Bacterial responses to stress - polyphosphate (POLYP) is a ubiquitous biomolecule in many gram-negative bacteria that plays a central role in bacterial stress resistance

The National Institute of General Medical Sciences awarded the Board of Trustees of Illinois State University $148,000 on August 3, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to investigate how polyphosphate protects gram-negative bacteria from membrane stress and to examine the impact of pharmacological PPK1 inhibition on biofilm-associated bacteria.

The project, titled "Bacterial Responses to Stress—Polyphosphate," will run through May 31, 2030, with work performed in Normal, Illinois. The research addresses the role of polyphosphate, a ubiquitous biomolecule in gram-negative bacteria that contributes to stress resistance, antibiotic tolerance, biofilm formation, and host colonization. The recipient will investigate three major aims: how polyphosphate production protects against polymyxin-induced membrane stress and whether polyphosphate reduces oxidative damage, supports membrane integrity, and promotes stress-responsive gene expression; the impact of mesalamine-mediated inhibition of polyphosphate kinase 1 (PPK1) on biofilm-associated bacteria; and the mechanism by which Staphylococcus aureus stimulates polyphosphate synthesis in other bacteria. Preliminary data suggest that mesalamine, an FDA-approved drug, acts as a potent PPK1 inhibitor and may sensitize bacteria to antimicrobials, offering a bacteria-specific therapeutic strategy.

Composed of phosphate chains linked by phosphoanhydride bonds, POLYP supports survival against phagocytic oxidants, contributes to antibiotic tolerance, and enhances biofilm formation, motility, and host colonization. We recently showed that POLYP also promotes antagonism of gram-positive competitors, enabling pseudomonas aeruginosa to kill staphylococcus aureus. POLYP is synthesized by POLYP kinase 1 (PPK1), which transfers the terminal phosphate from ATP onto POLYP chains. Because eukaryotes synthesize POLYP through alternative mechanisms and lack PPK1, targeting this enzyme offers a bacteria-specific therapeutic strategy. We previously identified the FDA-approved drug mesalamine as a potent PPK1 inhibitor, eliciting phenotypes that closely mimic POLYP-deficient mutants. Pharmacological inhibition of PPK1 may thus sensitize bacteria to antimicrobials and enhance host clearance. Despite these broad effects, mechanistic details of POLYP function remain poorly understood. We present preliminary data suggesting that POLYP protects gram-negative bacteria from membrane stress and that mesalamine treatment alleviates this protection. Compromised POLYP production in polymyxin-stressed bacteria increases oxidative stress and destabilizes the cell envelope. Additionally, we found that S. Aureus secretes an unknown compound into the extracellular environment that stimulates POLYP synthesis in several bacteria, including P. Aeruginosa. This proposal addresses three major goals. Aim 1 will investigate how POLYP production protects against polymyxin-induced membrane stress. We will test whether POLYP reduces oxidative damage, supports membrane integrity, and promotes stress-responsive gene expression. Aim 2 will examine the impact of mesalamine-mediated PPK1 inhibition on biofilm-associated bacteria. We will assess biofilm survival, quantify POLYP-dependent tolerance, and evaluate polymyxin/mesalamine combination therapies for synergistic effects. Aim 3 seeks to identify the S. Aureus secretion factor that stimulates POLYP production in P. Aeruginosa. Biochemical fractionation, genetic screening, and enzymatic assays will be used to define this interspecies signaling mechanism. Collectively, this work will elucidate a fundamental bacterial stress defense system with broad relevance to multidrug-resistant gram-negative bacteria. By defining how POLYP protects against membrane stress and contributes to polymyxin tolerance, our studies may establish mesalamine or related inhibitors as effective adjuvant therapies. Furthermore, discovery of a signaling mechanism by which S. Aureus triggers POLYP synthesis in P. Aeruginosa will provide new insights into microbial interactions that shape infection dynamics. Importantly, this project will also enrich the research environment at a regional undergraduate institution by engaging students in impactful research at the interface of microbiology and biochemistry.