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

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Abstract

The C3f peptide is a by-product of the 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. The occurrence of two histidine residues in the amino acid sequence of the peptide suggested an enhancement of its antimicrobial activity at lower pH and in the presence of metal cations, particularly zinc cations. Since such conditions can be realized in inflammatory foci, the study of the dependence of C3f activity on pH and the presence of metal cations provides an opportunity to assess the biological significance of the antimicrobial properties of the peptide. The peptide C3f and its analogs with histidine substitutions 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; the presence of Zn2+ did not affect the conformation of the peptide. In the present work, it was shown that C3f can also exhibit antimicrobial activity against Gram-negative bacteria, in particular, Pseudomonas aeruginosa ATCC 27583. The action of the peptide on Ps. aeruginosa and Listeria monocytogenes EGD is accompanied by impairment of the barrier function of bacterial membranes. Zn2+ ions, unlike Cu2+ ions, enhanced the antimicrobial activity of C3f against L. monocytogenes, with 4- and 8-fold molar excess of Zn2+ being no more effective than a 20% excess. The activity of the C3f analogs was also enhanced to some extent by zinc ions. Thus, we hypothesize a histidine-independent formation of C3f–Zn2+ complexes leading to an increase in the total charge and antimicrobial activity of the peptide. In the presence of 0.15 M NaCl, C3f lost its activity regardless of the presence of Zn2+, indicating a minor role of C3f as an endogenous antimicrobial peptide. The presence of C3f abolished the bactericidal effect of Zn2+ against the zinc-sensitive Escherichia coli strain ESBL 521/17, indirectly confirming the interaction of the peptide with Zn2+. The activity of C3f against Micrococcus luteus A270, but not against L. monocytogenes, increased with decreasing pH. In this work, we show the significance of factors such as pH and metal cations in realizing the activity of antimicrobial peptides based on the example of C3f.

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I. A. Krenev

Institute of Experimental Medicine

Email: berlov.mn@iemspb.ru
Russian Federation, 197022, Saint Petersburg

E. V. Egorova

Institute of Experimental Medicine; Saint Petersburg State University

Email: berlov.mn@iemspb.ru
Russian Federation, 197022, Saint Petersburg; 199034, Saint Petersburg

M. M. Khaydukova

Institute of Experimental Medicine; Research Institute of Hygiene, Occupational Pathology and Human Ecology

Email: berlov.mn@iemspb.ru
Russian Federation, 197022, Saint Petersburg; 192019, Saint Petersburg

A. D. Mikushina

Institute of Experimental Medicine; Alferov University

Email: berlov.mn@iemspb.ru4
Russian Federation, 197022, Saint Petersburg; 194021, Saint Petersburg

Ya. A. Zabrodskaya

Institute of Experimental Medicine; Smorodintsev Research Institute of Influenza; Peter the Great Saint Petersburg Polytechnic University

Email: berlov.mn@iemspb.ru
Russian Federation, 197022, Saint Petersburg; 197376, Saint Petersburg; 195251, Saint Petersburg

A. S. Komlev

Institute of Experimental Medicine

Email: berlov.mn@iemspb.ru
Russian Federation, 197022, Saint Petersburg

I. E. Eliseev

Institute of Experimental Medicine; Alferov University

Email: berlov.mn@iemspb.ru
Russian Federation, 197022, Saint Petersburg; 194021, Saint Petersburg

O. V. Shamova

Institute of Experimental Medicine; Saint Petersburg State University

Email: berlov.mn@iemspb.ru
Russian Federation, 197022, Saint Petersburg; 199034, Saint Petersburg

M. N. Berlov

Institute of Experimental Medicine; Saint Petersburg State University

Author for correspondence.
Email: berlov.mn@iemspb.ru
Russian Federation, 197022, Saint Petersburg; 199034, Saint Petersburg

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Supplementary files

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2. Supplementary
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3. Fig. 1. CD spectra of peptide C3f in the absence and presence of ZnCl2

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4. Fig. 2. Inferred secondary structure of C3f

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5. Fig. 3. Antimicrobial activity of C3f peptide and zinc ions against M. luteus, St. aureus and Ps. aeruginosa. a - Bacterial survival in the presence of C3f; b - Bacterial survival in the presence of Zn2+

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6. Fig. 4. Antimicrobial activity of C3f and its analogues against L. monocytogenes in the absence and presence of metal ions. a - Bacterial survival in the presence of Zn2+ and Cu2+; b - effect of C3f depending on the presence of Zn2+ and 0.15 M NaCl; c - effect of C3f at different molar excess of Zn2+; d - effect of C3f depending on the presence of Cu2+; e and f - effect of C3f[H/S] and C3f[H/K] in the absence and presence of Zn2+

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7. Fig. 5. Antimicrobial activity of ZnCl2 against E. coli ESBL 521/17 in the absence and presence of 20% molar excess of C3f

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8. Fig. 6. Antimicrobial activity of peptides at different pH values. a - Action of C3f against L. monocytogenes; b - Action of HNP against L. monocytogenes; c and d - Action of C3f and C3f[H/K] against M. luteus

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9. Fig. 7. Effect of peptides on membrane permeability of bacterial cells detected by fluorescence of propidium iodide complex with nucleic acids

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