Role of sarA and agr in Insulin Modulation of Staphylococcus aureus Biofilm Formation

*Corresponding author: Balbina J. Plotkin. Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Role of sarA and agr in Insulin Modulation of Staphylococcus aureus Biofilm Formation Balbina J. Plotkin 1, *, Monika I. Konaklieva 2, and Ira Sigar 1 1 Department of Microbiology and Immunology, Midwestern University, Downers Grove, IL 60515. 2 Department of Chemistry, American University, 4400 Massachusetts Ave. NW, Washington, DC, 20016-8014, USA. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 093-096 Publication history: Received on 25 August 2025; revised on 05 October 2025; accepted on 07 October 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.24.1.0860 Abstract The focus of this study is to determine whether insulin, quorum signaling and biofilm formation are under regulation of agr and sarA regulation. Deletion strains agr-, sarA -, and agr-sarAas well as their parent strain 8325-4 were tested with and without glucose (0.1% and 0.2%) and/or physiologic levels of insulin (2µU/mL, 20µU/mL, and 200µU/mL). In the absence of sarA and agr, there was a marked suppression of biofilm levels in the presence of insulin, indicating that insulin modulation of biofilm formation appears to be regulated, in part, by sarA and agr. Keywords: Glucose; Quorum Compound; Signaling Compound; Hormone 1. Introduction The ability of S. aureus to form biofilms is essential in its establishment and maintenance of infectious processes, e.g., foot ulcers in individuals with type 2 diabetes. S. aureus biofilm formation during infection in individuals with uncontrolled type 2 diabetes occurs in the presence of insulin and glucose. Bacterial infections, including surgical site infections (SSIs), are a common and serious complication of diabetes. Staphylococcus aureus is a major cause of SSI in diabetic patients [1,2]. However, the role of insulin and glucose in diabetes predisposing to staphylococcal infection is not fully elucidated. Biofilm formation and stability are dependent on the environment. Promotion, or inhibition, of biofilm formation occurs in response to available nutrients, including pathway metabolites, quorum-signaling compounds, and the genetic profile of the organism [3]. Temporal expression of many of the virulence determinants, including biofilm in S. aureus, is under the control of several genetic loci, including agr and sarA [4-6]. The accessory gene regulator (agr) is a critical system that controls population density-associated gene expression (quorum-sensing). At low cell densities, low-agr activity is associated with biofilm formation activity. When the quorum population density is reached, the agr system is activated, decreasing cell-surface colonization factors. The Staphylococcus accessory regulator A (SarA) system is a global regulator that promotes biofilm formation, such as the icaRA and bap operons, adhesion, and toxin production. SarA modulation of gene expression depends on environmental conditions. SarA enhances bacterial colonization, particularly in device-related and chronic infections. Conversely, in sarA mutants, the increased protease production can disrupt biofilms. SarA also activates the agr quorum sensing system to coordinate gene expression. SarA's role in biofilm formation is epistatic to agr. When agr is highly expressed, SarA's repression of proteases is the overriding factor that promotes biofilm formation. For example, in MRSA strains, such as USA300, which have high agr expression, sarA is required for robust biofilm development. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 093-096 94 2. Materials and Methods Glucose was dissolved and diluted in Luria Broth (LB) for use. Insulin (Humulin) was diluted in LB for use. Overnight cultures were inoculated into homologous LB to a final concentration of 105CFU/ml, then added to various concentrations of glucose and/or insulin in honey-comb plates (200µL/well). The plates were incubated for 24 hrs (37 °C , shaking). Controls consisted of organisms grown in LB alone. After incubation, the plates were washed, dried, and stained with crystal violet (300µl). Bound stain was dissolved in absolute alcohol (300µl) with absorbance determined (580nm). Controls consisted of organisms grown in LB alone. Experiments were done in quintuplets and repeated once. Data were evaluated by analysis of variance (ANOVA; GraphPad InStat 3.06 for Windows, GraphPad Software Inc.). Mean values were considered significantly different at p < 0.05 (*). Figure 1 Effect of insulin and glucose on biofilm formation by S. aureus. A. Parent strain 8325-4 biofilm formation in response to insulin and glucose. B. S. aureus agr-sarAbiofilm formation in response to insulin and glucose. C. S. aureus agrbiofilm formation in response to insulin and glucose. D. S. aureus sarAbiofilm formation in response to insulin and glucose. * SEM p < 0.05 as compared to homologous control 3. Results and Discussion Biofilm formation in S. aureus is regulated, in part, by sarA and agr via the extracellular signal of Agr, a posttranslationally modified peptide containing a thiolactone [7] (Figure 1 A-D). Previous studies have shown that insulin is an inter-kingdom quorum signaling molecule that, together with glucose, modulates biofilm formation [8-11]. This response of S. aureus is glucose and insulin-concentration specific [12]. Whether the insulin effect is also under the regulation of sarA in epistatic coordination with agr has not been determined. For the parent strain, 8325-4, the World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 093-096 95 presence of insulin alone did not significantly affect biofilm formation, as has been reported previously for quality control and clinical S. aureus isolates (Figure 1A) [12]. However, the addition of glucose 0.2% significantly increased biofilm production as compared to medium & Staphylococcus alone or glucose 0.1%. When glucose 0.2% and insulin 20µU/mL are given together, a synergistic positive effect on biofilm formation is measured. In contrast, insulin did not affect the double negative S. aureus agr-sarAstrain. Without agr and sarA (Figure 1B), S. aureus still responded to glucose in a concentration-specific manner with regard to its biofilm formation. Glucose at 0.2% resulted in significantly (p < 0.05) increased biofilm production. In contrast, the presence of sarA and absence of agr- (Figure 1C) restored the response to glucose 0.2% and insulin (20µU/mL or 200µU/mL) to that similar to the parent strain, but not to the addition of glucose 0.2% alone. Interestingly, the presence of agr together with the deletion of sarA (Figure 1D) resulted in a pattern of biofilm formation that, while significantly less (4-6-fold) than that measured for the double negative strain agr-sarA-, the pattern of biofilm formation in response to glucose was similar. Glucose 0.2% significantly increased biofilm production as compared to medium & staphylococcus at 0.1% glucose. Insulin, regardless of concentration, had no effect. 4. Conclusion The findings from this study indicate that sarA, in a glucose concentration-specific manner, plays a role in the regulation of S. aureus response to insulin modulation of biofilm formation. Furthermore, although agr is reported to be important in S. aureus response to endogenous quorum signaling compounds, it appears to be independent of insulin-mediated biofilm formation signaling response in S. aureus to glucose. In addition, biofilm formation appears to be regulated, in part, by sarA and agr. Taken together, these findings indicate that insulin is an inter-kingdom quorum signaling compound in S. aureus that plays a role in modulating biofilm formation, partially under the control of sarA. Compliance with ethical standards Acknowledgments We acknowledge the intramural funding support of Midwestern University, Office of Research and Sponsored Programs. Disclosure of conflict of interest The authors declare no conflicts of interest. Funding This research received no external funding. References [1] Ambrosch, A.; Haefner, S.; Jude, E.; Lobmann, R. Diabetic foot infections: microbiological aspects, current and future antibiotic therapy focusing on methicillin-resistant Staphylococcus aureus. International Wound Journal 2011, 8, 567-577, doi:10.1111/j.1742-481X.2011.00849.x. [2] Gupta, S.; Poret, A.J.; Hashemi, D.; Eseonu, A.; Yu, S.H.; D'Gama, J.; Neel, V.A.; Lieberman, T.D. Cutaneous surgical wounds have distinct microbiomes from intact skin. Microbiol Spectr 2023, 11, e03300-03322. [3] Schembri, M.A.; Kjærgaard, K.; Klemm, P. Global gene expression in Escherichia coli biofilms. Molecular Microbiology 2003, 48, 253-267, doi:10.1046/j.1365-2958.2003.03432.x. [4] Fujimoto, D.F.; Higginbotham, R.H.; Sterba, K.M.; Maleki, S.J.; Segall, A.M.; Smeltzer, M.S.; Hurlburt, B.K. Staphylococcus aureus SarA is a regulatory protein responsive to redox and pH that can support bacteriophage lambda integrase-mediated excision/recombination. Mol Microbiol 2009, 74, 1445-1458, doi:10.1111/j.13652958.2009.06942.x. [5] Podkowik, M.; Perault, A.I.; Putzel, G.; Pountain, A.; Kim, J.; Dumont, A.; Zwack, E.; Ulrich, R.J.; Karagounis, T.K.; Zhou, C., et al. Quorum-sensing agr system of Staphylococcus aureus primes gene expression for protection from lethal oxidative stress. eLife Sciences Publications, Ltd: 2024; 10.7554/elife.89098.3. [6] Chien, Y.-t.; Cheung, A.L. Molecular Interactions between Two Global Regulators, sar and agr, in Staphylococcus aureus *. Journal of Biological Chemistry 1998, 273, 2645-2652, doi:https://doi.org/10.1074/jbc.273.5.2645. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 093-096 96 [7] Le, K.Y.; Otto, M. Quorum-sensing regulation in staphylococci—an overview. Frontiers in Microbiology 2015, Volume 6 - 2015, doi:10.3389/fmicb.2015.01174. [8] Conlon, J. Evolution of the insulin molecule: insights into structure-activity and phylogenetic relationships. Peptides 2001, 22, 1183-1193. [9] Klosowska, K.; Plotkin, B. Human insulin modulation of Escherichia coli adherence and chemotaxis. American Journal of Infectious Diseases 2006, 2, 197-200. [10] LeRoith, D.; Shiloach, J.; Heffron, R.; Rubinovitz, C.; Tanenbaum, R.; Roth, J. Insulin-related material in microbes: similarities and differences from mammalian insulins. Can. J. Biochem. Cell Biol. 1985, 63, 839-849. [11] Plotkin, B.; Wu, Z.; Ward, K.; Nadella, S.; Green, J.; ., B.R. Effect of human insulin on the formation of catheterassociated E. coli biofilms. Open Journal of Urology, 2014, 4, 49-56., doi:doi: 10.4236/oju.2014.45009. [12] Plotkin, B.J.; Halkyard, S.; Spoolstra, E.; Micklo, A.; Kaminski, A.; Sigar, I.M.; Konaklieva, M.I. The Role of the Insulin/Glucose Ratio in the Regulation of Pathogen Biofilm Formation. Biology (Basel) 2023, 12, doi:10.3390/biology12111432.