Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in <em>Pseudomonas aeruginosa</em>

Abstract

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Host cell-based screening assays for identification of molecules targeting Pseudomonas aeruginosa cyclic di-GMP signaling and biofilm formation

“…(C) The robust z' values for bioluminescent, cytotoxicity, and GFP are calculated according to the formula provided in the section "Materials and methods." for high-throughput screens (Rugjee et al, 2016). Calculate robust z' using the formula:…”

“…Evaluate the uniformity and reproducibility of the assay using robust z ’ analysis, which is the most common quality metric reported for high-throughput screens ( Rugjee et al, 2016 ). Calculate robust z ’ using the formula:…”

Covalent attachment and Pro‐Pro endopeptidase (PPEP‐1)‐mediated release of Clostridium difficile cell surface proteins involved in adhesion

“…The development and application of fluorescence‐based reporters has opened some new research avenues. For example, fluctuations of the c‐di‐GMP concentration in vivo were detected by a GFP reporter assay using the promoter of cdrA, a large adhesin whose expression, in P. aeruginosa , is highly increased in the presence of c‐di‐GMP (Rybtke et al ., ; Baraquet and Harwood, ; Rugjee et al ., ). Additionally, the development of riboswitch‐based biosensors has provided an excellent tool to quantify c‐di‐GMP in living cells (Kellenberger et al ., ).…”