A Multi-Scale Study of Antimicrobial Peptide Action Beyond Steady-State Growth - PROJECT SUMMARY In this project, we address a critical and unresolved challenge in antimicrobial peptide (AMP) research: understanding how AMPs perform under physiologically relevant conditions that mimic the bacterial states found during infections. AMPs are a unique class of immune-derived molecules that kill bacteria by physically disrupting membranes and also function as immunomodulators, impacting host responses to infection and tissue damage. Despite decades of progress in AMP research, including the discovery of natural peptides, characterization of AMP–lipid interactions, and investigations into their immunological functions, no AMP has reached routine clinical use. A key knowledge gap is how AMPs act on cells that are embedded in dense structures or limited by nutrient and growth constraints. Historically, most laboratory assays have focused on exponentially growing, planktonic cells—conditions that fail to capture the complexity of real infections. Real infections involve dense bacterial communities, biofilms, and metabolically inactive subpopulations that are tolerant to conventional antibiotics. AMPs are widely believed to overcome this limitation and kill non- growing cells due to their membrane-targeting mechanisms. However, this claim remains largely untested in rigorous, quantitative models. Even basic questions—such as whether AMPs retain potency against stationary- phase or dormant bacteria—remain controversial. To advance the field, we must develop protocols and tools that shift experimentation toward infection-relevant structures and states, generating data that define AMP activity in those contexts. This project integrates custom microfluidics, single-cell live imaging, and transcriptomic analysis to evaluate AMP performance in structured and metabolically diverse bacterial populations. Using Escherichia coli and Bacillus subtilis as model organisms, we will pursue three aims: (1) develop microfluidic platforms to grow dense bacterial colonies and biofilms and quantify AMP diffusion and killing profiles; (2) compare AMP susceptibility of growing and stationary-phase cells at the single-cell level; and (3) use RNA-sequencing to characterize transcriptional responses to AMP treatment across different metabolic states. The results will directly address how AMPs perform under suboptimal growth conditions. Ultimately, these efforts will help shift the AMP field beyond theoretical potential toward practical application. The tools developed in this work will be broadly applicable to other antimicrobial studies, enabling systematic testing of efficacy against dormant cells, biofilms, and intracellular targets. In parallel, this project will train undergraduate and graduate students in California State University, Northridge, in advanced microbiological and quantitative techniques, including microfluidics, microscopy, and transcriptomics. This training aligns with the SuRE program’s mission to expand access to research opportunities and build a diverse biomedical workforce.
