Structural and Regulatory Changes in PBP4 Trigger Decreased β-Lactam Susceptibility in Enterococcus faecalis

Area of Science:

• Microbiology
• Infectious Diseases
• Antibiotic Resistance

Background:

• Enterococcus faecalis is a significant cause of hospital-acquired and community infections.
• Antibiotic resistance in E. faecalis presents therapeutic challenges.
• Penicillin and ampicillin resistance in E. faecalis is uncommon but linked to PBP4 alterations.

Purpose of the Study:

• To investigate the mechanisms of reduced susceptibility to beta-lactam antibiotics in an E. faecalis vancomycin-susceptible E. faecalis strain (LS4828).
• To analyze genetic and protein alterations in penicillin-binding protein 4 (PBP4) associated with resistance.
• To understand the impact of these alterations on antibiotic binding affinity and protein stability.

Main Methods:

• Isolation and characterization of E. faecalis strain LS4828 from a prosthetic joint infection.
• Antimicrobial susceptibility testing (MIC determination) for penicillins and carbapenems.
• DNA sequencing of the pbp4 gene and its promoter region.
• Protein purification and characterization of PBP4 variants (affinity, stability via differential scanning fluorimetry).
• Gene expression analysis (RT-PCR) and protein quantification (Western blot).

Main Results:

• E. faecalis LS4828 exhibited increased minimum inhibitory concentrations (MICs) for penicillins and carbapenems compared to wild-type.
• LS4828 possessed two point mutations in pbp4 (V223I, A617T) and an upstream adenine deletion affecting the promoter.
• Purified PBP4 from LS4828 showed reduced affinity for Bocillin FL, with the A617T mutation being key.
• PBP4 variants were destabilized, and LS4828 displayed increased pbp4 transcription and higher PBP4 protein quantities.

Conclusions:

• Reduced susceptibility to beta-lactam antibiotics in E. faecalis LS4828 results from combined increased PBP4 expression and active site remodeling.
• The adenine deletion upstream of pbp4 is associated with increased gene transcription.
• These findings offer insights into beta-lactam antibiotic interactions with low-affinity PBP4 and have implications for treating enterococcal infections.