RNA-dependent lipid remodeling by bacterial multiple peptide resistance factors

Abstract

Multiple peptide resistance (MprF) virulence factors control cellular permeability to cationic antibiotics by aminoacylating inner membrane lipids. It has been shown previously that one class of MprF can use Lys-tRNA Lys to modify phosphatidylglycerol (PG), but the mechanism of recognition and possible role of other MprFs are unknown. Here, we used an in vitro reconstituted lipid aminoacylation system to investigate the two phylogenetically distinct MprF paralogs (MprF1 and MprF2) found in the bacterial pathogen Clostridium perfringens. Although both forms of MprF aminoacylate PG, they do so with different amino acids; MprF1 is specific for Ala-tRNA Ala , and MprF2 utilizes Lys-tRNA Lys . This provides a mechanism by which the cell can fine tune the charge of the inner membrane by using the neutral amino acid alanine, potentially providing resistance to a broader range of antibiotics than offered by lysine modification alone. Mutation of tRNA Ala and tRNA Lys had little effect on either MprF activity, indicating that the aminoacyl moiety is the primary determinant for aminoacyl-tRNA recognition. The lack of discrimination of the tRNA is consistent with the role of MprF as a virulence factor, because species-specific differences in tRNA sequence would not present a barrier to horizontal gene transfer. Taken together, our findings reveal how the MprF proteins provide a potent virulence mechanism by which pathogens can readily acquire resistance to chemically diverse antibiotics.alanine ͉ lysine ͉ tRNA

Quality control despite mistranslation caused by an ambiguous genetic code

“…2). This in vivo finding contributes to the discussion of the posttransfer editing mechanism by class II aaRSs and the binding of misacylated aa-tRNA to EF-Tu (42). In the absence of complete tRNA editing, and driven by our missense selection system, the misacylated aa-tRNA is then presented to the ribosome by EF-Tu.…”

Focal targeting by human β-defensin 2 disrupts localized virulence factor assembly sites in Enterococcus faecalis

“…Many bacteria encode MprF to mediate resistance to cationic antimicrobials (27,33,34). E. faecalis contains two paralogs of this gene, mprF1 and mprF2.…”

Deciphering the tRNA-dependent lipid aminoacylation systems in bacteria: Novel components and structural advances

“…31 It has been established in vitro that some aaPGSs exhibit promiscuity for aa-tRNA recognition and can utilize multiple aa-tRNAs, while some are specific for a unique aa-tRNA. 32 For example, the LysPGS from Agrobacterium tumefaciens and the AlaPGS from C. perfringens specifically use Lys-tRNA and Ala-tRNA respectively, while the aaPGS from Bacillus subtilis utilizes both Lys- and Ala-tRNA as amino acid donors in vitro .…”

“…A minihelix encompassing just the acceptor stem and TYC stem loop of tRNA Ala is as efficiently recognized as full length tRNA by AlaPGS. 31 Further investigations with the AlaPGS from P. aeruginosa revealed that a microhelix (a hairpin consisting of 7 base pairs) is the minimal substrate for AlaPGS activity. An in-depth study showed that the Ala moiety and the fifth base pair of the tRNA helix constitute the major elements for efficient recognition of Ala-tRNA Ala .…”