Biomedical subjects

Nagraj Mani

Publications and source records attributed to Nagraj Mani.

8 recordsLinked to original sources

PubMed2006

Dual targeting of GyrB and ParE by a novel aminobenzimidazole class of antibacterial compounds.

A structure-guided drug design approach was used to optimize a novel series of aminobenzimidazoles that inhibit the essential ATPase activities of bacterial DNA gyrase and topoisomerase IV and that show potent activities against a variety of bacterial pathogens. Two such compounds, VRT-125853 and VRT-752586, were characterized for their target specificities and preferences in bacteria. In metabolite incorporation assays, VRT-125853 inhibited both DNA and RNA synthesis but had little effect on protein synthesis. Both compounds inhibited the maintenance of negative supercoils in plasmid DNA in Escherichia coli at the MIC. Sequencing of DNA corresponding to the GyrB and ParE ATP-binding regions in VRT-125853- and VRT-752586-resistant mutants revealed that their primary target in Staphylococcus aureus and Haemophilus influenzae was GyrB, whereas in Streptococcus pneumoniae it was ParE. In Enterococcus faecalis, the primary target of VRT-125853 was ParE, whereas for VRT-752586 it was GyrB. DNA transformation experiments with H. influenzae and S. aureus proved that the mutations observed in gyrB resulted in decreased susceptibilities to both compounds. Novobiocin resistance-conferring mutations in S. aureus, H. influenzae, and S. pneumoniae were found in gyrB, and these mutants showed little or no cross-resistance to VRT-125853 or VRT-752586 and vice versa. Furthermore, gyrB and parE double mutations increased the MICs of VRT-125853 and VRT-752586 significantly, providing evidence of dual targeting. Spontaneous frequencies of resistance to VRT-752586 were below detectable levels (<5.2x10(-10)) for wild-type E. faecalis but were significantly elevated for strains containing single and double target-based mutations, demonstrating that dual targeting confers low levels of resistance emergence and the maintenance of susceptibility in vitro.

Anti-Bacterial Agents↗

PubMed2006

Regulation of toxin and bacteriocin gene expression in Clostridium by interchangeable RNA polymerase sigma factors.

The production of major extracellular toxins by pathogenic strains of Clostridium botulinum, Clostridium tetani and Clostridium difficile, and a bacteriocin by Clostridium perfringens is dependent on a related group of RNA polymerase sigma-factors. These sigma-factors (BotR, TetR, TcdR and UviA) were shown to be sufficiently similar that they could substitute for one another in in vitro DNA binding and run-off transcription experiments. In cells, however, the sigma-factors fell into two subclasses. BotR and TetR were able to direct transcription of their target genes in a fully reciprocal manner. Similarly, UviA and TcdR were fully interchangeable. Neither BotR nor TetR could substitute for UviA or TcdR, however, and neither UviA nor TcdR could direct transcription of the natural targets of BotR or TetR. The extent of functional interchangeability of the sigma-factors was attributed to the strong conservation of their subregion 4.2 sequences and the conserved -35 sequences of their target promoters, while restrictions on interchangeability were attributed to variations in their subregion 2.4 sequences and the target site -10 sequences. The four sigma-factors have been assigned to group 5 of the sigma(70) family and seem to have arisen from a common ancestral protein that may have co-evolved with the genes whose transcription they direct. A fifth Clostridiumsigma-factor, sigma(Y) of Clostridium acetobutylicum, resembles the TcdR family, but was not functionally interchangeable with members of this family.

Amino Acid Sequence↗

PubMed2006

In vitro characterization of the antibacterial spectrum of novel bacterial type II topoisomerase inhibitors of the aminobenzimidazole class.

Antibiotics with novel mechanisms of action are becoming increasingly important in the battle against bacterial resistance to all currently used classes of antibiotics. Bacterial DNA gyrase and topoisomerase IV (topoIV) are the familiar targets of fluoroquinolone and coumarin antibiotics. Here we present the characterization of two members of a new class of synthetic bacterial topoII ATPase inhibitors: VRT-125853 and VRT-752586. These aminobenzimidazole compounds were potent inhibitors of both DNA gyrase and topoIV and had excellent antibacterial activities against a wide spectrum of problematic pathogens responsible for both nosocomial and community-acquired infections, including staphylococci, streptococci, enterococci, and mycobacteria. Consistent with the novelty of their structures and mechanisms of action, antibacterial potency was unaffected by commonly encountered resistance phenotypes, including fluoroquinolone resistance. In time-kill assays, VRT-125853 and VRT-752586 were bactericidal against Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, and Haemophilus influenzae, causing 3-log reductions in viable cells within 24 h. Finally, similar to the fluoroquinolones, relatively low frequencies of spontaneous resistance to VRT-125853 and VRT-752586 were found, a property consistent with their in vitro dual-targeting activities.

Anti-Bacterial Agents↗

PubMed2006

Isolation of RNA polymerase from Clostridium difficile and characterization of glutamate dehydrogenase and rRNA gene promoters in vitro and in vivo.

Clostridium difficile is the primary causative agent of antibiotic-associated diarrheal disease. To facilitate molecular genetic analysis of gene expression in this organism, methods were developed to study transcriptional regulation in vitro and in vivo. That is, C. difficile RNA polymerase was partially purified and shown to bind to and initiate transcription in vitro from bona fide C. difficile promoters for rRNA and glutamate dehydrogenase genes. In addition, primer extension analyses and a beta-glucuronidase reporter system were used to quantitate transcription from these promoters in vivo. With these tools in hand, it is now possible to characterize the behavior of any C. difficile gene in vivo and to study the regulation of its expression in detail.

Base Sequence↗

PubMed2005

Transcription activation of a UV-inducible Clostridium perfringens bacteriocin gene by a novel sigma factor.

Expression of the plasmid-encoded Clostridium perfringens gene for bacteriocin BCN5 was shown to depend in vivo and in vitro on the activity of UviA protein. UviA, also plasmid-encoded, proved to be an RNA polymerase sigma factor and was also partly autoregulatory. The uviA gene has two promoters; one provided a UviA-independent, basal level of gene expression while the stronger, UviA-dependent promoter was only utilized after the cell experienced DNA damage. As a result, BCN5 synthesis is induced by treatment with UV light or mitomycin C. UviA is related to a special class of sigma factors found to date only in Clostridium species and responsible for activating transcription of toxin genes in Clostridium difficile, Clostridium tetani, and Clostridium botulinum.

Bacterial Proteins↗

PubMed2004

Inhibition of antibiotic efflux in bacteria by the novel multidrug resistance inhibitors biricodar (VX-710) and timcodar (VX-853).

Inhibitors of mammalian multidrug efflux, such as the plant alkaloid reserpine, are also active in potentiating antibiotic activity by inhibiting bacterial efflux. Based on this precedent, two novel mammalian multiple drug resistance inhibitors, biricodar (VX-710) and timcodar (VX-853), were evaluated for activity in a variety of bacteria. Both VX-710 and VX-853 potentiated the activity of ethidium bromide (EtBr), a model efflux substrate, against three clinically significant gram-positive pathogens: Staphylococcus aureus, Enterococcus faecalis, and Streptococcus pneumoniae. Similar to reserpine, VX-710 and VX-853 directly blocked EtBr efflux in S. aureus. Furthermore, these compounds were effective in lowering the MICs of several clinically used antibiotics, including fluoroquinolones, suggesting that VX-710 and VX-853 are representatives of a new class of bacterial efflux inhibitors with the potential for use in combination therapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

Environmental response and autoregulation of Clostridium difficile TxeR, a sigma factor for toxin gene expression.

TxeR, a sigma factor that directs Clostridium difficile RNA polymerase to recognize the promoters of two major toxin genes, was shown to stimulate its own synthesis. Whether expressed in C. difficile, Clostridium perfringens, or Escherichia coli, TxeR stimulated transcription of fusions of the txeR promoter region to reporter genes. As is the case for the tox genes, txeR expression was responsive to the cellular growth phase and the constituents of the medium. That is, the level of expression in broth culture was low during the exponential growth phase, but rapidly increased as cells approached the stationary phase. In the presence of excess glucose, expression from the txeR promoter was repressed. The results support a model for toxin gene expression in which synthesis of TxeR is induced by specific environmental signals. The increased level of TxeR then permits high-level expression of the toxin genes. The study of txeR gene regulation in C. difficile was made possible by introduction of a mobilizable, replicative plasmid via conjugation with E. coli.

Bacterial Proteins↗

PubMed2002

A pathway-specific cell based screening system to detect bacterial cell wall inhibitors.

A pathway-specific cell-based screen is described to detect compounds that inhibit the biosynthesis of the cell wall of bacteria. The basis for detection is the discovery that the beta-lactamase gene from Citrobacterfreundii, cloned into Escherichia coli, is induced when cells are exposed to known cell wall inhibitors, and not just beta-lactam-based antibiotics. In a wild type host, cell wall inhibitors such as moenomycin, vancomycin, and ramoplanin, which are excluded by the outer membrane, only induce at high concentrations. However, these compounds, as well as fosfomycin, cycloserine, and cefoxitin, induce at concentrations at or below the MIC of a host carrying the envA-mutation, which causes a defect in the outer membrane. As additional proof that induction of beta-lactamase is the direct result of cell wall inhibition, a host strain carrying a temperature-sensitive mutation in the murG gene, whose product converts the cell wall intermediate Lipid I, to Lipid II, also induced beta-lactamase at the restrictive temperature. A protocol is described for screening samples in high-throughput mode.

Anti-Bacterial Agents↗