Characterization of Structural Properties and Antimicrobial Activity of the C3f Peptide of Complement System

The C3f peptide is a by-product of regulation of the activated complement system with no firmly established function of its own. We have previously shown that C3f exhibits moderate antimicrobial activity against some Gram-positive bacteria in vitro. Presence of two histidine residues in the amino acid sequence of the peptide suggests enhancement of its antimicrobial activity at lower pH and in the presence of metal cations, particularly zinc cations. Since such conditions could be realized in inflammatory foci, the study of dependence of C3f activity on pH and presence of metal cations could provide an opportunity to assess biological significance of antimicrobial properties of the peptide. The peptide C3f and its analogs with histidine residues substituted by lysines or serines, C3f[H/K] and C3f[H/S], were prepared by solid-phase synthesis. Using CD spectroscopy, we found that C3f contained a β-hairpin and unstructured regions; presence of Zn²⁺ did not affect conformation of the peptide. In the present work, it was shown that C3f could also exhibit antimicrobial activity against Gram-negative bacteria, in particular, Pseudomonas aeruginosa ATCC 27583. Exposure of P. aeruginosa and Listeria monocytogenes EGD to the peptide was accompanied by disruption of the barrier function of bacterial membranes. Zn²⁺ ions, unlike Cu²⁺ ions, enhanced antimicrobial activity of C3f against L. monocytogenes, with 4- and 8-fold molar excess of Zn²⁺ being no more effective than a 20% excess. Activity of the C3f analogs was also enhanced to some extent by the zinc ions. Thus, we hypothesize existence of the histidine-independent formation of C3f-Zn²⁺ complexes leading to increase in the total charge and antimicrobial activity of the peptide. In the presence of 0.15 M NaCl, C3f lost its antimicrobial activity regardless of the presence of Zn²⁺, indicating an insignificant role of C3f as an endogenous antimicrobial peptide. Presence of C3f eliminated bactericidal effect of Zn²⁺ against the zinc-sensitive Escherichia coli strain ESBL 521/17, indirectly confirming interaction of the peptide with Zn²⁺. Activity of C3f against Micrococcus luteus A270 increased with decreasing pH, while effect of pH on the C3f activity against L. monocytogenesis was more complex. In this work, we show significance of the factors such as pH and metal cations in realization of antimicrobial activity of peptides based on the example of C3f.