Lantibiotic Smb: Characterization of the immunity protein, identification of a novel receptor-like protein, and a new perspective on regulation

Abstract

Dental caries, commonly known as tooth decay, is a chronic disease that develops slowly and requires formation of biofilm on tooth surfaces, commonly known as dental plaque. Dental plaque is a highly complex multispecies biofilm containing over ~700 different microorganisms. In this microbiota, Streptococcus mutans is considered to be the primary etiological agent for cariogenesis. To colonize and maintain its dominance over competing non-cariogenic species, S. mutans secretes various antimicrobial peptides called bacteriocins. S. mutans produces two types of bacteriocins: linear unmodified peptides known as non-lantibiotics and extensively modified nonlinear peptides called lantibiotics. S. mutans GS-5 strain is a highly virulent isolate that has been extensively used for genetic and biochemical studies. This strain produces a broad-spectrum lantibiotic called Smb. This lantibiotic is one of the arsenals that S. mutans GS-5 uses to shift the established bacterial flora associated with dental health towards the flora associated with dental caries. A lantibiotic producer strain must contain a self-protection mechanism to protect itself from the lantibiotic-mediated damage. Immunity mechanisms against Smb have not been identified. A previous report by Kuramitsu's group described SmbG, a putative ABC transporter with a peptidase domain, as the immunity protein for Smb. This proposed function of SmbG in providing immunity is not supported by their experimental data. In this study we show that an ABC-transporter encoded by SmbFT functions as an immunity complex. We show that GS-5 becomes sensitized to Smb upon deletion of smbT, which makes the ABC transporter non-functional. We demonstrate that SmbFT can confer protection against Smb when expressed heterologously in four different sensitive streptococci. We also demonstrate that SmbFT can confer protection against structurally similar two-petide lantibiotics such as haloduracin. We conclude that SmbFT truly displays immunity function and confers protection against Smb and structurally similar lantibiotics. Lantibiotics are potent bactericidal agents and usually functional at nanomolar range, whereas other antimicrobial peptides are effective at micromolar concentrations. This fact indicates that the interplay between the lantibiotics and the target organisms must be specific and perhaps it occurs through receptor-mediated interaction. However, to date, no such receptor molecules have been identified for any lantibiotics. In this study we identify in S. pyogenes (a human pathogen) a membrane-bound protein that exhibits a receptor-like function for Smb. This protein, which we named LsrS, belongs to CAAX-protease family. LsrS is widely present in streptococci including S. mutans and is highly conserved. Deletion of the LsrS homolog in sensitive S. mutans strains makes them refractory to Smb inhibition. However, neither LsrS nor its homolog can recognize other structurally similar two-peptide lantibitics. Nevertheless, this is the first protein that displays a receptor-like function for any lantibiotics. It is of great importance to understand how the producer strain regulates the expression of its immunity protein to counteract the cognate lantibiotic produced by the cell as well as by the neighbours. An auto-sensing mechanism may exist to maintain a constant ratio of the immunity protein and the lantibiotic. Little is known about the transcriptional regulation of the smb operon. Unlike most of the other lantibiotic loci that encode their own regulatory factors, smb locus does not encode any factor that can function as an auto-regulator. We provide experimental evidences that Smb peptides function as signaling molecules and auto regulate the smb operon through some yet to be discovered regulators. We attempted to identify the unknown regulators by transposon mutagenesis and identified an operon that seems to be involved in activation of smb operon. Further analysis indicated that a transcriptional regulator encoded within the operon indeed regulates smb production. Our results show that a new regulator and perhaps a new regulatory pathway might control smb expression. Lantibiotics, such as Smb, are highly potent, stable, and active at nanomolar concentrations. Because of the stability and potency lantibiotics are widely used in food industry as preservative. Few other lantibiotics are in clinical trial with the prospective to be used as antimicrobial agents in the healthcare industry. Since Smb can inhibit many pathogenic streptococci, it has the potential to be used as an antimicrobial agent in food and/or healthcare industry.