Characterization of a Multipeptide Lantibiotic Locus in Streptococcus pneumoniae

Abstract

Bacterial communities are established through a combination of cooperative and antagonistic interactions between the inhabitants. Competitive interactions often involve the production of antimicrobial substances, including bacteriocins, which are small antimicrobial peptides that target other community members. Despite the nearly ubiquitous presence of bacteriocin-encoding loci, inhibitory activity has been attributed to only a small fraction of gene clusters. In this study, we characterized a novel locus (the pld locus) in the pathogen Streptococcus pneumoniae that drives the production of a bacteriocin called pneumolancidin, which has broad antimicrobial activity. The locus encodes an unusual tandem array of four inhibitory peptides, three of which are absolutely required for antibacterial activity. The three peptide sequences are similar but appear to play distinct roles in regulation and inhibition. A modification enzyme typically found in loci encoding a class of highly modified bacteriocins called lantibiotics was required for inhibitory activity. The production of pneumolancidin is controlled by a two-component regulatory system that is activated by the accumulation of modified peptides. The locus is located on a mobile element that has been found in many pneumococcal lineages, although not all elements carry the pld genes. Intriguingly, a minimal region containing only the genes required for pneumolancidin immunity was found in several Streptococcus mitis strains. The pneumolancidin-producing strain can inhibit nearly all pneumococci tested to date and provided a competitive advantage in vivo. These peptides not only represent a unique strategy for bacterial competition but also are an important resource to guide the development of new antimicrobials.

Importance: Successful colonization of a polymicrobial host surface is a prerequisite for the subsequent development of disease for many bacterial pathogens. Bacterial factors that directly inhibit the growth of neighbors may provide an advantage during colonization if the inhibition of competitors outweighs the energy for production. In this work, we found that production of a potent antimicrobial called pneumolancidin conferred a competitive advantage to the pathogen Streptococcus pneumoniae. S. pneumoniae secreting pneumolancidin inhibits a wide array of Gram-positive organisms, including all but one tested pneumococcal strain. The pneumolancidin genetic locus is of particular interest because it encodes three similar modified peptides (lantibiotics), each of which has a distinct role in the function of the locus. Lantibiotics represent a relatively untapped resource for the development of clinically useful antibiotics which are desperately needed. The broad inhibitory activity of pneumolancidin makes it an ideal candidate for further characterization and development.

FIG 1

Inhibitory activity and genetic structure of the pld lantibiotic locus. (A) Overlay assays were performed using either P174 wild-type or deletion mutants of either upstream regulators of the blp or cib locus or a deletion of the pldM gene that was identified in the transposon mutagenesis screen. A TIGR4 strain was used as the overlay strain. (B) The pld locus of P174 and the corresponding loci of S. mitis and S. pneumoniae ATCC 700669 are shown. The percent amino acid identity between the predicted proteins found in S. mitis B6 and P174 homologues is noted above the B6 ORFs. Presumed functional designations are indicated by the color of the ORF. Regions of DNA homology between sequences are in grey. (C) Amino acid alignment demonstrating the homology between predicted structural proteins PldA1 to 4. The proposed signal peptide sequence cleavage point is shown with an arrow. Shared amino acid residues in the functional peptide are highlighted in yellow. Amino acid residues in red are sites of possible modification catalyzed by PldM. (D) Deletions of various genes in the pld locus of P174 were constructed and assayed for inhibitory and signaling activity as well as immunity to WT lantibiotic. Inhibition and signal secretion were tested by stabbing the strain of interest and overlaying with the sensitive indicator strain TIGR4 or the reporter strain P174pldM-lacZ, respectively. The chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) was included in the overlay mixture for signaling assay. Immunity was determined by stabbing P174 and overlaying with each of the deletion mutants.

FIG 2

Deletion of the lantibiotic peptides in either P174 or the hyperinducible P174act background. In-frame, unmarked peptide deletion mutants were constructed and assayed for inhibitory and signaling activity as well as immunity to WT lantibiotic. Evidence of inhibition and signal secretion was tested for by stabbing the strain of interest and overlaying with the sensitive indicator strain TIGR4 or the reporter strain P174pldM-lacZ, respectively. Immunity was determined by stabbing P174 and overlaying with each of the deletion mutants. Peptide deletion mutants were made in either the P174 background (A) or the P174act background (B). (C) Phenotypic complementation was assayed using P174actΔpldA3 and P174actΔpldA1. Both strains were stabbed progressively more closely to each other and the plate subsequently overlaid with TIGR4. Pictures were taken at a higher magnification (×2) than other overlays to better appreciate the inhibitory effect.

FIG 3

A hyperinducible strain contains a mutation that is affecting the promoter upstream of pldFE. (A) Response to exogenous peptides was tested in either P174pldM-lacZ or P174actpldM-lacZ. P174 was stabbed multiple times into TSA plates and overlaid with either reporter. (B) Location of the single base pair mutation resulting in the hyperinducible phenotype in the intergenic region between pldA4 and pldFE. The site of the mutation is marked by an asterisk. The 4-bp region shown in red was deleted in strain P174Δ4bp. The proposed TATA box preceding the pldF ORF is underlined. The distance to the start codon of pldF is indicated by “N.” (C) Overlay assays assessing inhibition and immunity phenotype of the 4-bp deletion that included the site of the activating mutation. For inhibition, the P174Δ4bp strain was stabbed and overlaid with TIGR4. For immunity, P174 was stabbed onto a TSA plate and overlaid with the P174Δ4bp strain.

FIG 4

Transcriptional activity of pldF-lacZ fusion strains in either P174 or P174act background. (A) Schematics of the pldF-lacZ reporter strains are shown after plasmid integration. Dotted lines denote the plasmid-derived sequence; the lacZ gene is shown as a light blue arrow. An asterisk depicts the site of the Act mutation. To the right of the corresponding schematic of the locus is the phenotype of each construct grown on TSA plates containing X-Gal in which 5 µl of crude P174act-derived supernatant was added to the center of a lawn for induction. Response to supernatants was evidenced by the blue halos. (B) Transcriptional activity of the promoter driving lacZ was assessed in broth-grown organisms using strains P174, P174pldF-lacZ, P174P_actpldF-lacZ, and P174actP_174pldF-lacZ. Twofold dilutions of crude P174act derived supernatant were added to the strains at an OD at 620 nm of 0.2 and induced for 1.5 h. Activity was determined by calculating Miller units. To account for endogenous β-galactosidase activity, wild-type P174 was included.

FIG 5

Competitive advantage of the pld locus in vivo. Mice were either colonized with P174ΔpldK or treated with sterile PBS at day 0. At day 3, mice were challenged intranasally with either P174 or P174ΔpldA1-4. Nasal washes were obtained 3 days postinoculation, and the number of CFU was calculated by differential plating. Medians and interquartile ranges are shown. The dotted line indicates the limit of detection (LOD). Statistical analysis was performed using an unpaired Mann-Whitney test. *, P < 0.05; **, P < 0.01.