Intra- and interspecies signaling between Streptococcus salivarius and Streptococcus pyogenes mediated by SalA and SalA1 lantibiotic peptides

Abstract

Streptococcus salivarius 20P3 produces a 22-amino-acid residue lantibiotic, designated salivaricin A (SalA), that inhibits the growth of a range of streptococci, including all strains of Streptococcus pyogenes. Lantibiotic production is associated with the sal genetic locus comprising salA, the lantibiotic structural gene; salBCTX genes encoding peptide modification and export machinery proteins; and salYKR genes encoding a putative immunity protein and two-component sensor-regulator system. Insertional inactivation of salB in S. salivarius 20P3 resulted in abrogation of SalA peptide production, of immunity to SalA, and of salA transcription. Addition of exogenous SalA peptide to salB mutant cultures induced dose-dependent expression of salA mRNA (0.2 kb), demonstrating that SalA production was normally autoregulated. Inactivation of salR encoding the response regulator of the SalKR two-component system led to reduced production of, and immunity to, SalA. The sal genetic locus was also present in S. pyogenes SF370 (M type 1), but because of a deletion across the salBCT genes, the corresponding lantibiotic peptide, designated SalA1, was not produced. However, in S. pyogenes T11 (M type 4) the sal locus gene complement was apparently complete, and active SalA1 peptide was synthesized. Exogenously added SalA1 peptide from S. pyogenes T11 induced salA1 transcription in S. pyogenes SF370 and in an isogenic S. pyogenes T11 salB mutant and salA transcription in S. salivarius 20P3 salB. Thus, SalA and SalA1 are examples of streptococcal lantibiotics whose production is autoregulated. These peptides act as intra- and interspecies signaling molecules, modulating lantibiotic production and possibly influencing streptococcal population ecology in the oral cavity.

Figures

FIG. 1

Genetic structures of the sal loci in S. salivarius 20P3 (A) and S. pyogenes SF370 (M type 1) (B). Open reading frames are depicted by arrows which show the numbers of aa residues in the deduced polypeptides. The sites of insertional inactivation with plasmid pFW5 constructs used to generate strains UB1309, UB1310, and UB1308 are shown. PCR-amplified segments of DNA used for hybridization probes or cloning in pFW5 are indicated as follows: I, 0.3-kb salA probe; II, 1.48-kb salB fragment; III, 1.16-kb salKR fragment. Symbols: ⧫, putative promoter; ●, inverted repeat.

FIG. 2

Comparison of the inferred aa sequences of the precursor SalA peptide in S. salivarius 20P3 and SalA1 peptide in S. pyogenes SF370. The arrow indicates the site of proteolytic cleavage (after residue 26) to generate the 22-aa SalA or SalA1 propeptides. The residues in boldface type are those likely to be involved in thioether bridge formation.

FIG. 3

Northern analysis of SalA1 gene transcription in S. pyogenes probed with a salA1 fragment corresponding to salA fragment I (Fig. 1B). Lane 1, S. pyogenes T11 grown in M17CS medium; lane 2, S. pyogenes T11 grown in S. pyogenes UB1308 salB culture medium; lane 3, S. pyogenes SF370 grown in M17CS medium; lane 4, S. pyogenes SF370 grown in strain T11 culture medium; lane 5, S. pyogenes UB1308 salB grown in M17CS medium; lane 6, S. pyogenes UB1308 salB grown in strain T11 culture medium. The position of the salA1 mRNA transcript (0.2 kb) is indicated by an arrow. The lanes contained equivalent amounts of total RNA (10 μg).

FIG. 4

Autoinduction of SalA in S. salivarius 20P3: Northern analysis of salA mRNA transcripts from streptococci grown at 37°C for 30 min in M17CS medium (lane 1) and in M17CS medium containing purified SalA peptide at concentrations of 0.05 pmol/ml (lane 2), 0.5 pmol/ml (lane 3), and 5 pmol/ml (lane 4). The lanes contained equivalent amounts of total RNA (10 μg).

FIG. 5

RT-PCR analysis of sal locus transcripts from S. salivarius 20P3. cDNA was generated from mRNA by using the oligonucleotide SalRterm. (A) Lanes 2 to 5 PCRs performed with primers SalAF and SalXR; lanes 7 to 10, PCRs performed with primers SalY2S and SalRterm; lanes 2 and 7, cDNA template generated by RT; lanes 3 and 8, RNA controls (no RT); lanes 4 and 9, chromosomal DNA template; lanes 5 and 10, no-template controls. (B) PCRs performed with primers SalAF and SalRR. Lane 2, cDNA template generated by RT; lane 3, RNA control; lane 4, chromosomal DNA; lane 5, no template. The molecular mass markers (panel A, lanes 1 and 6; panel B, lane 1) were λ DNA HindIII fragments (23.1, 9.41, 6.56, 4.36, 2.32, and 2.02 kb).

FIG. 6

Northern analysis of salA gene transcription in S. salivarius UB1309 salB probed with salA fragment I (Fig. 1A). mRNAs were extracted from S. salivarius UB1309 cells grown at 37°C for 4 h. Lane 1, cells grown in their own cell-free spent culture medium; lane 2, cells grown in fresh M17CS medium containing 5 pmol of purified SalA/ml; lane 3, cells grown in cell-free S. salivarius 20P3 spent culture medium; lane 4, cells grown in cell-free S. pyogenes T11 spent culture medium.

FIG. 7

Model for autoregulation of SalA production in S. salivarius and S. pyogenes. The product of the salA gene, preproSalA peptide, is modified by the membrane-associated SalBC polypeptides and then exported with leader peptide cleavage by the membrane-integral SalTX polypeptides. Extracellular lantibiotic peptide from S. salivarius (SalA) or from S. pyogenes (SalA1) is sensed at the cell surface, possibly by the two-component SalKR system, and transcription of the salA promoter is upregulated via a phosphorylated (P) regulatory protein (R). Only the bacteria that respond to extracellular SalA peptide by producing active SalA are immune to the inhibitory effects of the lantibiotic peptide, but the mechanism of immunity is unknown.