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.

Keywords: Krebs/tricarboxylic acids cycle; Staphylococcus pseudintermedius; antibacterial; bacteriocin; cytotoxic; nuclear magnetic resonance (NMR); succinyl-coenzyme A; succinylation.

Figure 1

RP-HPLC chromatogram from purification of BacSp222 and its succinylated forms (denoted by black, red and green characters, respectively). The peptide sequence and the succinylation places are presented in the insert. fM denotes the formylated methionine residue at the N-terminus.

Figure 2

Results of NMR studies of the structure of unmodified and succinylated forms of BacSp222. Panel (A) illustrates the superimposition of the most representative structures of unmodified BacSp222 (cyan) and suc-K20-BacSp222 (red). The side chain of lysine 20 in both structures is represented as sticks–hydrogens white, nitrogens blue, carbons of unmodified lysine cyan, and carbons of modified lysine orange. The superimposition of ¹H-¹⁵N HSQC spectra and ¹H-¹³C HSQC spectra tuned to aromatic carbons for unmodified BacSP222 (green) and suc-K20-BacSp222 (red) is presented in panels (B),(C), respectively.

Figure 3

Comparison of the cytotoxic activity of different forms of bacteriocin BacSp222 against human neutrophils. The cells were isolated from healthy volunteers’ blood and immediately exposed to various concentrations of BacSp222 forms for 4 h: unmodified BacSp222, suc-K20-BacSp222, or suc-K11/K20-BacSp222. After incubation, the cells were centrifuged, and the supernatants were transferred to a fresh plate for further analyses. (A) Morphological changes were analysed using an inverted light microscope. The circles indicate activated neutrophils. (B) The viability of the cells was determined using an ATPlite assay. (C) LDH activity in the medium indicates cell membrane damage in the presence of various forms of bacteriocin. * p < 0.05.

Figure 4

Cytotoxicity of different BacSp222 forms against murine monocyte-macrophage cells RAW 264.7 and murine primary dermal fibroblasts MDF. The cells were incubated for 4 h in a medium containing various concentrations of BacSp222 forms: unmodified BacSp222 and suc-K20-BacSp222. After incubation, the culture media were transferred on a fresh plate for further analyses. (A) The viability of the cells was measured using the MTT method. (B) The LDH activity in the medium indicates cell membrane damage in the presence of the various forms of bacteriocin. * p < 0.05.

Figure 5

Results of in vitro studies of nonenzymatic succinylation of BacSp222. The unmodified form of BacSp222 was incubated with donors of the butanedioic group in different conditions. Next, the amount of BacSp222 forms was determined by RP-HPLC. (A) Ability of physiological donors of the butanedioic group to modify BacSp222. (B) Influence of the presence of other molecules containing amino groups on the modification of BacSp222 by suc-CoA. Effect of the reaction time (C) and pH of the solution (D) on the degree of BacSp222 succinylation.

Figure 6

Effect of environmental factors on production of BacSp222 and its post-translationally modified forms by strain Sp222. The bacteria were cultured for various time intervals in different conditions; next, the levels of all BacSp222 forms were determined in the post-culture media by RP-HPLC. (A) Effect of the access to nutrients on the biosynthesis of all BacSp222 forms. The successive panels illustrate the effect of culture temperature (B), pH of the culture (C), carbon source (D), and biological stress (presence of nitric oxide) (E) on the amount of modified BacSp222forms.