Bacterial DNA-dependent RNA Polymerase Research Articles

Transcription activation in Escherichia coli and Salmonella.

Promoter-specific activation of transcript initiation provides an important regulatory device in Escherichia coli and Salmonella. Here, we describe the different mechanisms that operate, focusing on how they have evolved to manage the "housekeeping" bacterial transcription machinery. Some mechanisms involve assisting the bacterial DNA-dependent RNA polymerase or replacing or remodeling one of its subunits. Others are directed to chromosomal DNA, improving promoter function, or relieving repression. We discuss how different activators work together at promoters and how the present complex network of transcription factors evolved.

Structural basis of the mycobacterial stress-response RNA polymerase auto-inhibition via oligomerization

Self-assembly of macromolecules into higher-order symmetric structures is fundamental for the regulation of biological processes. Higher-order symmetric structure self-assembly by the gene expression machinery, such as bacterial DNA-dependent RNA polymerase (RNAP), has never been reported before. Here, we show that the stress-response σB factor from the human pathogen, Mycobacterium tuberculosis, induces the RNAP holoenzyme oligomerization into a supramolecular complex composed of eight RNAP units. Cryo-electron microscopy revealed a pseudo-symmetric structure of the RNAP octamer in which RNAP protomers are captured in an auto-inhibited state and display an open-clamp conformation. The structure shows that σB is sequestered by the RNAP flap and clamp domains. The transcriptional activator RbpA prevented octamer formation by promoting the initiation-competent RNAP conformation. Our results reveal that a non-conserved region of σ is an allosteric controller of transcription initiation and demonstrate how basal transcription factors can regulate gene expression by modulating the RNAP holoenzyme assembly and hibernation.

M. tuberculosis Transcription Machinery: A Review on the Mycobacterial RNA Polymerase and Drug Discovery Efforts.

Mycobacterium tuberculosis (MTB) is the main source of tuberculosis (TB), one of the oldest known diseases in the human population. Despite the drug discovery efforts of past decades, TB is still one of the leading causes of mortality and claimed more than 1.5 million lives worldwide in 2020. Due to the emergence of drug-resistant strains and patient non-compliance during treatments, there is a pressing need to find alternative therapeutic agents for TB. One of the important areas for developing new treatments is in the inhibition of the transcription step of gene expression; it is the first step to synthesize a copy of the genetic material in the form of mRNA. This further translates to functional protein synthesis, which is crucial for the bacteria living processes. MTB contains a bacterial DNA-dependent RNA polymerase (RNAP), which is the key enzyme for the transcription process. MTB RNAP has been targeted for designing and developing antitubercular agents because gene transcription is essential for the mycobacteria survival. Initiation, elongation, and termination are the three important sequential steps in the transcription process. Each step is complex and highly regulated, involving multiple transcription factors. This review is focused on the MTB transcription machinery, especially in the nature of MTB RNAP as the main enzyme that is regulated by transcription factors. The mechanism and conformational dynamics that occur during transcription are discussed and summarized. Finally, the current progress on MTB transcription inhibition and possible drug target in mycobacterial RNAP are also described to provide insight for future antitubercular drug design and development.

Pharmacology and early ADMET data of corallopyronin A, a natural product with macrofilaricidal anti-wolbachial activity in filarial nematodes

Corallopyronin A (CorA), a natural product antibiotic of Corallococcus coralloides, inhibits the bacterial DNA-dependent RNA polymerase. It is active against the essential Wolbachia endobacteria of filarial nematodes, preventing development, causing sterility and killing adult worms. CorA is being developed to treat the neglected tropical diseases onchocerciasis and lymphatic filariasis caused by Wolbachia-containing filariae. For this, we have completed standard Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) studies. In Caco-2 assays, CorA had good adsorption values, predicting good transport from the intestines, but may be subject to active efflux. In fed-state simulated human intestinal fluid (pH 5.0), CorA half-life was >139 minutes, equivalent to the stability in buffer (pH 7.4). CorA plasma-stability was >240 minutes, with plasma protein binding >98% in human, mouse, rat, dog, mini-pig and monkey plasma. Clearance in human and dog liver microsomes was low (35.2 and 42 µl/min/mg, respectively). CorA was mainly metabolized via phase I reactions, i.e., oxidation, and to a minimal extent via phase II reactions. In contrast to rifampicin, CorA does not induce CYP3A4 resulting in a lower drug-drug-interaction potential. Apart from inhibition of CYP2C9, no impact of CorA on enzymes of the CYP450 system was detected. Off-target profiling resulted in three hits (inhibition/activation) for the A3 and PPARγ receptors and COX1 enzyme; thus, potential drug-drug interactions could occur with antidiabetic medications, COX2 inhibitors, angiotensin AT1 receptor antagonists, vitamin K-antagonists, and antidepressants. In vivo pharmacokinetic studies in Mongolian gerbils and rats demonstrated excellent intraperitoneal and oral bioavailability (100%) with fast absorption and high distribution in plasma. No significant hERG inhibition was detected and no phototoxicity was seen. CorA did not induce gene mutations in bacteria (Ames test) nor chromosomal damage in human lymphocytes (micronucleus test). Thus, CorA possesses an acceptable in vitro early ADMET profile; supported by previous in vivo experiments in mice, rats and Mongolian gerbils in which all animals tolerated CorA daily administration for 7-28 days. The non-GLP package will guide selection and planning of regulatory-conform GLP models prior to a first-into-human study.

RISE OF RIFAXIMIN IN PAEDIATRIC INFECTIOUS DIARRHOEA

The incidence and risk of morality is high under the age of five years is due to diarrhoea. In India twenty percentages of paediatric deaths is due to diarrhoeal disease. It is the second cause of leading mortality in children after acute respiratory infections. Following on from the GEMS study when we looked at the data in the management of bacterial diarrhoea the primary treatments included oral rehydration solution, oral zinc probiotics and antibiotics. Anecdotally we noted that a lot of children were treated with oral antibiotics and the majority of them were treated with fluoroquinolones like Ofloxacin and drugs like Metronidazole there are multiple side-effects associated with the usage of fluoroquinolones and a major side-effect of that is the risk to the growing cartilage. We also noticed that irrational combinations such as imidazole like metronidazole have shown more problems than solutions .usage of Ofloxacin in children should be discouraged for all the side-effects plus the infectivity in the management of bacterial diarrhoea. The duration of treatment have paved the way for usage of drugs such as new molecules in paediatric i.e. rifaximin comes under the group of oral non-absorbable GI antibiotics broad-spectrum agents with antibacterial activity against gram positive and gram negative aerobes and Anaerobes makes Rifaximin a bactericidal drug in nature. Rifaximin is particularly recommended in children above the age of two years in acute infectious diarrhoea the mechanism of action of rifaximin is by inhibition of bacterial protein synthesis by reversibly binding to bacterial DNA dependent RNA polymerase. In this review we discussed the “phrma” behind rifaximin, clinical experience of rifaximin in children and indications and dosage of rifaximin in paediatric.

Elaborations on Corallopyronin A as a Novel Treatment Strategy Against Genital Chlamydial Infections.

Ascending Chlamydia trachomatis infection causes functional damage to the fallopian tubes, which may lead to ectopic pregnancy and infertility in women. Treatment failures using the standard regimens of doxycycline and azithromycin have been observed. We tested the polyketide-derived α-pyrone antibiotic Corallopyronin A (CorA) that inhibits the bacterial DNA dependent RNA polymerase and has strong activity against various extracellular and some intracellular bacteria. Extensive testing in cell culture infection models and in an ex vivo human fallopian tube model under different oxygen concentrations was performed to assess the anti-chlamydial efficacy of CorA at physiological conditions. CorA showed high efficacy against C. trachomatis (MICN/H: 0.5 μg/mL for serovar D and L2), C. muridarum (MICN/H: 0.5 μg/mL), and C. pneumoniae (MICN/H: 1 μg/mL) under normoxic (N) and hypoxic (H) conditions. Recoverable inclusion forming units were significantly lower already at 0.25 μg/mL for all tested chlamydiae. CorA at a concentration of 1 μg/mL was also effective against already established C. trachomatis and C. pneumoniae infections (up to 24 h.p.i.) in epithelial cells, while efficacy against C. muridarum was limited to earlier time points. A preliminary study using a C. muridarum genital infection model revealed corresponding limitations in the efficacy. Importantly, in an ex vivo human fallopian tube model, the growth of C. trachomatis was significantly inhibited by CorA at concentrations of 1–2 μg/mL under normoxic and hypoxic conditions. The overall high efficacies of CorA against C. trachomatis in cell culture and an ex vivo human fallopian tube model under physiological oxygen concentrations qualifies this drug as a candidate that should be further investigated.

Antimicrobial profile and clinical evidence of fidaxomicin (Dafclir®), a therapeutic agent for Clostridioides (Clostridium) difficile infection

Fidaxomicin (Dafclir® Tablets 200 mg) is a member of a novel class of oral, 18-membered macrocyclic antibiotic agents used for the treatment of patients with Clostridioides (Clostridium) difficile (C. difficile) infection (CDI), approved in Japan in July 2018. Preclinical studies demonstrated that fidaxomicin inhibits RNA synthesis by bacterial DNA-dependent RNA polymerase derived from C. difficile, shows antibacterial activities against C. difficile clinically isolated in Japan, and is bactericidal against C. difficile. Fidaxomicin was less likely to disrupt gut microflora due to its narrow antimicrobial spectrum, showing antibacterial activities against limited gram-positive bacteria including C. difficile, but not against gram-negative bacteria, as determined by minimum inhibitory concentration (MIC) measurement against American Type Culture Collection strains. Fidaxomicin inhibited spore production, subsequent spore recovery/outgrowth after removal of fidaxomicin, outgrowth to vegetative cells, and toxin production under fidaxomicin at lower MIC. Additionally, it had protective effects on lethal CDI in animal models. In clinical studies conducted in Europe, US, and Japan, fidaxomicin 200 mg twice daily for 10 days showed higher clinical cure, higher global cure (cure with no recurrence), and lower recurrence rate compared with oral vancomycin 125 mg four times daily for 10 days. Adverse events observed in the fidaxomicin group were similar to those in the vancomycin group, and no clinically important findings regarding safety and tolerability were reported. In conclusion, in vitro, in vivo and clinical evidence indicate that fidaxomicin is an effective treatment for C. difficile, with limited disruption to gut microflora, for adult patients with CDI in Japan.

Studying transcription initiation by RNA polymerase with diffusion-based single-molecule fluorescence.

Over the past decade, fluorescence-based single-molecule studies significantly contributed to characterizing the mechanism of RNA polymerase at different steps in transcription, especially in transcription initiation. Transcription by bacterial DNA-dependent RNA polymerase is a multistep process that uses genomic DNA to synthesize complementary RNA molecules. Transcription initiation is a highly regulated step in E. coli, but it has been challenging to study its mechanism because of its stochasticity and complexity. In this review, we describe how single-molecule approaches have contributed to our understanding of transcription and have uncovered mechanistic details that were not observed in conventional assays because of ensemble averaging.

Function of the Loading Module in CorI and of the O -Methyltransferase CorH in Vinyl Carbamate Biosynthesis of the Antibiotic Corallopyronin A

Corallopyronin A is a promising in vivo active antibiotic, currently undergoing preclinical evaluation. This myxobacterial compound interferes with a newly identified drug target site, i.e., the switch region of the bacterial DNA-dependent RNA-polymerase (RNAP). Since this target site differs from that of known RNAP inhibitors such as the rifamycins, corallopyronin A shows no cross-resistance with other antibacterial agents. Corallopyronin A is a polyketide synthase- and nonribosomal peptide synthetase-derived molecule whose structure and biosynthesis is distinguished by several peculiarities, such as the unusual vinyl carbamate functionality whose formation involves carbonic acid as an unprecedented C1-starter unit. Using in vitro experiments the nature of this starter molecule was revealed to be the methyl ester of carbonic acid. Biochemical investigations showed that methylation of carbonic acid is performed by the O-methyltransferase CorH. These experiments shed light on the biosynthesis of the Eastern chain of α-pyrone antibiotics such as corallopyronin A.

Inactivation of the Bacterial RNA Polymerase Due to Acquisition of Secondary Structure by the ω Subunit

The widely conserved ω subunit encoded by rpoZ is the smallest subunit of Escherichia coli RNA polymerase (RNAP) but is dispensable for bacterial growth. Function of ω is known to be substituted by GroEL in ω-null strain, which thus does not exhibit a discernable phenotype. In this work, we report isolation of ω variants whose expression in vivo leads to a dominant lethal phenotype. Studies show that in contrast to ω, which is largely unstructured, ω mutants display substantial acquisition of secondary structure. By detailed study with one of the mutants, ω6 bearing N60D substitution, the mechanism of lethality has been deciphered. Biochemical analysis reveals that ω6 binds to β' subunit in vitro with greater affinity than that of ω. The reconstituted RNAP holoenzyme in the presence of ω6 in vitro is defective in transcription initiation. Formation of a faulty RNAP in the presence of mutant ω results in death of the cell. Furthermore, lethality of ω6 is relieved in cells expressing the rpoC2112 allele encoding β'2112, a variant β' bearing Y457S substitution, immediately adjacent to the β' catalytic center. Our results suggest that the enhanced ω6-β' interaction may perturb the plasticity of the RNAP active center, implicating a role for ω and its flexible state.

Targeting Bacterial RNA Polymerase σ70 for Development of Broadspectrum Antisense Antibacterials

Bacterial DNA-dependent RNA polymerase (RNAP) is the central enzyme among the progress of transcription. The ubiquity, structural and functional similarities among different bacterial species make it an attractive target for developing protein-level anti-RNAP bactericidal agents with broad spectrum. However, the practical value of RNAP inhibitors is limited by an extensively observed single mutation in rpoB gene (encoding the β subunit of RNAP), resulting in completely resistant strains. Antisense antibacterials (also called RNA silencers in bacteria) may present an unusual opportunity for developing broad-spectrum sequence-selective nucleic acid based therapeutics. This review will first present a brief state of knowledge on progress and problems in targeting RNAP. Special emphasis will then be given to introduce the advantageous features of RNAP σ70 as a potential target for broad-spectrum antisense inhibition. Characteristics of the antisense antibacterial strategy (including antisense mechanism, basic chemistry involved in nucleic acid analogs, their anti-infection applications in vitro and in vivo) will also be thoroughly described. Notably, our exploration on targeting σ70 gene in gram-negative and gram-positive bacteria respectively for realization of broad-spectrum antisense bactericidal effect will be elaborated on. This firstly demonstrates peptide nucleic acid (PNA)-peptide conjugate as a successful example of broad-spectrum antisense antibacterial. At the end, we will also highlight a few promising targets and delivery strategies that favor the possible development of broad-spectrum antisense antibacterials.

Myxopyronin induced bacterial RNA polymerase dynamics and computer aided drug discovery

Bacterial DNA‐dependent RNA polymerase (RNAP) is composed of a core of five subunits with the composition, 2α, β, β′, and ω. Each subunit has a highly conserved sequence among bacteria that is not shared among eukaryotes, characteristics that allow development of broad‐spectrum antibiotics. Here, we use two 175ns molecular dynamics simulations of MYX‐bound and unbound RNAP to show that MYX binding likely leads to an overall decrease in RNAP mobility and shortens the average distance and root mean square fluctuations between the β and β′ domains by one angstrom. These results are consistent with the view that MYX binding prevents opening of the DNA binding gate formed by the β and β′ domains, disallowing DNA binding and leading to inhibition. Additionally, we report a set of computationally predicted inhibitors found by integrating the molecular dynamics generated RNAP conformational distributions with a physics‐based virtual screen of the NCI diversity set.

Rifaximin pharmacology and clinical implications

Rifaximin is a semisynthetic, rifamycin-based non-systemic antibiotic, with a low gastrointestinal absorption and a good antibacterial activity. The antibacterial action covers Gram-positive and Gram-negative organisms, both aerobes and anaerobes. Its antimicrobial action is based on its property to bind to the β-subunit of bacterial DNA-dependent RNA polymerase inhibiting, thereby, the bacterial RNA synthesis. Rifaximin contributes to restore gut microflora imbalance, becoming an important therapeutic agent in several organic and functional gastrointestinal diseases such as hepatic encephalopathy, small intestine bacterial overgrowth, inflammatory bowel disease and colonic diverticular disease. This antibiotic has the advantage of low microbial resistance and few systemic adverse events and is safe in all patient populations, including young children.

Rifalazil and Other Benzoxazinorifamycins in the Treatment of Chlamydia‐Based Persistent Infections

Rifalazil is a benzoxazinorifamycin which inhibits bacterial DNA-dependent RNA polymerase. The benzoxazine ring endows benzoxazinorifamycins with unique physical and chemical characteristics which favor the use of rifalazil and derivatives in treating diseases caused by the obligate intracellular pathogens of the genus chlamydia. Minimal inhibitory concentrations of benzoxazinorifamycins against chlamydia are in the pg/mL range. These compounds have potential as monotherapeutic agents to treat chlamydia-associated disease because they retain activity against chlamydia strains resistant to currently approved rifamycins such as rifampin. A pivotal clinical trial with rifalazil has been initiated for the treatment of peripheral arterial disease. The rationale for this innovative use of rifalazil, including the association of C. pneumoniae in atherosclerotic plaque formation, as well as rifalazil's potency and efficacy against chlamydia in both preclinical and clinical studies, is discussed. Other benzoxazino derivatives may have utility as stand-alone topical antibacterials or combination antibacterials to treat serious Gram-positive infections. None of the benzoxazinorifamycins examined to date induce the cytochrome P450 3A4 enzyme. This is in contrast to currently approved rifamycins which are strong inducers of P450 enzymes, resulting in drug-drug interactions that limit the clinical utility of this drug class.

Interaction of bacterial DNA-dependent RNA polymerase with promoters

Transcription initiation is the most regulated step in the transcription cycle and directly affects the overall level of gene expression. Bacterial RNA polymerase (RNAP) requires a σ subunit to recognize the basal conserved promoter elements and melt DNA at the transcription start site. However, various domains and structural elements of the RNAP core enzyme directly contact promoter DNA in the promoter complex, suggesting that the σ subunit is not the only determinant of the recognition of promoter DNA and stability of the promoter complex formed by the RNAP holoenzyme. The review summarizes data on the role of the structural elements of the bacterial RNAP core enzyme in promoter recognition.

Low-molecular-weight inhibitors of bacterial DNA-dependent RNA polymerase

In this review, current data on low molecular weight inhibitors of bacterial RNA polymerases, both classical and those recently discovered, are summarised. This area has progressed rapidly in recent years largely due to availability of high-resolution structures of RNA polymerase and its complexes with inhibitors. The structural information, together with biochemical data, allows to understand molecular mechanisms of transcription inhibition by rifampicin, sorangicin, tagetitoxin, streptolydigin, and microcin J25. In its turn, the mechanistic understanding of the action of these inhibitors provides better understanding of transcription mechanism and RNA polymerase structure-function.

Pharmacological treatment of bacterial infections of the respiratory tract

Treatment of bacterial infections of the respiratory tract should allow for factors such as the patient’s history, the treatment situation and the result of any bacteriological diagnosis. Epiglottitis attributable to Haemophilus influenzae is treated with cefotaxime (a cephalosporin inhibitor of bacterial cell wall synthesis) or with chloramphenicol (an inhibitor of bacterial protein synthesis). Exacerbations of chronic bronchitis are treated with broad spectrum penicillins (inhibitors of bacterial cell wall synthesis), tetracyclines or macrolides (both inhibitors of bacterial protein synthesis). Community acquired pneumonias are treated with penicillins or a macrolide such as erythromycin. If community acquired pneumonia is severe, a combination of a macrolide and a cephalosporin is indicated. When Staphylococcus aureus is the suspected cause of severe community acquired pneumonia, flucloxacillin (a penicillin that is resistant to β-lactamase) should be added to the treatment regimen. Suspected atypical pneumonia is treated with a macrolide or an antibiotic from the tetracycline group (inhibitors of bacterial protein synthesis). If Legionella pneumophila is the suspected causative organism in severe community acquired pneumonia, rifampicin (an inhibitor of bacterial DNA-dependent RNA polymerase) should be used in combination with a macrolide. Hospital acquired pneumonia is treated with broad-spectrum cephalosporins or anti-pseudomonal penicillins, such as ticarcillin or piperacillin. In severe cases, an aminoglycoside inhibitor of bacterial protein synthesis (e.g. gentamicin) may be used. The treatment of tuberculosis requires specialized knowledge and involves the use of combinations of rifampicin with inhibitors of tubercular mycolic acid synthesis (e.g. isoniazid or pyrazinamide) or with an inhibitor of tubercular arabinosyl transferase (e.g. ethambutol).

Stereochemical Assignment of Intermediates in the Rifamycin Biosynthetic Pathway by Precursor-Directed Biosynthesis

Natural and semisynthetic rifamycins are clinically important inhibitors of bacterial DNA-dependent RNA polymerase. Although the polyketide-nonribosomal peptide origin of the naphthalene core of rifamycin B is well established, the absolute and relative configuration of both stereocenters introduced by the first polyketide synthase module is obscured by aromatization of the naphthalene ring. To decode the stereochemistry of the rifamycin polyketide precursor, we synthesized all four diastereomers of the biosynthetic substrate for module 2 of the rifamycin synthetase in the form of their N-acetylcysteamine (SNAC) thioester. Only one diastereomer was turned over in vivo into rifamycin B, thus establishing the absolute and relative configuration of the native biosynthetic intermediates.

A Novel Genomic Approach Identifies Bacterial DNA-Dependent RNA Polymerase as the Target of an Antibacterial Oligodeoxynucleotide, RBL1

Rapid emergence of antibiotic-resistant bacterial pathogens limits the applicability of existing drugs, which has created an urgent need for novel antibiotics preferably with entirely new mechanisms of action. Oligodeoxynucleotides (ODNs) and their modified forms have been shown to inhibit bacterial gene expression, representing a potential for developing highly specific and efficacious antibacterial agents. In this study, a tetracycline (Tet)-inducible, randomized single-stranded DNA (ssDNA) expression library was constructed and screened for conditional growth-defective or lethal phenotypes in an Escherichia coli system. From approximately 5000 transformants screened, 12 bacterial colonies were identified with either growth-defective or lethal phenotypes. One clone, CY01, with a lethal phenotype was selected and sequenced, and the ODN sequence that it generates was designated as RBL-1. Because RBL-1 shows no significant homologies to any bacterial gene sequence, a potential RBL-1 targeting protein was isolated by affinity purification. Using mass spectrometry analysis, this protein was identified as bacterial DNA-dependent RNA polymerase (RNAP). RBL-1 was further shown to effectively inhibit RNA polymerase activity in vitro. The usage of this randomized ssDNA expression library screening technology to selectively modulate production and/or function of proteins may provide a powerful strategy in both identifying novel gene targets for antibiotic discovery and developing novel antibacterial agents.

Treatment of chronic infections with rifamycins: is resistance likely to follow?

Rifampin and related rifamycins are the most important drugs in use for the treatment of tuberculosis ([2][1]). Rifamycins interfere with bacterial DNA-dependent RNA polymerase and are potent bactericidal agents. Rifampin and its analogues kill actively multiplying extracellular organisms,