RNA-dependent lipid remodeling by bacterial multiple peptide resistance factors

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Multiple peptide resistance (MprF) proteins are crucial virulence factors that confer resistance to cationic antibiotics by modifying inner membrane lipids.
• Previous studies identified MprF enzymes that aminoacylate phosphatidylglycerol (PG) using Lys-tRNA(Lys), but the recognition mechanisms and roles of other MprF paralogs remained unclear.

Purpose of the Study:

• To investigate the substrate specificity and mechanism of two distinct MprF paralogs (MprF1 and MprF2) from Clostridium perfringens using an in vitro reconstituted lipid aminoacylation system.
• To elucidate the role of aminoacyl-tRNA recognition in MprF-mediated antibiotic resistance.

Main Methods:

• Utilized an in vitro reconstituted lipid aminoacylation system to study MprF1 and MprF2 activity.
• Investigated the amino acid and tRNA specificity of MprF1 and MprF2.
• Performed mutations on tRNA(Ala) and tRNA(Lys) to assess their impact on MprF activity.

Main Results:

• Both MprF1 and MprF2 were found to aminoacylate phosphatidylglycerol (PG).
• MprF1 specifically utilizes Ala-tRNA(Ala), while MprF2 uses Lys-tRNA(Lys), demonstrating distinct amino acid specificities.
• Mutations in tRNA(Ala) and tRNA(Lys) had minimal impact on MprF activity, indicating the aminoacyl moiety is the primary recognition determinant.

Conclusions:

• The dual specificity of MprF paralogs allows for fine-tuning of the inner membrane charge, potentially enhancing resistance to a wider spectrum of antibiotics.
• The lack of tRNA discrimination by MprF facilitates horizontal gene transfer, contributing to the spread of antibiotic resistance.
• These findings highlight MprF proteins as potent virulence factors enabling pathogens to acquire resistance to diverse antibiotics.