Reduction in membrane phosphatidylglycerol content leads to daptomycin resistance in Bacillus subtilis

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Daptomycin (DAP) is a critical antibiotic for Gram-positive bacterial infections.
• Understanding DAP resistance mechanisms is vital for combating antibiotic resistance.
• Bacillus subtilis serves as a model organism for studying bacterial physiology and antibiotic interactions.

Purpose of the Study:

• To investigate the genetic and molecular basis of daptomycin resistance in Bacillus subtilis.
• To identify key mutations conferring high-level DAP resistance and cross-resistance to other cell wall targeting antibiotics.
• To elucidate the functional consequences of these mutations on bacterial cell membrane properties and antibiotic susceptibility.

Main Methods:

• Serial passage of Bacillus subtilis 168 to generate DAP-resistant mutants.
• Phenotypic characterization of DAP-resistant isolates, including antibiotic susceptibility profiling.
• Whole-genome sequencing to identify mutations in resistant strains.
• Genetic reconstruction experiments, including allelic replacement, to confirm gene function.
• Transcriptomic and proteomic analyses to assess global cellular changes.

Main Results:

• An evolved isolate, Dap(R)1, exhibited 30-fold DAP resistance and cross-resistance to vancomycin, moenomycin, and bacitracin.
• Genome sequencing identified 44 point mutations, with three key mutations in mreB, relA, and pgsA identified in an intermediate resistant strain.
• Genetic reconstruction demonstrated that the pgsA(A64V) mutation is primarily responsible for DAP resistance.
• Reduced phosphatidylglycerol levels due to pgsA mutation decrease membrane negative charge, impairing Ca(2+)-DAP complex binding.

Conclusions:

• The primary mechanism of DAP resistance involves alterations in cell membrane lipid composition, specifically reduced phosphatidylglycerol.
• Mutations in pgsA significantly contribute to DAP resistance by modifying membrane charge and reducing antibiotic interaction.
• Pleiotropic effects and additional mutations may modulate resistance levels or compensate for fitness costs.