The Physiology and Transport of Guanidinium (Gdm+) and Metformin Metabolites of Bacterial Guanidinium Exporters (Gdx)

Abstract

Membrane proteins are a crucial interface between the intracellular and extracellular environments and play a vital role in microbial pathogenesis, ranging from dental plaque buildup to complications in medical transplants. The necessity for new antimicrobial strategies to combat persistent microbial infections has never been more urgent. Microbial resistance can be often facilitated by horizontal gene transfer (HGT). Small Multidrug Resistance (SMR) transporters are frequently found in multidrug resistant gene arrays in environments such as wastewater and human-adjacent ecosystems. Recent research implicates certain SMR transporters in metabolism of the pharmaceutical metformin by bacterial consortia. These ~100 residue proteins assemble as antiparallel dimers with four transmembrane helices per monomer that are deeply embedded in the membrane. My thesis focuses on the role of these SMR transporters in evolutionary adaptation and transport of the previously overlooked metabolite, guanidinium (Gdm+). Therefore, the work presented in each chapter and appendix, involve the study of membrane proteins to gain better insight into how bacteria adapt and evolve to selective pressures through in vitro and in vivo assays. Recent evidence has shown that bacteria can sense Gdm+, use it as a nitrogen source, and export it using guanidinium exporters (SMRGdx), underlining the importance of Gdm+ homeostasis in bacterial physiology. In this thesis, I show that both genomic and plasmid-associated SMR transporters export byproducts of microbial metformin metabolism, with particularly high export efficiency for guanylurea. Biochemical, electrophysiological, biophysical, and structural biological approaches were used to characterize the transport and binding kinetics of four representative SMRGdx homologues. A guanylurea-bound x-ray crystal structure for Gdx-Clo, a recently characterized SMR homologue, established a functional framework that will inform future mechanistic studies of this model transport protein. Our findings demonstrate how native transport physiologies are co-opted to contend with new selective pressures. This thesis also explores the biological origins and physiological impacts of Gdm+ on bacteria, revealing that accumulation negatively affects the fitness of E.coli strains with a genetic deletion of the guanidinium exporters. Through competition assays between the strains, we exploited the distinct differences in phenotypes to identify metabolic pathways potentially involved in Gdm+ production. We also investigated the impact of Gdm+ accumulation on other cellular pathways, particularly those related to virulence, through assays involving planktonic growth, resistance, swimming, and biofilm formation. Our findings suggest a Gdm+ link to nitrogen availability and virulence, pathways that appear to be particularly sensitive to this metabolite. The sensitivity exposes a potential vulnerability in microbes that could serve as an alternative strategy for combating antimicrobial resistance. Further research is essential to elucidate the exact mechanistic basis for Gdm+ sensitivity.