Investigation of Bacterial Cell Surface Accessibility and Peptidoglycan Interactions

Antibiotic resistance is a threat to public health that demands to be met with either new and innovative antibiotics or alternative therapies. The occurrence of antibiotic resistant bacteria is a multifactorial process but often involves improper use of antibiotics or sharing of genetic components that allow bacteria to survive when challenged with antimicrobial agents. Specific resistance mechanisms usually fall under one of the following categories: inactivation of drug, target modification, active efflux, and limiting uptake. All of the antibiotics that have been assembled in arsenal against bacterial pathogens during the “Golden Age” of antibiotic discovery have been met with resistance and many are now losing clinical efficiency. The ongoing antibiotic crisis continues to worsen as many challenges impede further drug discovery and development. In order to develop new treatment options, a more in-depth understanding of bacterial biology and resistance mechanisms is needed.

An unappreciated mechanism of resistance that relates to limiting uptake is accessibility. It is evident that bacteria are actively protecting the cell surface, the site of many prominent antigens and binding sites, and that there is a lapse in understanding of the requirements to reach the surface of those cells. This dissertation defense will describe a novel assay that aims to assess accessibility to the surface of live cells in a systematic and high-throughput way. Additionally, modulations to the bacterial cell surface were introduced in order to determine what factors, if any, impede accessibility. The assay workflow relies on site specific incorporation a free thiol handle onto the peptidoglycan layer of the cell wall and use of a library of compounds that range in size and flexibility with a bioorthogonal binding moiety and a fluorescent reporter molecule that can be tracked via flow cytometry. The assay is able to quantitatively report on the ability of each library member to reach the bacterial cell wall. An application of the accessibility assay, to a screen of a transposon mutant library in effort to reveal genes that impact accessibility, will also be detailed.

A novel assay platform called SaccuFlow that allows for a high-throughput and quantitative investigation into bacterial peptidoglycan interactions with a range of molecules will also be discussed. For the first time whole native isolated peptidoglycan or sacculi from any organism is demonstrated to be compatible with flow cytometry analysis which is key to the assay. The utility of the assay is demonstrated in a high-throughput pilot screen for inhibitors of an important Staphylococcus aureus enzyme, sortase A which covalently installs bacterial proteins onto the cell wall. Sortase A is a promising target for anti-virulence drug discovery.