Structure, Biosynthesis, and Biological Activity of Succinylated Forms of Bacteriocin BacSp222

Abstract

BacSp222 is a multifunctional peptide produced by Staphylococcus pseudintermedius 222. This 50-amino acid long peptide belongs to subclass IId of bacteriocins and forms a four-helix bundle molecule. In addition to bactericidal functions, BacSp222 possesses also features of a virulence factor, manifested in immunomodulatory and cytotoxic activities toward eukaryotic cells. In the present study, we demonstrate that BacSp222 is produced in several post-translationally modified forms, succinylated at the ε-amino group of lysine residues. Such modifications have not been previously described for any bacteriocins. NMR and circular dichroism spectroscopy studies have shown that the modifications do not alter the spatial structure of the peptide. At the same time, succinylation significantly diminishes its bactericidal and cytotoxic potential. We demonstrate that the modification of the bacteriocin is an effect of non-enzymatic reaction with a highly reactive intracellular metabolite, i.e., succinyl-coenzyme A. The production of succinylated forms of the bacteriocin depends on environmental factors and on the access of bacteria to nutrients. Our study indicates that the production of succinylated forms of bacteriocin occurs in response to the changing environment, protects producer cells against the autotoxicity of the excreted peptide, and limits the pathogenicity of the strain.

Bacteriocin BacSp222 and Its Succinylated Forms Exhibit Proinflammatory Activities Toward Innate Immune Cells

“…BacSp222 and its succinylated forms (suc-K20-BacSp222 and suc-K11/K20-BacSp222) were purified from bacterial post-culture medium by reversed-phase high-pressure liquid chromatography (RP-HPLC) essentially as described earlier. 15 The bacteriocin deprived of N-terminal formyl-methionine (-fM-BacSp222) was obtained after overnight digestion of BacSp222 using cyanogen bromide and 70% (v/v) trifluoroacetic acid (TFA). After the reaction, the modified peptide was purified using RP-HPLC as described above.…”

“…The LDH release and the level of ATP were determined according to the protocol described earlier. 15 …”

“…Nuclear magnetic resonance (NMR) studies have shown that the peptide is globular and forms a bundle composed of four helices, as in the case of the other staphylococcal subclass IId of bacteriocins: aureocin A53, lacticin Q, entrocin 7A/JSA, and enterocin 7B/JSB. 14 , 15 However, despite the significant similarities in the structure of these molecules, their size, amino acid composition, formylation at the N-terminus, and physicochemical properties, all these bacteriocins have quite different amino acid sequences. In addition, our recent study has shown that BacSp222 is produced in three forms: one unmodified and two posttranslationally modified, named suc-K20-BacSp222 and suc-K11/K20-BacSp222.…”

“…Our studies revealed that the production of such modified forms occurs in response to environmental changes, protects the cells of the producing bacteria from autotoxicity of the secreted bacteriocin and limits the pathogenicity of the strain. 15 The principal biological activity of BacSp222 is bactericidal action against Gram-positive bacteria, especially staphylococci. However, in the context of the present work, it is particularly interesting that BacSp222 also demonstrates some features of a virulence factor and an immunomodulatory peptide.…”

Bacteriocin Producing Streptococcus agalactiae Strains Isolated from Bovine Mastitis in Brazil

“…Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria. For instance, such compounds may grant bacteriocin-producing bacteria advantages in replication and settlement to different sites [ 38 , 39 ]. Thus, these substances have similar characteristics to bacterial virulence factors.…”