Production of the Bsa lantibiotic by community-acquired Staphylococcus aureus strains

Abstract

Lantibiotics are antimicrobial peptides that have been the focus of much attention in recent years with a view to clinical, veterinary, and food applications. Although many lantibiotics are produced by food-grade bacteria or bacteria generally regarded as safe, some lantibiotics are produced by pathogens and, rather than contributing to food safety and/or health, add to the virulence potential of the producing strains. Indeed, genome sequencing has revealed the presence of genes apparently encoding a lantibiotic, designated Bsa (bacteriocin of Staphylococcus aureus), among clinical isolates of S. aureus and those associated with community-acquired methicillin-resistant S. aureus (MRSA) infections in particular. Here, we establish for the first time, through a combination of reverse genetics, mass spectrometry, and mutagenesis, that these genes encode a functional lantibiotic. We also reveal that Bsa is identical to the previously identified bacteriocin staphylococcin Au-26, produced by an S. aureus strain of vaginal origin. Our examination of MRSA isolates that produce the Panton-Valentine leukocidin demonstrates that many community-acquired S. aureus strains, and representatives of ST8 and ST80 in particular, are producers of Bsa. While possession of Bsa immunity genes does not significantly enhance resistance to the related lantibiotic gallidermin, the broad antimicrobial spectrum of Bsa strongly indicates that production of this bacteriocin confers a competitive ecological advantage on community-acquired S. aureus.

FIG. 1.

In silico analysis of bsa and homologous genes. (A) Visual representation of epidermin and Bsa (Bsa_Au-26, Bsa, strain COL Bsa [Bsa_COL], and Bsa_ET3-1) loci. Numbers represent the percentages of identity to the corresponding bsa gene (except for bsaA1, bsaX, and bsaA2, which are compared in greater detail in panels B and C). Homologous genes share a color (white indicates the absence of homologues). Dashed lines indicate unsequenced regions within the Bsa_Au-26 operon. Three products (i, ii, and iii) amplified from within this region in S. aureus strain 26 are indicated. The locus tags indicated correspond to those for lukE. Bsa* represents the identical Bsa genes in strains MW2 (accession no. NC_003923), FPR3757 (accession no. NC_007793), USA300_TCH1516 (accession no. NC_010079), NCTC8325-4 (accession no. NC_007795), Newman (accession no. NC-009641), and MSSA476 (accession no. NC_002953). Accession numbers for the COL and ET3-1 genomes are NC_002951 and NC_007622, respectively. (B) Alignment of the predicted amino acid sequences of the Bsa and Bsa_ET3-1 structural propeptides with those of other epidermin-like and related peptides. (C) Alignment of the predicted amino acid sequences of BsaX and BsaX_ET3-1 [BsaX(RF122)]. The sole difference is indicated by an arrow. (D) Predicted BsaA2 structure (modified residues are shaded in black).

FIG. 2.

Results from agar-based antimicrobial assays with representative Bsa⁺ strains. (A) Results from agar well diffusion assays highlighting the activities of partially purified Bsa produced by S. aureus NCTC8325 against C. testudinoris DPC 6273 (i), S. epidermidis DPC 6293 (ii), Psychrobacter sp. strain DPC 6277 (iii), M. luteus NCIMB9278 (iv), M. luteus DPC 6275 (v), and M. oxydans DPC 6277 (vi). (B) Results from agar well diffusion assays with cell-free supernatant from S. aureus ET3-1 using M. luteus NCIMB9278 as an indicator. (C) Results from deferred antagonism assays assessing the activities of S. aureus RN4220 and RN4220::pOri280bsaB against M. luteus NCIMB9278.

FIG. 3.

CMS analysis of S. aureus isolates. Gray box, data for S. aureus strain 26 and NCTC8325-4; white box, data for CA-MRSA strains. Masses (expressed in daltons) corresponding to the predicted mass of BsaA2 are indicated by a larger font.

FIG. 4.

Sensitivity of S. aureus strains to gallidermin produced by S. gallinarum DSMZ 4616 (as assessed by deferred antagonism agar diffusion assays).