PROJECT SUMMARY/ABSRACT Bacterial antibiotic resistance is a substantial public health threat, currently responsible for more than one million deaths globally and predicted to escalate dramatically in the coming decades. As multidrug resistant strains continue to emerge, it is imperative that we identify new antibiotic targets, design new antibiotics, and/or find ways to resuscitate the activity of existing drugs for which resistance has become widespread. For example, the aminoglycosides are a historically important class of ribosome-targeting antibiotics whose use is threatened by both well-established mechanisms (e.g. aminoglycoside modifying enzymes) and the more recent emergence of acquired aminoglycoside-resistance 16S rRNA methyltransferases. This latter group of enzymes chemically modify the 16S rRNA in the ribosome decoding center (where mRNA is decoded) in the small ribosomal (30S) subunit, conferring resistance to entire classes of aminoglycosides, including the most recently approved drugs (e.g. plazomicin). Identifying ways to counter this mechanism of aminoglycoside resistance is thus of paramount importance. In addition to being the target of the acquired aminoglycoside-resistance methyltransferases, the ribosomal decoding center is also modified by intrinsic (housekeeping) methyltransferases which may also influence aminoglycoside action. Loss of the intrinsic methyltransferases RsmH and RsmI, for example, is associated with increased susceptibility to aminoglycosides. However, we currently do not know how these two enzymes recognize the 30S subunit to methylate the same target residue (16S rRNA C1402), the full extent of how they influence aminoglycoside susceptibility, and whether they impact aminoglycoside-resistance methyltransferase activity and thus the resistance these acquired enzymes confer. In this project, I will test my overall hypothesis that loss of methylation of C1402 by RsmH and RsmI induces rRNA structural rearrangements that both increase aminoglycoside susceptibility and reduce resistance methyltransferase activity. I will test this hypothesis through the following three complementary Specific Aims. Aim 1–I will determine the mechanism of dual C1402 modifications by RsmH and RsmI using high resolution cryo-EM studies complemented with mutagenesis studies and in vitro methylation assays. Aim 2–I will define how C1402 modifications influence aminoglycoside susceptibility for a broad panel of aminoglycosides using a combination of microbiological assays along with high-resolution structural studies and molecular dynamics simulations. Aim 3–I will define the effects of C1402 methylation on acquired aminoglycoside-resistance methyltransferase activity and ability to confer resistance using in vitro methylation assays in addition to a range of microbiological studies. Defining how RsmI and RsmH function and how they influence aminoglycoside susceptibility and resistance will enhance our understanding of intrinsic dec...