Detailed mutational analysis of Vga(A) interdomain linker: implication for antibiotic resistance specificity and mechanism

Abstract

Detailed mutational analysis examines the roles of individual residues of the Vga(A) linker in determining the antibiotic resistance phenotype. It defines a narrowed region of residues 212 to 220 whose composition determines the resistance specificity to lincosamides, pleuromutilins, and/or streptogramins A. From the analogy with the recently described function of the homologous ABC-F protein EttA as a translational factor, we infer that the Vga(A) linker interacts with the ribosome and directly or indirectly affects the binding of the respective antibiotic.

FIG 1

Amino acid sequence variability of Vga(A)* variants mined from the NCBI protein database. Only variable positions are shown. Amino acid residues that differ from those in Vga(A) are indicated by a gray background. Specific residues important for antibiotic specificity of Vga(A) and Vga(A)_LC are indicated by light green and light bronze background, respectively (4). In the Resistance phenotype^a column, the superscript letter a indicates that the resistance phenotype as published in the relevant reference is shown as follows: SgA, streptogramins A; L, lincosamides; P, pleuromutilins; CLI, clindamycin. The superscript letter b indicates that pleuromutilins were not tested, and the superscript letter c indicates that only CLI was tested.

FIG 2

Efficiency of the Vga(A)* variants prepared and analyzed in this study. (A) Scheme of the Vga(A)_LCba protein used in preparing the mutant forms of Vga(A). Mutated amino acids (X) at positions 212, 219, 220, and 226 are indicated. (B) MICs, resistance phenotypes, and consensus sequence logos. The amino acid (AA) combinations at positions 212, 219, 220, and 226 are shown. The sequence patterns of the original Vga(A) and Vga(A)_LC are underlined. The superscript number indicates that the predicted MIC values for the missing variant LGTG are shown. Polar amino acids are shown in green in the sequence logo. (C) Impact of the individual positions to the level of resistance to a particular antibiotic. Results are expressed as the relative ratio of the mean MIC for variants with a particular residue.

FIG 3

(A) EttA (blue) bound to the 50S ribosomal subunit in complex with P-tRNA^fMet (red) (RCSB Protein Database [PDB] accession numbers 3J5S [19] and 3I9C [24] and binding sites of clindamycin (green) (PDB accession number 3OFZ [25]) and SgA and dalfopristin (purple) (PDB accession number 1SM1 [21]). The arm subdomain of EttA (bright blue) and the PtlM domain (dark blue) are indicated. The enlargement shows a detailed view of the PtlM domain interacting with the stem of P-tRNA^fMet in the direction toward bound antibiotics. (B) Significant Pfam-A matches of the Vga(A) linker with ABC_tran_2 domain and sequence alignment with the PtlM domain of EttA. The hidden Markov model sequence (HMM), match between HMM and Vga(A) (MATCH), and posterior probability (PP) (the degree of confidence in each individual aligned residue) are given. Amino acid residues of Vga(A) and EttA that are identical (red) and the antibiotic specificity site of Vga(A) (green) are indicated. The bright blue lines represent the regions of contact of PtlM with P-site tRNA, and the gray lines represent regions of contact of PtlM with 23S rRNA (19).