Development and Validation of a High-Throughput Cell-Based Screen To Identify Activators of a Bacterial Two-Component Signal Transduction System

Abstract

iCpxRA is a two-component signal transduction system (2CSTS) found in many drug-resistant Gram-negative bacteria. In response to periplasmic stress, CpxA autophosphorylates and donates a phosphoryl group to its cognate response regulator, CpxR. Phosphorylated CpxR (CpxR-P) upregulates genes involved in membrane repair and downregulates multiple genes that encode virulence factors, which are trafficked across the cell membrane. Mutants that constitutively activate CpxRA in Salmonella enterica serovar Typhimurium and Haemophilus ducreyi are avirulent in mice and humans, respectively. Thus, the activation of CpxRA has high potential as a novel antimicrobial/antivirulence strategy. Using a series of Escherichia coli strains containing a CpxR-P-responsive lacZ reporter and deletions in genes encoding CpxRA system components, we developed and validated a novel cell-based high-throughput screen (HTS) for CpxRA activators. A screen of 36,000 compounds yielded one hit compound that increased reporter activity in wild-type cells. This is the first report of a compound that activates, rather than inhibits, a 2CSTS. The activity profile of the compound against CpxRA pathway mutants in the presence of glucose suggested that the compound inhibits CpxA phosphatase activity. We confirmed that the compound induced the accumulation of CpxR-P in treated cells. Although the hit compound contained a nitro group, a derivative lacking this group retained activity in serum and had lower cytotoxicity than that of the initial hit. This HTS is amenable for the screening of larger libraries to find compounds that activate CpxRA by other mechanisms, and it could be adapted to find activators of other two-component systems. The increasing prevalence of multidrug-resistant Gram-negative bacteria has prompted urgent calls for new antibiotics (1). Escherichia coli sequence type 131, a clonal group that expresses extended-spectrum ␤-lactamases (ESBLs) and quinolone resistance, has emerged as a major cause of community-and health care-associated urinary tract infections in the United States (2-4). The Klebsiella pneumoniae carbapenemase (KPC) has rendered some strains of K. pneumoniae resistant to all ␤-lactams, while the New Delhi metallo-(NDM-1) ␤-lactamase-containing plasmid has rendered some strains of E. coli and K. pneumoniae panresistant (5-9). These developments raise the specter that several common infections, such as urinary tract infections due to E. coli or K. pneumoniae, may soon be caused by organisms that are virtually untreatable (5,8,9).The traditional approach to discover antibiotics has been to screen libraries of natural or synthetic products for bacterial killing activity in culture. Unfortunately, this strategy has yielded no new targets or classes of drugs for Gram-negative bacteria over the past 50 years (10-12). More contemporary approaches are aimed at identifying inhibitors of novel targets essential for growth or virulence. Attractive targets include bacterial two-component signal transduction systems (2CSTS), which ty...

Evaluation of CpxRA as a Therapeutic Target for Uropathogenic Escherichia coli Infections

“…When E. coli is grown in peptide-based media containing an excess of rapidly metabolized carbon sources such as glucose, CpxR accepts phosphate groups from small-molecule phosphate donors such as acetyl phosphate (20)(21)(22). In such media, cpxA deletion mutants, which lack CpxR phosphatase activity, accumulate phosphorylated CpxR, resulting in activation of the system (20).…”

The MisR Response Regulator Is Necessary for Intrinsic Cationic Antimicrobial Peptide and Aminoglycoside Resistance in Neisseria gonorrhoeae

“…In other Gram-negative organisms, response regulators can be phosphorylated spontaneously by the phosphoryl donor molecule acetyl phosphate, leading to activation/repression of target genes (53,54). This process is mediated by the activity of the Pta-AckA metabolic pathway and is robust when E. coli cells are grown in the presence of 0.4% glucose (55,56), the very same concentration of glucose present in gonococcal GCB agar and broth (27). Since the Pta-AckA pathway is genetically intact in N. gonorrhoeae strains FA 1090 (http: //www.genome.ou.edu/gono.html; http://www.kegg.jp/pathway /ngo01200) and FA19 (GenBank accession number CP012026), it is possible that gonococcal MisR could be constitutively phosphorylated by acetyl phosphate during growth in standard laboratory media.…”

Both MisR (CpxR) and MisS (CpxA) Are Required for Neisseria gonorrhoeae Infection in a Murine Model of Lower Genital Tract Infection

“…CpxRA activation primarily affects genes encoding envelope-localized and/or -associated factors, many of which could be potential virulence determinants or vaccine targets; characterization of MisRS-downregulated targets could identify novel gonococcal virulence determinants and vaccine targets. Pharmacological activation of CpxRA is being pursued as a novel antimicrobial/antivirulence strategy for drugresistant Gram-negative pathogens (73); as antimicrobial-resistant gonococcal strains are emerging at an alarming rate, it will be interesting to explore whether pharmacological activation or inhibition of MisRS could be an antimicrobial/antivirulence strategy for the gonococcus. Infection of mice with a meningococcal misR mutant stimulates a protective immune response, suggesting that a misR mutant could serve as a live attenuated vaccine for meningococcal infection (74); the potential of misR and misS mutants as live attenuated vaccines for gonococcal infection is also worth investigating.…”