Graduate Student Aarshi Singh

"Insights into Gram-Negative Bacteria: Innovative Membrane Mimics and Secreted Vesicles"

Antimicrobial resistance, specifically in gram-negative bacteria, constitutes a great threat to the healthcare system, since these pathogens are increasingly becoming resistant to the current last resort antibiotics. Gram-negative bacteria are distinguished from gram-positive bacteria by their dual membrane structure, where the outer membrane provides an additional barrier from the environment and antibiotic threats. Analyzing the outer membrane poses challenges due to its complex composition. Moreover, small outer membrane segments are secreted as vesicles called outer membrane vesicles (OMVs), adding further complexity in membrane analysis. New methods that allow for the mimicking of the outer membrane and its incorporation in the analytical methods, as well as exploring OMVs to harness their full potential are required. Due to their ability to trigger an immune response, OMVs are sought as therapeutic agents. However, due to their inherent heterogeneity, their further use and potential remains limited. Addressing nanoscale heterogeneity can be challenging and expensive to detect. To address this, here we developed a multivariate single OMV analysis approach, utilizing fluorescence microscopy, enabling the detection of size- based heterogeneities. With the escalating antimicrobial resistance, new antibiotic design approaches are required. Antimicrobial peptides are next-generation antibiotics that interact with the membrane of the bacteria, however, there are limitations in studying their efficacy since mimicking the bacteria membrane is complicated. Here, we will describe two distinct methods that we have developed to create bacterial membrane mimics using either OMVs or chaotropic agents. Additionally, we will demonstrate how the resultant bilayers can be used to test antimicrobial peptides interaction. These novel methods for bacterial membrane mimics and multivariate single OMV analysis, advance the current understanding of the outer membrane and contribute to the development of future therapeutic strategies.