DMS-Guided Structural Biology Approaches to Durable Streptogramin A Antibiotics

The growing prevalence of multidrug resistant pathogens is drastically diminishing our supply of effective antibiotics, in turn increasing the risk associated with bacterial infections. A contributing factor to this issue is how rapidly bacteria adapt to restore the resistant phenotype. Regardless of the strength of the antibiotic concoction, the capability of evolved resistance will always remain a lingering threat. This highlights an important question: How can we design antibiotics that are more resilient to evolved resistance? I have studied this question in the context of group A streptogramin (SA) analogs, antibiotics which bind the peptidyl transferase center (PTC) of the bacterial ribosome and are deactivated by resistance enzymes like VatA, which adds an acetyl group to the macrocycle and prevents ribosomal binding.In Chapter 1, I explore how we can overcome VatA-mediated resistance by leveraging newly developed techniques in the Seiple lab that can create fully synthetic SA analogs with great diversity. Using a combination of cryogenic electron microscopy (Cryo-EM) and molecular biology techniques, we have developed a pipeline to optimize and identify new SA antibiotics that overcome resistance mediated by wild type VatA while not compromising ribosomal inhibition. Our iterative design strategy led us to identify several SA analogs that overcome resistance mediated by wild type VatA and show improved ribosomal binding. Our best performer, analog 47, was shown in a murine thigh model of infection to be 20X more effective at reducing the bacterial load of VatA-expressing S. aureus than flopristin, a comparative semisynthetic SA derivative. Crystallography shows that analog 47 reshuffles the binding site of VatA, which may explain the reduced kcat/Km of VatA at acetylating this analog compared to flopristin. Finally, energy calculations show that binding of 47 in the ribosome is of lower energy than in VatA. Despite these encouraging results, it is important to remember that, given a selective pressure, bacteria will evolve to restore their ability to grow and proliferate without hindrance in the new environment. In our case, this means that treatment of analog 47 on wild type VatA-expressing bacteria would encourage the directed evolution of VatA variants that restore the ability to deactivate the new antibiotic. Therefore, in Chapter 2, I endeavor to explore the effects of single amino acid mutations in VatA on the survival of bacteria grown in the presence of our best SA analogs and their stepwise scaffolds using Deep Mutational Scanning (DMS) and Next-Generation Sequencing (NGS). Our results revealed several strong gain of function mutations in the C-terminal domain, a region distal to the active site. We hypothesize that mutations in this region, which shows poor sequence conservation to other related Vat enzymes, likely contribute to the stability and folding of VatA. Both of these chapters highlight our efforts to improve the durability of SA antibiotics. As demonstrated in Chapter 1, success in improving SA antibiotics’ performance can be further magnified by administering them with their binding partner: group B streptogramins (SB). When used together, SA and SB act synergistically and induce a bactericidal effect. In Chapter 3, I discuss recent work exploring the potential for these two antibiotics to perform click chemistry on the ribosome and form a covalently linked molecule. Throughout my graduate career, I have also implemented Cryo-EM in various collaborations to study the interactions between analogs of other antibiotic classes and the E. coli ribosome. I highlight my contributions to these works in Chapter 4