Uncovering silver proteome enables in-depth deciphering its molecular mechanisms

Abstract

Silver compounds have been historically used for the treatment of burns, ulcerations, and infected wounds. Silver and silver nanoparticles (AgNPs) are nowadays widely used in healthcare, food industry, hard surface materials and textiles. Despite the broad-spectrum antimicrobial activities of silver, its internal usage is restricted owing to the toxicity. Strategies to enhance its efficacy are highly desirable and rely heavily on understanding of its molecular mechanism of action. However, up to now, no direct silver-binding proteins have been mined at proteome-wide scale due to the limitations of current metalloproteomic approaches, which hinders systemic studies on the biological pathways interrupted by silver. A novel approach named LC-GE-ICP-MS is developed and validated to match metal and proteins with improved resolution. For the first time, 34 Ag+-binding proteins are identified in the model Gram-negative bacterium E. coli. Further analysis with bioinformatics, metabolomics and biochemical tests demonstrated that silver primarily targets enzymes involved in the early steps of Krebs and glucose-alanine cycles, subsequently perturbs oxidative stress defense systems via inactivation of key antioxidant enzymes in E. coli. Importantly, co-administration of metabolites involved in Krebs and glucose-alanine cycles significantly potentiates silver efficacy to E. coli. Silver proteome including 38 Ag+-binding proteins in the whole cell scale of S. aureus were further separated and identified by the newly designed LC-GE-ICP-MS. Comparison of Ag+-binding protein profiles and identities in Gram-positive and Gram-negative bacteria showed that both similarity and discrepancy were observed. Even though Ag+ targets both glycolysis and TCA pathways in Gram-positive and Gram-negative bacteria, the identified Ag+-binding proteins involved in these pathways in two bacteria are distinct. The binding of Ag+ to global regulators of CcpA and CodY were identified for the first time in S. aureus. The antibacterial activities of AgNPs with three sizes (N10, N42 and N102) showed that AgNPs with smaller diameters exhibit higher bactericidal activity in both Gram-positive S. aureus and Gram-negative E. coli cells. Exploration of AgNPs binding proteins in both bacteria demonstrated that N10 and Ag+ have similar profiles of silver-binding proteins in S. aureus and E. coli. While for N42 and N102, both of them preferentially bind to proteins with smaller MWs and relatively lower intensities. The same identity but different 107Ag intensity of the silver-binding proteins in AgNPs- and Ag+-treated bacteria revealed that AgNP with smaller sizes are able to bind to more proteins with relatively higher 107Ag signals, which might contribute to the higher antibacterial activity of small AgNPs. Even though N10 have a lower release of Ag+ than AgNO3, both high and low concentrations of N10 induced similar metabolic alterations as Ag+ did in E. coli and even more profound metabolic changes in S. aureus, demonstrating the nano-effect originated from the specific chemical and physical properties of AgNPs could contribute the antibacterial activities. The study in this thesis provides a strategy to enhance silver bactericidal effects, offers a novel and general approach for deciphering molecular mechanism of silver and other metallodrugs in various pathogens and cells to facilitate the development of new therapeutics.