Recombinant yeast and human cells as screening tools to search for antibacterial agents targeting the transcription termination factor Rho.

The alarming issue of antibiotic resistance expansion requires a continuous search for new and efficient antibacterial agents. Here we describe the design of new tools to screen for target-specific inhibitors of the bacterial Rho factor directly inside eukaryotic cells. Rho factor is a global regulator of gene expression which is essential to most bacteria, especially Gram-negative. Since Rho has no functional or structural homolog in eukaryotes, it constitutes a valuable and well known bacterial target as evidenced by its inhibition by the natural antibiotic, Bicyclomycin. Our screening tools are based on perturbation of mRNA processing and packaging reactions in the nucleus of eukaryotic cells by the RNA-dependent helicase/translocase activity of bacterial Rho factor leading to a growth defect phenotype. In this approach, any compound that impedes Rho activity should restore growth to yeast or human cells expressing Rho protein, providing valuable means to screen for target-specific antibacterial agents within the environment of a eukaryotic cell. The yeast tool expressing E. coli Rho factor was validated using Bicyclomycin as the control antibacterial agent. The validation of the screening tool was further extended with a stable human cell line expressing Rho factor conditionally. Finally, we show that Rho factors from different bacterial pathogens can also be designed as yeast-based screening tools which can reveal subtle variations in the functional features of the proteins.

Regulation of Ca2+-independent smooth muscle contraction by alternative staurosporine-sensitive kinase.

It is well known that inhibition of myosin phosphatase induces smooth muscle contraction in the absence of Ca2+. We characterized the kinase(s) which plays a role in Ca2+-independent, microcystin-LR-induced contraction in permeabilized smooth muscle of the rabbit portal vein. Assessments of various protein kinase inhibitors revealed this kinase(s) (1) was sensitive to staurosporine (1 microM), but resistant to other agents including wortmannin (10 microM), Y-27632 ((R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide+ ++, 100 microM). HA1077 (1-(5-isoquinolinylsulfonyl)-homopiperazine, 100 microM), H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, 100 microM), and calphostin C (100 microM), and (2) induced phosphorylation of 20 kDa myosin light chain at serine-19. We concluded that other kinases exist which phosphorylate myosin light chain at serine-19 and induce Ca2+-independent smooth muscle contraction, distinct from Rho-associated kinase, myosin light chain kinase, and protein kinase C.

Effects of bicyclomycin on RNA- and ATP-binding activities of transcription termination factor Rho.

Bicyclomycin is a commercially important antibiotic that has been shown to be effective against many gram-negative bacteria. Genetic and biochemical evidence indicates that the antibiotic interferes with RNA metabolism in Escherichia coli by inhibiting the activity of transcription termination factor Rho. However, the precise mechanism of inhibition is not completely known. In this study we have used in vitro transcription assays to analyze the effects of bicyclomycin on the termination step of transcription. The Rho-dependent transcription termination region located within the hisG cistron of Salmonella typhimurium has been used as an experimental system. The possible interference of the antibiotic with the various functions of factor Rho, such as RNA binding at the primary site, ATP binding, and hexamer formation, has been investigated by RNA gel mobility shift, photochemical cross-linking, and gel filtration experiments. The results of these studies demonstrate that bicyclomycin does not interfere with the binding of Rho to the loading site on nascent RNA. Binding of the factor to ATP is not impeded, on the contrary, the antibiotic appears to decrease the apparent equilibrium dissociation constant for ATP in photochemical cross-linking experiments. The available evidence suggests that this decrease might be due to an interference with the correct positioning of ATP within the nucleotide-binding pocket leading b an inherent block of ATP hydrolysis. Possibly, as a consequence of this interference, the antibiotic also prevents ATP-dependent stabilization of Rho hexamers.

Bicyclomycin sensitivity and resistance affect Rho factor-mediated transcription termination in the tna operon of Escherichia coli.

The growth-inhibiting drug bicyclomycin, known to be an inhibitor of Rho factor activity in Escherichia coli, was shown to increase basal level expression of the tryptophanase (tna) operon and to allow growth of a tryptophan auxotroph on indole. The drug also relieved polarity in the trp operon and permitted growth of a trp double nonsense mutant on indole. Nine bicyclomycin-resistant mutants were isolated and partially characterized. Recombination data and genetic and biochemical complementation analyses suggest that five have mutations that affect rho, three have mutations that affect rpoB, and one has a mutation that affects a third locus, near rpoB. Individual mutants showed decreased, normal, or increased basal-level expression of the tna operon. All but one of the resistant mutants displayed greatly increased tna operon expression when grown in the presence of bicyclomycin. The tna operon of the wild-type drug-sensitive parent was also shown to be highly expressed during growth with noninhibitory concentrations of bicyclomycin. These findings demonstrate that resistance to this drug may be required by mutations at any one of three loci, two of which appear to be rho and rpoB.