Biophysical modeling
Fluorescent recovery after photobleaching (FRAP) is a common experimental technique used to determine the diffusion coefficient of a molecule through a medium. The most prevalent modeling used to determine this diffusion coefficient is phenomenological, often assuming that the recovery curve takes the form of an exponential function and using values such as the mobile or immobile fraction and time it takes to reach half the maximum intensity to describe the diffusion. While one can still extract useful information from this type of analysis, the effective diffusion coefficient from fitting an exponential to the data does not reveal deeper information about the mechanisms leading to the diffusion patterns observed. We propose a newly derived model that allows more mechanistic understanding of the diffusion observed as the fluorescence recovers. The model accounts for potential binding of the molecule to either one or two separate entities in the medium that can alter the diffusion of the molecule, generating 2-state and 3-state models. Though previous models have also incorporated binding effects, our model is a simpler method to derive the fluorescent recovery in such cases and provides a framework that can be extended to even more scenarios of binding events. Applying these models to more complex FRAP experiments will lead to greater information gained per experiment. Lastly, we leverage these models to support the hypothesis that positively-charged antibiotics can be sequestered by sputum biopolymers and bacteriophage.
Articles
Q Chen, PC Cai, THW Chang, E Burgener, MJ Kratochvil, A Gupta, A Hargill, PR Secor, JE Nielsen, AE Barron, SC Heilshorn, AJ Spakowitz, PL Bollyky. “Pf bacteriophages hinder sputum antibiotic diffusion via electrostatic binding.” Science Advances.
Despite great progress in the field, chronic Pseudomonas aeruginosa (Pa) infections remain a major cause of mortality in patients with cystic fibrosis (pwCF), necessitating treatment with antibiotics. Pf is a filamentous bacteriophage produced by Pa and acts as a structural element in Pa biofilms. Pf presence has been associated with antibiotic resistance and poor outcomes in pwCF, although the underlying mechanisms are unclear. We have investigated how Pf and sputum biopolymers impede antibiotic diffusion using pwCF sputum and fluorescent recovery after photobleaching. We demonstrate that tobramycin interacts with Pf and sputum polymers through electrostatic interactions. We also developed a set of mathematical models to analyze the complex observations. Our analysis suggests that Pf in sputum reduces the diffusion of charged antibiotics due to a greater binding constant associated with organized liquid crystalline structures formed between Pf and sputum polymers. This study provides insights into antibiotic tolerance mechanisms in chronic Pa infections and may offer potential strategies for novel therapeutic approaches.