topoisomerase IV-catalyzed decatenation were dramatically stimulated by potassium glutamate or aspartate (500- and 50-fold by 700 and 350 mM glutamate, respectively), whereas topoisomerase IV-dependent DNA relaxation was inhibited 3-fold by 350 mM glutamate. The relevance of the effect of dicarboxylic amino acids on the activities of type II topoisomerases is discussed with regard to the intracellular osmolite composition of S. aureus.

scholarly journals Streptococcus pneumoniae DNA Gyrase and Topoisomerase IV: Overexpression, Purification, and Differential Inhibition by Fluoroquinolones

1999 ◽  
Vol 43 (5) ◽  
pp. 1129-1136 ◽  
Author(s):  
Xiao-Su Pan ◽  
L. Mark Fisher
Keyword(s):  
Streptococcus Pneumoniae ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Topoisomerase Iv ◽  
T7 Promoter ◽  
Bacterial Killing ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Comparable Activity

ABSTRACT Streptococcus pneumoniae gyrA and gyrBgenes specifying the DNA gyrase subunits have been cloned into pET plasmid vectors under the control of an inducible T7 promoter and have been separately expressed in Escherichia coli. Soluble 97-kDa GyrA and 72-kDa GyrB proteins bearing polyhistidine tags at their respective C-terminal and N-terminal ends were purified to apparent homogeneity by one-step nickel chelate column chromatography and were free of host E. coli topoisomerase activity. Equimolar amounts of the gyrase subunits reconstituted ATP-dependent DNA supercoiling with comparable activity to gyrase of E. coli and Staphylococcus aureus. In parallel, S. pneumoniae topoisomerase IV ParC and ParE subunits were similarly expressed in E. coli, purified to near homogeneity as 93- and 73-kDa proteins, and shown to generate efficient ATP-dependent DNA relaxation and DNA decatenation activities. Using the purified enzymes, we examined the inhibitory effects of three paradigm fluoroquinolones—ciprofloxacin, sparfloxacin, and clinafloxacin—which previous genetic studies with S. pneumoniae suggested act preferentially through topoisomerase IV, through gyrase, and through both enzymes, respectively. Surprisingly, all three quinolones were more active in inhibiting purified topoisomerase IV than gyrase, with clinafloxacin showing the greatest inhibitory potency. Moreover, the tested agents were at least 25-fold more effective in stabilizing a cleavable complex (the relevant cytotoxic lesion) with topoisomerase IV than with gyrase, with clinafloxacin some 10- to 32-fold more potent against either enzyme, in line with its superior activity againstS. pneumoniae. The uniform target preference of the three fluoroquinolones for topoisomerase IV in vitro is in apparent contrast to the genetic data. We interpret these results in terms of a model for bacterial killing by quinolones in which cellular factors can modulate the effects of target affinity to determine the cytotoxic pathway.

scholarly journals Tumor-Progressive Mechanisms Mediating miRNA–Protein Interaction

2021 ◽  
Vol 22 (22) ◽  
pp. 12303
Author(s):  
Hiroaki Konishi ◽  
Hiroki Sato ◽  
Kenji Takahashi ◽  
Mikihiro Fujiya
Keyword(s):  
Cancer Progression ◽  
Regulatory System ◽  
Mrna Translation ◽  
Cellular Protein ◽  
Clinical Applications ◽  
Cancer Prognosis ◽  
Cellular Functions ◽  
Previous Decade ◽  
Extracellular Mirnas ◽  
Development And Differentiation

MicroRNAs (miRNAs) are single-stranded short-chain RNAs that are endogenously expressed in vertebrates; they are considered the fine-tuners of cellular protein expression that act by modifying mRNA translation. miRNAs control tissue development and differentiation, cell growth, and apoptosis in cancer and non-cancer cells. Aberrant regulation of miRNAs is involved in the pathogenesis of various diseases including cancer. Numerous investigations have shown that the changes in cellular miRNA expression in cancerous tissues and extracellular miRNAs enclosed in exosomes are correlated with cancer prognosis. Therefore, miRNAs can be used as cancer biomarkers and therapeutic targets for cancer in clinical applications. In the previous decade, miRNAs have been shown to regulate cellular functions by directly binding to proteins and mRNAs, thereby controlling cancer progression. This regulatory system implies that cancer-associated miRNAs can be applied as molecular-targeted therapy. This review discusses the roles of miRNA–protein systems in cancer progression and its future applications in cancer treatment.

scholarly journals Type I interferon promotes alveolar epithelial type II cell survival during pulmonary Streptococcus pneumoniae infection and sterile lung injury in mice

2016 ◽  
Vol 46 (9) ◽  
pp. 2175-2186 ◽  
Author(s):  
Barbara B. Maier ◽  
Anastasiya Hladik ◽  
Karin Lakovits ◽  
Ana Korosec ◽  
Rui Martins ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Lung Injury ◽  
Cell Survival ◽  
Type I Interferon ◽  
Type I ◽  
Type Ii ◽  
Alveolar Epithelial ◽  
Type Ii Cell

scholarly journals Toxin-Antitoxin Genes of the Gram-Positive Pathogen Streptococcus pneumoniae: So Few and Yet So Many

2012 ◽  
Vol 76 (4) ◽  
pp. 773-791 ◽  
Author(s):  
Wai Ting Chan ◽  
Inma Moreno-Córdoba ◽  
Chew Chieng Yeo ◽  
Manuel Espinosa
Keyword(s):  
Streptococcus Pneumoniae ◽  
Biofilm Formation ◽  
Bacterial Infections ◽  
Host Cells ◽  
Molecular Characteristics ◽  
Type Ii ◽  
Pneumococcal Infections ◽  
Interesting Phenomenon ◽  
Content Type ◽  
Toxin Protein

SUMMARYPneumococcal infections cause up to 2 million deaths annually and raise a large economic burden and thus constitute an important threat to mankind. Because of the increase in the antibiotic resistance ofStreptococcus pneumoniaeclinical isolates, there is an urgent need to find new antimicrobial approaches to triumph over pneumococcal infections. Toxin-antitoxin (TA) systems (TAS), which are present in most living bacteria but not in eukaryotes, have been proposed as an effective strategy to combat bacterial infections. Type II TAS comprise a stable toxin and a labile antitoxin that form an innocuous TA complex under normal conditions. Under stress conditions, TA synthesis will be triggered, resulting in the degradation of the labile antitoxin and the release of the toxin protein, which would poison the host cells. The three functional chromosomal TAS fromS. pneumoniaethat have been studied as well as their molecular characteristics are discussed in detail in this review. Furthermore, a meticulous bioinformatics search has been performed for 48 pneumococcal genomes that are found in public databases, and more putative TAS, homologous to well-characterized ones, have been revealed. Strikingly, several unusual putative TAS, in terms of components and genetic organizations previously not envisaged, have been discovered and are further discussed. Previously, we reported a novel finding in which a unique pneumococcal DNA signature, the BOX element, affected the regulation of the pneumococcalyefM-yoeBTAS. This BOX element has also been found in some of the other pneumococcal TAS. In this review, we also discuss possible relationships between some of the pneumococcal TAS with pathogenicity, competence, biofilm formation, persistence, and an interesting phenomenon called bistability.

scholarly journals Exchangeable Chaperone Modules Contribute to Specification of Type I and Type II Hsp40 Cellular Function

2004 ◽  
Vol 15 (2) ◽  
pp. 761-773 ◽  
Author(s):  
Chun-Yang Fan ◽  
Soojin Lee ◽  
Hong-Yu Ren ◽  
Douglas M. Cyr
Keyword(s):  
In Vivo Studies ◽  
Cell Physiology ◽  
Type I ◽  
Type Ii ◽  
Cellular Functions ◽  
Chaperone Function ◽  
Sequence Identity ◽  
Carboxyl Terminal ◽  
High Degree

Hsp40 family members regulate Hsp70s ability to bind nonnative polypeptides and thereby play an essential role in cell physiology. Type I and type II Hsp40s, such as yeast Ydj1 and Sis1, form chaperone pairs with cytosolic Hsp70 Ssa1 that fold proteins with different efficiencies and carry out specific cellular functions. The mechanism by which Ydj1 and Sis1 specify Hsp70 functions is not clear. Ydj1 and Sis1 share a high degree of sequence identity in their amino and carboxyl terminal ends, but each contains a structurally unique and centrally located protein module that is implicated in chaperone function. To test whether the chaperone modules of Ydj1 and Sis1 function in the specification of Hsp70 action, we constructed a set of chimeric Hsp40s in which the chaperone domains of Ydj1 and Sis1 were swapped to form YSY and SYS. Purified SYS and YSY exhibited protein-folding activity and substrate specificity that mimicked that of Ydj1 and Sis1, respectively. In in vivo studies, YSY exhibited a gain of function and, unlike Ydj1, could complement the lethal phenotype of sis1Δ and facilitate maintenance of the prion [RNQ+]. Ydj1 and Sis1 contain exchangeable chaperone modules that assist in specification of Hsp70 function.

scholarly journals Engineering the Specificity of Antibacterial Fluoroquinolones: Benzenesulfonamide Modifications at C-7 of Ciprofloxacin Change Its Primary Target in Streptococcus pneumoniae from Topoisomerase IV to Gyrase

2000 ◽  
Vol 44 (2) ◽  
pp. 320-325 ◽  
Author(s):  
Fabiana L. Alovero ◽  
Xiao-Su Pan ◽  
Julia E. Morris ◽  
Ruben H. Manzo ◽  
L. Mark Fisher
Keyword(s):  
Streptococcus Pneumoniae ◽  
Resistance Mutation ◽  
Dna Supercoiling ◽  
Quinolone Resistance ◽  
Primary Target ◽  
Topoisomerase Iv ◽  
Enhanced Activity

ABSTRACT We have examined the antipneumococcal mechanisms of a series of novel fluoroquinolones that are identical to ciprofloxacin except for the addition of a benzenesulfonylamido group to the C-7 piperazinyl ring. A number of these derivatives displayed enhanced activity againstStreptococcus pneumoniae strain 7785, including compound NSFQ-105, bearing a 4-(4-aminophenylsulfonyl)-1-piperazinyl group at C-7, which exhibited an MIC of 0.06 to 0.125 μg/ml compared with a ciprofloxacin MIC of 1 μg/ml. Several complementary approaches established that unlike the case for ciprofloxacin (which targets topoisomerase IV), the increased potency of NSFQ-105 was associated with a target preference for gyrase: (i) parC mutants of strain 7785 that were resistant to ciprofloxacin remained susceptible to NSFQ-105, whereas by contrast, mutants bearing a quinolone resistance mutation in gyrA were four- to eightfold more resistant to NSFQ-105 (MIC of 0.5 μg/ml) but susceptible to ciprofloxacin; (ii) NSFQ-105 selected first-step gyrAmutants (MICs of 0.5 μg/ml) encoding Ser-81-to-Phe or -Tyr mutations, whereas ciprofloxacin selects parC mutants; and (iii) NSFQ-105 was at least eightfold more effective than ciprofloxacin at inhibiting DNA supercoiling by S. pneumoniae gyrase in vitro but was fourfold less active against topoisomerase IV. These data show unequivocally that the C-7 substituent determines not only the potency but also the target preference of fluoroquinolones. The importance of the C-7 substituent in drug-enzyme contacts demonstrated here supports one key postulate of the Shen model of quinolone action.

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