Suicin 3908, a new lantibiotic produced by a strain of Streptococcus suis serotype 2 isolated from a healthy carrier pig

Abstract

While Streptococcus suis serotype 2 is known to cause severe infections in pigs, it can also be isolated from the tonsils of healthy animals that do not develop infections. We hypothesized that S. suis strains in healthy carrier pigs may have the ability to produce bacteriocins, which may contribute to preventing infections by pathogenic S. suis strains. Two of ten S. suis serotype 2 strains isolated from healthy carrier pigs exhibited antibacterial activity against pathogenic S. suis isolates. The bacteriocin produced by S. suis 3908 was purified to homogeneity using a three-step procedure: ammonium sulfate precipitation, cationic exchange HPLC, and reversed-phase HPLC. The bacteriocin, called suicin 3908, had a low molecular mass; was resistant to heat, pH, and protease treatments; and possessed membrane permeabilization activity. Additive effects were obtained when suicin 3908 was used in combination with penicillin G or amoxicillin. The amino acid sequence of suicin 3908 suggested that it is lantibiotic-related and made it possible to identify a bacteriocin locus in the genome of S. suis D12. The putative gene cluster involved in suicin production by S. suis 3908 was amplified by PCR, and the sequence analysis revealed the presence of nine open reading frames (ORFs), including the structural gene and those required for the modification of amino acids, export, regulation, and immunity. Suicin 3908, which is encoded by the suiA gene, exhibited approximately 50% identity with bovicin HJ50 (Streptococcus bovis), thermophilin 1277 (Streptococcus thermophilus), and macedovicin (Streptococcus macedonicus). Given that S. suis 3908 cannot cause infections in animal models, that it is susceptible to conventional antibiotics, and that it produces a bacteriocin with antibacterial activity against all pathogenic S. suis strains tested, it could potentially be used to prevent infections and to reduce antibiotic use by the swine industry.

Fig 1. Detection of sslA, the structural gene of suicin 90–1330, in S. suis 3908 and 94–623, two bacteriocin-producing strains.

Lane 1, DNA molecular weight markers; lanes 2 and 4, S. suis 3908; lanes 3 and 5, S. suis 94–623; lanes 2 and 3, sslA gene; lanes 4 and 5, gdh gene.

Fig 2. Inhibitory zones produced by S. suis 3908 when spotted on lawns of S. suis strains belonging to different sequence types (ST1, ST25, ST28) and that were isolated from diseased pigs.

Three independent experiments were performed. The inhibitory zones (in mm) are expressed as means ± standard deviations.

Fig 3. SDS-PAGE analysis of the purified bacteriocin produced by S. suis 3908.

Panel A. Gel stained with Coomassie blue. Panel B. Gel used to detect antibacterial activity by an overlay with the indicator strain S. suis MGGUS3. Lane 1: molecular weight markers; Lane 2: nisin A; Lane 3: suicin 3908.

Fig 4. Effect of suicin 3908 on the membrane permeabilization of S. suis MGGUS3 as determined using the SYTOX Green dye.

Ethanol (70%) was used as a positive control. Three assays were performed. The results are expressed as means ± standard deviations.

Fig 5. Genetic organization of the putative suicin 3908 gene cluster.

Fig 6. Comparison of the amino acid sequence of suicin 3908 with the amino acid sequence of the putative lantibiotic corresponding to gene SSUD12_1310 (accession # G7SEC9) in S. suis D12 and with other lantibiotics produced by S. bovis (bovicin HJ50; bovA), S. macedonicus (macedovicin; mdvA), and S. thermophilus (thermophilin 1177; tepA).