DNA enzyme generated by a novel single-stranded DNA expression vector inhibits expression of the essential bacterial cell division gene ftsZ

Rapid emergence of antibiotic-resistant bacterial pathogens has created urgent demand for the discovery and development of new antibacterial agents directed toward novel targets. Antisense oligodeoxynucleotides (AS-ODN) and their modified forms have been utilized to block gene expression in bacterial cells, showing potential for developing highly specific and efficacious antibacterial agents. In this study, a tetracycline-regulated expression vector was developed for generating single-stranded DNA (ssDNA) of a desired target sequence in bacterial cells. This inducible ssDNA expression vector was tested for producing a DNA enzyme designed to specifically cleave ftsZ mRNA. Our results indicate that the expressed DNA enzyme molecules not only repress ftsZ gene expression and but also inhibit bacterial cell proliferation. Although we believe that the cleavage of ftsZ mRNA by the expressed DNA enzyme molecules is responsible for the inhibitory effects on ftsZ gene expression and bacterial cell proliferation, the antisense mechanism could also be responsible for the biological effects. The ability of this ssDNA expression system to selectively modulate gene expression may provide a powerful strategy in determining the contribution of a given gene product to bacterial growth or pathogenesis and opens a new venue for developing antibacterial agents.     

Identification of a novel essential two-component signal transduction system, YhcSR, in Staphylococcus aureus

Two-component signal transduction systems play an important role in the ability of bacteria to adapt to various environments by sensing changes in their habitat and by altering gene expression. In this study, we report a novel two-component system, YhcSR, in Staphylococcus aureus which is required for bacterial growth in vitro. We found that the down-regulation of yhcSR expression by induced yhcS antisense RNA can inhibit and terminate bacterial growth. Moreover, without complementary yhcS or yhcR, no viable yhcS or yhcR gene replacement mutant was recoverable. Collectively, these results demonstrated that the YhcSR regulatory system is indispensable for S. aureus growth in culture. Moreover, induced yhcS antisense RNA selectively increased bacterial susceptibility to phosphomycin. These data suggest that YhcSR probably modulates the expression of genes critical for bacterial survival and may be a potential target for the development of novel antibacterial agents.     

New target for inhibition of bacterial RNA polymerase: 'switch region'

A new drug target - the 'switch region' - has been identified within bacterial RNA polymerase (RNAP), the enzyme that mediates bacterial RNA synthesis. The new target serves as the binding site for compounds that inhibit bacterial RNA synthesis and kill bacteria. Since the new target is present in most bacterial species, compounds that bind to the new target are active against a broad spectrum of bacterial species. Since the new target is different from targets of other antibacterial agents, compounds that bind to the new target are not cross-resistant with other antibacterial agents. Four antibiotics that function through the new target have been identified: myxopyronin, corallopyronin, ripostatin, and lipiarmycin. This review summarizes the switch region, switch-region inhibitors, and implications for antibacterial drug discovery.     

A new class of small molecule RNA polymerase inhibitors with activity against rifampicin-resistant Staphylococcus aureus

The RNA polymerase holoenzyme is a proven target for antibacterial agents. A high-throughput screening program based on this enzyme from Staphylococcus aureus had previously identified a 2-ureidothiophene-3-carboxylate as a low micromolar inhibitor. An investigation of the relationships between the structures of this class of compounds and their inhibitory- and antibacterial activities is described here, leading to a set of potent RNA polymerase inhibitors with antibacterial activity. Characterization of this bioactivity, including studies of the mechanism of action, is provided, highlighting the power of the reverse chemical genetics approach in providing tools to inhibit the bacterial RNA polymerase.     

Plasmid rolling circle replication: identification of the RNA polymerase-directed primer RNA and requirement for DNA polymerase I for lagging strand synthesis.

Plasmid rolling circle replication involves generation of single-stranded DNA (ssDNA) intermediates. ssDNA released after leading strand synthesis is converted to a double-stranded form using solely host proteins. Most plasmids that replicate by the rolling circle mode contain palindromic sequences that act as the single strand origin, sso. We have investigated the host requirements for the functionality of one such sequence, ssoA, from the streptococcal plasmid pLS1. We used a new cell-free replication system from Streptococcus pneumoniae to investigate whether host DNA polymerase I was required for lagging strand synthesis. Extracts from DNA polymerase I-deficient cells failed to replicate, but this was corrected by adding purified DNA polymerase I. Efficient DNA synthesis from the pLS1-ssoA required the entire DNA polymerase I (polymerase and 5'-3' exonuclease activities). ssDNA containing the pLS1-ssoA was a substrate for specific RNA polymerase binding and a template for RNA polymerase-directed synthesis of a 20 nucleotide RNA primer. We constructed mutations in two highly conserved regions within the ssoA: a six nucleotide conserved sequence and the recombination site B. Our results show that the former seemed to function as a terminator for primer RNA synthesis, while the latter may be a binding site for RNA polymerase.     

Validation of antibacterial mechanism of action using regulated antisense RNA expression in Staphylococcus aureus

Validation of antibiotic mode of action in whole bacterial cells is a key step for antibiotic drug discovery. In this study, one potential drug target, enoyl-acyl carrier protein reductase (FabI), an essential enzyme in the fatty acid biosynthesis pathway, was used to evaluate the feasibility of using a regulated antisense RNA interference approach to determine antibiotic mode of action. Antisense isogenic strains expressing antisense RNA to fabI were created using a tetracycline-regulated vector in Staphylococcus aureus. We demonstrated that down-regulation of FabI expression by induction of fabI antisense RNA induces a conditional lethal phenotype. In contrast, partial down-regulation gives a viable cell with a significant increase in sensitivity to FabI-specific inhibitors (i.e., a sensitized phenotype). More importantly, the mode of action for novel FabI inhibitors has been confirmed using this genetic approach in whole cell assay. These results indicate that controlled antisense technology provides a robust tool for defining and tracking the mode of action of novel antibacterial agents.     

A modular and optimized single marker system for generating Trypanosoma brucei cell lines expressing T7 RNA polymerase and the tetracycline repressor

Here, we present a simple modular extendable vector system for introducing the T7 RNA polymerase and tetracycline repressor genes into Trypanosoma brucei. This novel system exploits developments in our understanding of gene expression and genome organization to produce a streamlined plasmid optimized for high levels of expression of the introduced transgenes. We demonstrate the utility of this novel system in bloodstream and procyclic forms of Trypanosoma brucei, including the genome strain TREU927/4. We validate these cell lines using a variety of inducible experiments that recapture previously published lethal and non-lethal phenotypes. We further demonstrate the utility of the single marker (SmOx) TREU927/4 cell line for in vivo experiments in the tsetse fly and provide a set of plasmids that enable both whole-fly and salivary gland-specific inducible expression of transgenes.     

Control of segregation of chromosomal DNA by sex factor F in Escherichia coli. Mutants of DNA gyrase subunit A suppress letD (ccdB) product growth inhibition.

The letA (ccdA) and letD (ccdB) genes, located just outside the sequence essential for replication of the F plasmid, apparently contribute to stable maintenance of the plasmid. The letD gene product acts to inhibit partitioning of chromosomal DNA and cell division of the host bacteria, whereas the letA gene product acts to suppress the activity of the letD gene product. To identify the target of the letD gene product, temperature-sensitive growth-defective mutants were screened from bacterial mutants that had escaped the letD product growth inhibition that occurs in hosts carrying an FletA mutant. Of nine mutants analysed, three mutants were shown, by phage P1-mediated transduction and complementation analysis, to have mutations in the gyrA gene and the other six in the groE genes. The nucleotide sequence revealed that one of the gyrA mutants has a base change from G to A at position 641 (resulting in an amino acid change from Gly to Glu at position 214) of the gyrA gene. The mutant GyrA proteins produced by these gyrA(ts) mutants were trans-dominant over wild-type GyrA protein for letD tolerance. The wild-type GyrA protein, produced in excess amounts by means of a multicopy plasmid, overcame growth inhibition of the letD gene product. These observations strongly suggest that the A subunit of DNA gyrase is the target of the LetD protein.     

Local delivery of GM-CSF protects mice from lethal pneumococcal pneumonia

The growth factor GM-CSF has an important role in pulmonary surfactant metabolism and the regulation of antibacterial activities of lung sentinel cells. However, the potential of intra-alveolar GM-CSF to augment lung protective immunity against inhaled bacterial pathogens has not been defined in preclinical infection models. We hypothesized that transient overexpression of GM-CSF in the lungs of mice by adenoviral gene transfer (Ad-GM-CSF) would protect mice from subsequent lethal pneumococcal pneumonia. Our data show that intra-alveolar delivery of Ad-GM-CSF led to sustained increased pSTAT5 expression and PU.1 protein expression in alveolar macrophages during a 28-d observation period. Pulmonary Ad-GM-CSF delivery 2-4 wk prior to infection of mice with Streptococcus pneumoniae significantly reduced mortality rates relative to control vector-treated mice. This increased survival was accompanied by increased inducible NO synthase expression, antibacterial activity, and a significant reduction in caspase-3-dependent apoptosis and secondary necrosis of lung sentinel cells. Importantly, therapeutic treatment of mice with rGM-CSF improved lung protective immunity and accelerated bacterial clearance after pneumococcal challenge. We conclude that prophylactic delivery of GM-CSF triggers long-lasting immunostimulatory effects in the lung in vivo and rescues mice from lethal pneumococcal pneumonia by improving antibacterial immunity. These data support use of novel antibiotic-independent immunostimulatory therapies to protect patients against bacterial pneumonias.     

Downregulation of yidC in Escherichia coli by Antisense RNA Expression Results in Sensitization to Antibacterial Essential Oils Eugenol and Carvacrol

Background

The rising drug resistance in pathogenic bacteria and inefficiency of current antibiotics to meet clinical requirements has augmented the need to establish new and innovative approaches for antibacterial drug discovery involving identification of novel antibacterial targets and inhibitors. Being obligatory for bacterial growth, essential gene products are considered vital as drug targets. The bacterial protein YidC is highly conserved among pathogens and is essential for membrane protein insertion due to which it holds immense potential as a promising target for antibacterial therapy.

Methods/Principal Findings

The aim of this study was to explore the feasibility and efficacy of expressed antisense-mediated gene silencing for specific downregulation of yidC in Escherichia coli. We induced RNA silencing of yidC which resulted in impaired growth of the host cells. This was followed by a search for antibacterial compounds sensitizing the YidC depleted cells as they may act as inhibitors of the essential protein or its products. The present findings affirm that reduction of YidC synthesis results in bacterial growth retardation, which warrants the use of this enzyme as a viable target in search of novel antibacterial agents. Moreover, yidC antisense expression in E. coli resulted in sensitization to antibacterial essential oils eugenol and carvacrol. Fractional Inhibitory Concentration Indices (FICIs) point towards high level of synergy between yidC silencing and eugenol/carvacrol treatment. Finally, as there are no known YidC inhibitors, the RNA silencing approach applied in this study put forward rapid means to screen novel potential YidC inhibitors.

Conclusions/Significance

The present results suggest that YidC is a promising candidate target for screening antibacterial agents. High level of synergy reported here between yidC silencing and eugenol/carvacrol treatment is indicative of a potential antibacterial therapy. This is the first report indicating that the essential gene yidC is a therapeutic target of the antibacterial essential oils eugenol and carvacrol in E. coli.     

RPC40, a unique gene for a subunit shared between yeast RNA polymerases A and C

Yeast RNA polymerases A and C share an approximately equal to 40 kd subunit. We have identified, sequenced, and mutagenized in vitro the AC40 subunit gene. The RPC40 gene is unique in the yeast genome and is required for cell viability. This gene contains an open reading frame encoding a 37.6 kd protein having no significant homology with bacterial RNA polymerase subunits. The promoter region contains a 19 bp sequence also present in the largest subunit of RNA polymerase C. It also contains a well-conserved RPG box, a sequence found in the promoter region of many genes encoding the translational apparatus. A novel, plasmid-shuffling method was developed to isolate a large number of RPC40 ts mutants. One of these, ts4, was shown to be defective in the synthesis of RNA polymerases A and C at the restrictive temperature. In contrast, RNA polymerase B was made normally.     

Isolation of peptide ligands that inhibit glutamate racemase activity from a random phage display library

Several new antibacterial agents are currently being developed in response to the emergence of bacterial resistance to existing antibiotic substances. The new agents include compounds that interfere with bacterial membrane function. The peptidoglycan component of the bacterial cell wall is synthesized by glutamate racemase, and this enzyme is responsible for the biosynthesis of d-glutamate, which is an essential component of cell wall peptidoglycan. In this study, we screened a phage display library expressing random dodecapeptides on the surface of bacteriophage against an Escherichia coli glutamate racemase, and isolated specific peptide sequences that bind to the enzyme. Twenty-seven positive phage clones were analyzed, and seven different peptide sequences were obtained. Among them, the peptide sequence His-Pro-Trp-His-Lys-Lys-His-Pro-Asp-Arg-Lys-Thr was found most frequently, suggesting that this peptide might have the highest affinity to glutamate racemase. The positive phage clones and HPWHKKHPDRKT synthetic peptide were able to inhibit glutamate racemase activity in vitro, implying that our peptide inhibitors may be utilized for the molecular design of new potential antibacterial agents targeting cell wall synthesis.