Date of Award
Doctor of Philosophy (PhD)
Daptomycin, a last line-of-defense antibiotic for treating Gram-positive infections, is experiencing clinical failure against important infectious agents, including Corynebacterium striatum. The recent transition of daptomycin to generic antibiotic status is projected to dramatically increase availability, use, and clinical failure. Despite daptomycinճ more than 30-year history as an important antibiotic, four major questions were left unanswered. 1) How do bacteria become hyper-resistant to daptomycin? 2) What is the in vivo membrane target of daptomycin? 3) How does daptomycin interact with the membrane? 4) What is daptomycinճ mechanism of killing? These four questions have plagued the daptomycin field, and even now conflicting mechanisms have been reported in the literature. This thesis has focused on teasing apart the intricacies of these four questions.
To answer the first two questions, regarding 1) hyper-resistance to daptomycin and 2) the in vivo membrane target of daptomycin, we identified the mechanism of high-level daptomycin resistance in C. striatum, which helped us to identify phosphatidylglycerol as the in vivo target of daptomycin. Here, we confirm the genetic mechanism of high-level daptomycin resistance (HLDR, MIC > 256 g/mL) in C. striatum, which evolved within a patient during daptomycin therapy, a phenotype recapitulated in vitro. In all eight independent cases tested, loss of function mutations in phosphatidylglycerol synthase (pgsA2) were necessary and sufficient for high-level daptomycin resistance. Through lipidomic and biochemical analysis, we demonstrate that the activity of daptomycin is dependent on membrane phosphatidylglycerol (PG) concentration. Until now, the verification of PG as the in vivo target of daptomycin has proven difficult, as tested cell model systems were not viable without membrane PG. C. striatum becomes high-level daptomycin resistant by removing PG from the membrane and changing membrane composition to maintain viability. This work demonstrates that a single loss of function mutation in pgsA2, and the loss of membrane PG is necessary and sufficient to produce high-level resistance to daptomycin in C. striatum and result in daptomycinճ clinical failure.
Even though there have been numerous studies looking at daptomycinճ interaction with the membranes, several problems with the model systems and methods used have plagued the field, casting doubt and uncertainty on 3) how daptomycin interacts with the membrane, and 4) how daptomycin kills bacteria. Previous studies have used model membranes that are too large (giant unilamellar vesicles that are 100x bigger than bacteria), indirect measurements through fluorescence, unknown PG and acyl tail requirements for model systems, and chemically altered daptomycin that no longer behaves like daptomycin (as indicated by a drastic increase in minimum inhibitory concentration compared to native daptomycin). The data reported here demonstrates a shift in how we understand the interactions of daptomycin with the bacterial cell. Once daptomycin binds to the membrane, it is stably integrated in a calcium- and PG-dependent manner (~1:1 daptomycin to PG). It then recruits or co-localizes with other daptomycin molecules to form stable pores with an approximate Stokes radius of 8 nm, which is far larger than the previously reported ion channels. The resulting pore allows metabolites, ions and other intracellular components to exit the cell. When enough PG is present in the bacterial membranes, enough daptomycin pores can be formed to release necessary metabolites, ultimately resulting in cell death.
In summary, 1) bacteria become high-level resistant to daptomycin by removing daptomycinճ 2) in vivo target, phosphatidylglycerol (PG), from the membrane. 3) In the presence of PG, daptomycin binds and integrates into the membrane to form a large pore that 4) disrupts the membrane potential and allows intracellular metabolites, ions and other important molecules to leak out of the cell, ultimately killing the bacteria.
Chair and Committee
Rob Mitra, Shabaana A. Kahder, Paul H. Schlesinger, Carey-Ann D. Burnham,
Goldner, Nicholas Kevork, "The Mechanism of Hyper Daptomycin Resistance in Corynebacterium striatum and Daptomycinճ Mechanism of Action" (2018). Arts & Sciences Electronic Theses and Dissertations. 1623.