Browsing by Author "Supandy, Adeline"

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    Enolpyruvate transferase MurAAA149E, identified during adaptation of Enterococcus faecium to daptomycin, increases stability of MurAA–MurG interaction
    (Elsevier, 2023) Zhou, Yue; Utama, Budi; Pratap, Shivendra; Supandy, Adeline; Song, Xinhao; Tran, Truc T.; Mehta, Heer H.; Arias, Cesar A.; Shamoo, Yousif
    Daptomycin (DAP) is an antibiotic frequently used as a drug of last resort against vancomycin-resistant enterococci. One of the major challenges when using DAP against vancomycin-resistant enterococci is the emergence of resistance, which is mediated by the cell-envelope stress system LiaFSR. Indeed, inhibition of LiaFSR signaling has been suggested as a strategy to “resensitize” enterococci to DAP. In the absence of LiaFSR, alternative pathways mediating DAP resistance have been identified, including adaptive mutations in the enolpyruvate transferase MurAA (MurAAA149E), which catalyzes the first committed step in peptidoglycan biosynthesis; however, how these mutations confer resistance is unclear. Here, we investigated the biochemical basis for MurAAA149E-mediated adaptation to DAP to determine whether such an alternative pathway would undermine the potential efficacy of therapies that target the LiaFSR pathway. We found cells expressing MurAAA149E had increased susceptibility to glycoside hydrolases, consistent with decreased cell wall integrity. Furthermore, structure–function studies of MurAA and MurAAA149E using X-ray crystallography and biochemical analyses indicated only a modest decrease in MurAAA149E activity, but a 16-fold increase in affinity for MurG, which performs the last intracellular step of peptidoglycan synthesis. Exposure to DAP leads to mislocalization of cell division proteins including MurG. In Bacillus subtilis, MurAA and MurG colocalize at division septa and, thus, we propose MurAAA149E may contribute to DAP nonsusceptibility by increasing the stability of MurAA–MurG interactions to reduce DAP-induced mislocalization of these essential protein complexes.
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    Experimental Evolution as a Tool in Investigating Antibiotic Resistance and Antimicrobial Discovery
    (2023-04-20) Supandy, Adeline; Shamoo, Yousif
    The discovery of antibiotics was one of the greatest healthcare advances in history. However, its success is being challenged by the emergence of multidrug-resistant (MDR) pathogens. In addition to the misuse and overuse of antibiotics, the lack of a robust antibiotic development pipeline also contributes to the worldwide increase in antibiotic resistant pathogens. Due to these reasons, the rise of MDR pathogens has become one of the fastest growing medical problems of this century, leading to the urgent need for new strategies to control antibiotic resistance. Our lab has long been interested in using experimental evolution as a method to investigate approaches to slowing down antibiotic resistance. Adaptive evolution can be used to induce resistance in bacteria which will allow us to investigate the molecular and genetic mechanisms underlying antibiotic resistance. This knowledge can then be used as potential targets for drug development and potentially control the increase in antibiotic resistance. One of the rapidly emerging MDR pathogens, vancomycin-daptomycin resistant Enterococcus faecium, is an attractive bacterial strain to study using experimental evolution. As Daptomycin (DAP) is a drug of last resort being used to treat enterococcal infections, the combination of DAP with another antibiotic, Fosfomycin (FOS), has been proposed as a potential method to maintain DAP efficacy. Here, I provide evidence that DAP-FOS combination (DF) successfully delayed the emergence of DAP resistance in vitro. Genetic data indicate that resistance was acquired independently, with little evidence of significant cross-drug epistasis that could undermine a combinatorial approach. I also uncovered a novel FOS resistance mechanism, through changes in phosphoenolpyruvate (PEP) flux, that may potentially be shared with other bacterial species. Additionally, I also have evidence showing that E. faecium was able to develop DAP resistance through a variety of biochemical mechanisms and was able to employ different adaptive strategies. In addition to studying antibiotic resistance development, experimental evolution, in conjunction with microfluidics, can also be used as a platform for discovering new antimicrobials. By exposing an antibiotic producing strain (Producer) against its predator (Reporter), the Producer strain can be pressured to evolve and produce either a new antimicrobial or a modified known antibiotic that produces better activity to compete against its predator for resources. While the microfluidics platform can allow for high-throughput screening, a previous lab member had found that small molecules such as antibiotics can freely diffuse between the microdroplets. Antibiotics diffusing across the droplets will severely complicate our efforts to enrich for solely the antibiotic-producing strain, which is required for future studies. In this thesis, I will also be exploring the use of activated charcoal in suppressing the diffusion of small molecules. I was able to show that activated charcoal can successfully bind to a small quorum-sensing molecule that diffuses rapidly, AHL. In addition, I found that the sensitivity of the reporter system and media complexity drastically changes the amount of activated charcoal required to achieve significant suppression. Reporter system that are less sensitive and less complex media required less activated charcoal in the system to achieve suppression. I also have evidence showing that activated charcoal activity was retained in both bulk and microdroplet environments. Finally, I was also able to show that activated charcoal is also capable of binding antibiotics across different classes and molecular sizes. These findings support the rationale of adding activated charcoal to the microfluidic-based experimental evolution pipeline of discovering new antimicrobials.