Abstract
Antimicrobial resistance (AMR) is a global public health crisis, estimated to be associated with approximately 5 million deaths worldwide in 2019 alone. Despite this burden, the antibiotic discovery pipeline has slowed dramatically since the 1970s, and novel resistance phenotypes continue to emerge faster than new therapeutic agents can be brought to the clinic. Reflecting the urgency of this threat, the World Health Organization (WHO) has designated AMR among the most serious risks to global public health. A closer examination of the organisms identified by the WHO and the Centers for Disease Control and Prevention (CDC) as particularly concerning pathogens reveals that most are diderm bacteria, which possess two membrane layers in their cell envelope. The formidable cell envelopes of diderm bacteria are part of what makes them particularly challenging to target, because not only must an antibiotic effectively engage its target, but it must also reach the target site, most of which are intracellular. Consequently, a sound understanding of what drives molecules to permeate and accumulate effectively within the bacterial cellular milieu is critical for rational antibiotic design. However, current methods to decipher accumulation are limited in sensitivity, throughput, and/or physiological relevance, and these shortcomings have hampered efforts to establish meaningful structure-permeability relationships in the bacterial context. In this regard, the overarching goal of this thesis is to develop and apply novel methods to study and improve accumulation in diderm bacterial cells, with the aim of elucidating the chemical grammar that governs molecular permeability into these challenging pathogens.
Deciphering bacterial cell permeability begins with understanding the architecture of bacterial cell envelopes. Chapter 1 provides an overview and comparison of the cell envelopes of the three main bacterial groups – Gram-positive, Gram-negative, and mycobacteria. Importantly, the presence of vastly different cell envelope architectures across bacterial groups means that permeability rules established for one bacterial group do not necessarily transfer to another. In the same manner, rules governing the permeability of molecules across one layer of the cell envelope do not apply uniformly across other layers. As a result, approaches to improve accumulation into bacterial cells often need to be empirically assessed and iteratively optimized. Such efforts depend on the availability of relevant and reliable methods for measuring molecular accumulation. In this context, Chapter 2 examines the current landscape of strategies to improve accumulation into bacterial cells and outlines methods presently used to study it, highlighting their respective advantages and limitations.
The next chapters describe our efforts to develop high-throughput, physiologically relevant assays to study molecular accumulation in bacterial cells at subcellular resolution. Chapters 3 and 4 describe the development of bioluminescence-based assays to measure cytosolic accumulation of small molecules into Gram-negative bacteria and mycobacteria, respectively. These assays enable real-time, kinetically resolved tracking of cytosolic entry, offering valuable mechanistic insight into accumulation. For mycobacteria in particular, this work fills a longstanding methodological gap by enabling the direct study of cytosolic accumulation with defined subcellular localization.
Chapter 5 turns to the question of how molecular design can be used to overcome the mycomembrane, which is the principal permeability barrier in mycobacteria. We systematically investigated macrocyclization as a rational permeability strategy using an assay our lab recently reported - the Peptidoglycan Accessibility Click-Mediated Assessment (PAC-MAN) assay, which benefits from a smaller chemical tag. We showed for the first time that peptide macrocyclization generally improves accumulation beyond the mycomembrane. Furthermore, examination of two peptide antibiotics, tridecaptin A1 and griselimycin, revealed that macrocyclization plays a context-dependent role in enhancing both accumulation and antimicrobial activity against mycobacteria.
Finally, Chapter 6 describes a bioluminescence-based assay to simultaneously track the real-time uptake and intracellular unmasking of carboxylate esters in live mycobacterial cells. Systematic variation of the esterase-cleavable motif enabled us to quantitatively assess esterase substrate preferences and cytosolic uptake. Such analyses can help guide ester-based prodrug strategies against mycobacteria.
