Structural and regulatory changes in PBP4 trigger decreased ß-lactam susceptibility in Enterococcus faecalis Articles uri icon

authors

  • RICE, LOUIS B.
  • DESBONNET, CHARLENE
  • TAIT-KAMRADT, AMELIA
  • GARCIA SOLACHE, MONICA ADRIANA
  • LONKS, JOHN
  • MOON, THOMAS M.
  • D’ANDRÉA, ÉVERTON D.
  • PAGE, REBECCA
  • PETI, WOLFGANG

published in

publication date

  • May 2018

start page

  • e00361-18

issue

  • 2

volume

  • 9

International Standard Serial Number (ISSN)

  • 2161-2129

Electronic International Standard Serial Number (EISSN)

  • 2150-7511

abstract

  • Enterococcus faecalis strains resistant to penicillin and ampicillin are rare and have been associated with increases in quantities of low-affinity penicillin-binding protein 4 (PBP4) or with amino acid substitutions in PBP4. We report an E. faecalis strain (LS4828) isolated from a prosthetic knee joint that was subjected to long-term exposure to aminopenicillins. Subsequent cultures yielded E. faecalis with MICs of penicillins and carbapenems higher than those for wild-type strain E. faecalis JH2-2. Sequence analysis of the pbp4 gene of LS4828 compared to that of JH2-2 revealed two point mutations with amino acid substitutions (V223I, A617T) and deletion of an adenine from the region upstream of the predicted pbp4 −35 promoter sequence (UP region). Purified PBP4 from LS4828 exhibited less affinity for Bocillin FL than did PBP4 from JH2-2, which was recapitulated by purified PBP4 containing only the A617T mutation. Differential scanning fluorimetry studies showed that the LS4828 and A617T variants are destabilized compared to wild-type PBP4. Further, reverse transcription-PCR indicated increased transcription of pbp4 in LS4828 and Western blot analysis with polyclonal PBP4 antibody revealed greater quantities of PBP4 in LS4828 than in JH2-2 lysates and membrane preparations. Placing the promoter regions from LS4828 or JH2-2 upstream of a green fluorescent protein reporter gene confirmed that the adenine deletion was associated with increased transcription. Together, these data suggest that the reduced susceptibility to β-lactam antibiotics observed in E. faecalis LS4828 results from a combination of both increased expression and remodeling of the active site, resulting in reduced affinity for penicillins and carbapenems.

subjects

  • Biology and Biomedicine
  • Medicine
  • Pharmacy

keywords

  • antibiotic resistance; enterococcus; penicillin-binding proteins