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

Abstract

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.

Separation and Enrichment of Yeast Saccharomyces cerevisiae by Shape Using Viscoelastic Microfluidics

“…S accharomyces cerevisiae ( S. Cerevisiae ) is a single-celled eukaryotic microorganism, which is commonly known for its role as a workhorse in fermentative production of bread, beer, and wine at industrial scale. , S. Cerevisiae is also an important model organism , in modern cell biology research, which has been gaining appeal for studies of fundamental processes in cellular and molecular biology. Due to the importance of S. Cerevisiae in industry and basic biological research, a wide range of studies have been carried out, ranging from uncovering new avenues of fermentation capabilities and understanding basic genetics and physiology of eukaryotic cells and humans , to modifying genes randomly for enhanced functions or new tasks and studying healthcare problems of importance to human medicine and cancer. , …”

“…Due to the importance of S. Cerevisiae in industry and basic biological research, a wide range of studies have been carried out, ranging from uncovering new avenues of fermentation capabilities 5 and understanding basic genetics and physiology of eukaryotic cells and humans 6,7 to modifying genes randomly for enhanced functions or new tasks 8 and studying healthcare problems of importance to human medicine and cancer. 9,10 The variation in shape in the phases of the cell cycle is one of the most important noteworthy attributes for S. Cerevisiae. 11 S. Cerevisiae divides by budding, an entirely asexual form of reproduction.…”

A Fluorogenic Assay To Monitor Rho-Dependent Termination of Transcription

“…12,13 Rho-dependent termination (RDT) is essential in many bacteria, including pathogens, 14−16 and thus constitutes an interesting target for the development of new antibiotics. 12,17 A natural Rho inhibitor, bicyclomycin (BCM), was discovered in the 1970s and is currently used in veterinary medicine. 14 Beyond its role in the definition of transcript 3′-ends, 18−20 RDT contributes to the surveillance of transcription−translation coupling, 21,22 silencing of antisense 9,16 and foreign (horizontally acquired) DNA transcription, 23,24 prevention of transcriptional R-loops, 25,26 and avoidance of transcription− replication conflicts.…”

“…46 The assay should also prove to be useful for in vitro HTS and discovery of new antibiotics targeting Rho, thereby optimally complementing current in vivo screening assays. 17 ■ MATERIALS AND METHODS Materials. Unless specified otherwise, chemicals and enzymes were purchased from Sigma-Aldrich and New England Biolabs (NEB), respectively.…”

“…Rho-dependent termination (RDT) is essential in many bacteria, including pathogens, − and thus constitutes an interesting target for the development of new antibiotics. , A natural Rho inhibitor, bicyclomycin (BCM), was discovered in the 1970s and is currently used in veterinary medicine . Beyond its role in the definition of transcript 3′-ends, − RDT contributes to the surveillance of transcription–translation coupling, , silencing of antisense , and foreign (horizontally acquired) DNA transcription, , prevention of transcriptional R-loops, , and avoidance of transcription–replication conflicts. , RDT is also involved in a variety of conditional regulatory mechanisms governed by sRNAs, − proteins, , or riboswitches. − This great functional diversity coupled with the lack of simple sequence rules evoked above and some variability among species , significantly complicates the characterization of the RDT features and perimeter.…”