Many Gram-positive bacteria produce anti-bacterial peptides and small proteins, called bacteriocins. There are three main classes produced by Gram-positive lactic acid bacteria (LAB): class I bacteriocins are the lantibiotics, small (<4 kDa), post-translationally modified peptides containing unusual amino acids such as lanthionine; class II are small, unmodified, heat-stable bacteriocins (<10 kDa); class III include larger (>30 kDa) heat-labile proteins, such as murein hydrolases . Most bacteriocins are synthesized as precursors, which are matured and secreted, then target a specific bacterium and kill it by increasing its membrane permeability to various small molecules. Class II bacteriocins are subdivided into IIa, pediocin-like unmodified bacteriocins, IIb, two-peptide unmodified bacteriocins, IIc, formerly class V, where the N- and C-termini are covalently linked resulting in a cyclic structure, and class IId, non-pediocin, single, linear peptides . The genetics and biosynthesis of class IIa bacteriocins have been well studied , and these constitute one of the most important groups of antimicrobial peptides, due to their useful antibacterial properties. All known IIa bacteriocins are described as being active against Listeria and some have already been tested as food preservatives for controlling food-borne pathogens . Structurally, class IIa bacteriocins are related to each other being unstructured in aqueous solution, but with a central amphiphilic alpha-helical region when in lipid micelles or TFE [5–7]; they contain the characteristic conserved N-terminal YGNGVxCxxxxC sequence, though usually not the GxxxG motif(s) characteristic of IIb and IIc bacteriocins .
The class IIb two-peptide unmodified bacteriocins, for example plantaricin E/F , need the complementary action of both peptides to be active . These bacteriocins contain long amphiphilic alpha-helical stretches, and the two complementary peptides interact when exposed to membrane-like entities. The GxxxG motif is conserved in many two-peptide bacteriocins, and it is postulated that the two complementary peptides dimerize via a helix-helix interaction that involves this motif, to form the functionally active heterodimer . The dimer functions by creating a pore within the membrane through which small molecules leak out, and, typically, the genes encoding the two peptides are found adjacent to each other on the same operon .
The cyclic class IIc bacteriocins are characterized by being tryptophan-rich and lacking any GG, GxxxG or YGNGVxCxxxxC motifs, ; the class IId bacteriocins have none of these features but some have recently been found also to be circular, rather than linear, with conserved AxxhhN and AhhW/F motifs [12–14].
In order to neutralize the toxic effect of the peptide on the "producing" cell the genes encoding bacteriocins are generally co-transcribed with a cognate immunity protein. These small proteins (typically 88-115 amino acids) interact very tightly with a specific bacteriocin or pair of bacteriocins and protect the "producing" microbe from the toxic effect of its own bacteriocin [15, 16]. The immunity proteins usually show high specificity for their cognate bacteriocins [17, 18]. For each class IIa bacteriocin encoded in a genome there is a one to one relationship between bacteriocin and cognate immunity protein; whereas, in contrast, for each pair of class IIb two-peptide bacteriocins there is a single cognate immunity protein encoded in a genome . Structures of five immunity proteins have already been solved: ImB2 , EntA-im , PedB , PisI , and Mun-Im , all protective against IIa bacteriocins. As yet, no structures for immunity proteins protective against IIb or IIc bacteriocins have been solved.
The sequencing of bacterial genomes, including those from human pathogens, has revealed a number of genes which might potentially code for new bacteriocins and immunity proteins, suggesting that the use of these antimicrobial peptides is more widespread than previously thought and that bacteria might be targeting several different bacterial species using these toxins. Understanding which specific immunity protein neutralizes which bacteriocin toxin is important if these peptides are to be used as antimicrobials.
The Gram-positive bacterium Streptococcus pyogenes, closely related to LAB, is one of the most common human pathogens. It causes a wide range of both minor diseases such as pharyngitis, erysipelas and pyodermas, that are readily controlled by antibiotics, as well as major, often lethal, conditions such as acute rheumatic fever, necrotizing fasciitis and streptococcal toxic shock syndrome, in developing countries and in the western world . The search for new antibacterial agents effective against this species is thus urgent.
Other streptococcal species have been shown to secrete bacteriocin-like toxins, as there are reports of S. salivarius producing a variety of bacteriocin-like inhibitory substances showing in vitro inhibitory activity against S. pyogenes, including Salivaricin A [25, 26]. Such observations suggest that antibacterial toxins are playing a very important role in controlling the level of the S. pyogenes population in human microbiomes. Bacteriocin-like toxins and antitoxins may well have an impact on the development of new antibacterial strategies and treatments. A thorough understanding of the biology of the bacteriocins in combination with their immunity proteins is important for any possible therapeutic use of bacteriocins. The full sequences of LAB genomes provide opportunities to scan for the presence of toxins and their corresponding immunity proteins. However, the sequence alone may not be sufficient to identify these proteins.
Here we present the first structure of the protein from locus Spy_2152 (gene names taken from S. pyogenes M1 GAS), named pyogenecin immunity protein Sp-PIP, determined at 2.15 Å resolution. We provide structural and sequence analyses, and identify the putative corresponding bacteriocin-like toxins in the S. pyogenes genome, which are found to belong to the class IIb two-peptide bacteriocins.