Browsing by Author "Zhou, Yue"
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Item 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, YousifDaptomycin (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.Item Structural and Biochemical Studies of MurAA, an Enolpyruvate Transferase that Contributes to Cellular Fitness During Daptomycin Attack in Enterococcus faecium(2022-04-15) Zhou, Yue; Shamoo, Yousif; Tao, Yizhi JaneDaptomycin is an antibiotic frequently used as drug of last resort against Gram-positive Multi-Drug Resistant pathogens such as vancomycin-resistant enterococci. Unsurprisingly, as the use of daptomycin has increased, the number of pathogens evolving resistance has increased as well. Inhibition of the major antibiotic stress pathway (LiaFSR) responsible for daptomycin resistance has been suggested as a strategy to retain the efficacy of daptomycin. Such a strategy was shown to be effective in vitro, however alternative pathways to resistance were identified, including some with adaptive mutations in MurAA (MurAAA149E). MurAA catalyzes the first committed step in peptidoglycan biosynthesis. Cells expressing MurAAA149E has increased susceptibility to glycosyl hydrolases consistent with decreased cell wall integrity. High-resolution structures of MurAA in complex with the inhibitor, fosfomycin, and substrate, UDP-N-acetylglucosamine along with steady-state substrate kinetics, substrate and inhibitor binding assays indicated a modest decrease in MurAAA149E activity. Interestingly, MurAAA149E had a 16-fold increase in affinity for MurG. MurG performs the last intracellular step of peptidoglycan synthesis. Daptomycin attack leads to mislocalization of critical cell division proteins including MurG. In Bacillus subtilis, MurAA and MurG localize at division septa and thus MurAAA149E may contribute to cellular fitness during DAP exposure by increasing the stability of MurAA-MurG interactions at the division septa, but in doing so decreases cell resistance to attack by hydrolases. Our findings suggest that an adaptive strategy to circumvent LiaFSR inhibition through changes in MurAA may be ineffective in vivo as cellular hydrolases are an important component of the host response to pathogens.