Biochemical Characterization of Cardiolipin Synthase Mutations Associated with Daptomycin Resistance in Enterococci

Abstract

dDaptomycin (DAP) resistance in enterococci has been linked to mutations in genes that alter the cell envelope stress response (CESR) (liaFSR) and changes in enzymes that directly affect phospholipid homeostasis, and these changes may alter membrane composition, such as that of cardiolipin synthase (Cls). While Cls substitutions are observed in response to DAP therapy, the effect of these mutations on Cls activity remains obscure. We have expressed, purified, and characterized Cls enzymes from both Enterococcus faecium S447 (residues 52 to 482; Cls447a) and Enterococcus faecalis S613 (residues 53 to 483; Cls613a) as well as Cls variants harboring a single-amino-acid change derived from DAP-resistant isolates of E. faecium. E. faecium Cls447a and E. faecalis Cls613a are tightly associated with the membrane and copurify with their substrate, phosphatidylglycerol (PG), and product, cardiolipin (CL). The amount of PG that copurifies with Cls is in molar excess to protein, suggesting that the enzyme localizes to PG-rich membrane regions. Both Cls447a H215R and Cls447a R218Q showed an increase in V max (M CL/min/M protein) from 0.16 ؎ 0.01 to 0.26 ؎ 0.02 and 0.26 ؎ 0.04, respectively, indicating that mutations associated with adaptation to DAP increase Cls activity. Modeling of Cls447a to Streptomyces sp. phospholipase D indicates that the adaptive mutations Cls447a H215R and Cls447a R218Q are proximal to the phospholipase domain 1 (PLD1) active site and near the putative nucleophile H217. As mutations to Cls are part of a larger genomic adaptation process, increased Cls activity is likely to be highly epistatic with other changes to facilitate DAP resistance.

The action mechanism of daptomycin

“…Indeed, cardiolipin synthase mutants have been isolated from resistant mutants of Staphylococcus aureus 91 as well Enterococcus species. 99 Some of these mutants have been shown to have increased activity, 100 but their substitution into the genome of a daptomycin-susceptible strain did not detectably increase resistance. 101 A study that comprehensively characterized the membrane lipid compositions of daptomycin-resistant Staphylococcus aureus strains found the expected decrease in PG, as well as an increase in lysyl-PG, but no significant change in CL.…”

Resistencia a antibióticos de última línea en cocos Gram positivos: la era posterior a la vancomicina

“…El segundo gen corresponde al cls, el cual codifica para la sintetasa de cardiolipina (Cls) y ha sido postulado, asimismo, como responsable de la aparición de resistencia a la daptomicina en E. faecium y en S. aureus (véase el acápite anterior). Algunos estudios recientes han señalado que las sustituciones observadas en esta enzima incrementan su actividad catalítica, lo que podría actuar directamente sobre el fosfatidilglicerol de la membrana celular (117). El papel de los cambios en la Cls no está del todo claro y hay información contradictoria en la literatura científica.…”

A Current Perspective on Daptomycin for the Clinical Microbiologist

“…Paradoxically, introduction of this same mutation by allelic exchange into a daptomycin-susceptible E. faecium strain had no effect on the daptomycin MIC (162), although R281Q mutation has been observed in numerous daptomycin-NS E. faecium isolates ( Table 5). The activity of purified, recombinant Cls from E. faecium harboring R218Q and H215R mutations was assessed in vitro (167). Both enzymes were associated with increased cardiolipin production compared to that with the wild-type enzyme (167), but differences in cardiolipin content were not observed when daptomycin-NS and -susceptible E. faecium and E. faecalis strains were evaluated (168).…”

“…The Cls enzyme is comprised of two N-terminal putative transmembrane helices and two phosphatidylcholine-hydrolyzing phospholipase domains (PLDs). In Enterococcus (and Staphylococcus), each of the two PLD domains harbors a single conserved histidine kinase motif with an H217 putative active-site nucleophile (167). Various mutations to cls, predominantly in the region of the gene that encodes the N-terminal transmembrane helix, the linker region between the transmembrane domains and the PLD domains, and PLD1, have been observed in both in vivo-and in vitro-selected daptomycin-NS E. faecium and E. faecalis (Table 5).…”