Abstract
Innate immunity involving antimicrobial peptides represents an integrated and highly effective system of molecular and cellular mechanisms that protects host against infections. One of the most frequent hospital-acquired pathogens, Staphylococcus aureus, capable of producing proteolytic enzymes, which can degrade the host defence agents and tissue components. Numerous antimicrobial peptides derived from chromogranins, are secreted by nervous, endocrine and immune cells during stress conditions. These kill microorganisms by their lytic effect at micromolar range, using a pore-forming mechanism against Gram-positive bacteria, filamentous fungi and yeasts. In this study, we tested antimicrobial activity of chromogranin A-derived peptides (catestatin and cateslytin) against S. aureus and analysed S. aureus-mediated proteolysis of these peptides using HPLC, sequencing and MALDI-TOF mass spectrometry. Interestingly, this study is the first to demonstrate that cateslytin, the active domain of catestatin, is active against S. aureus and is interestingly resistant to degradation by S. aureus proteases.
Highlights
[1] They are naturally processed to produce numerous peptides with various biological activities. [2,3,4] During the past decade, our group characterized several new antimicrobial peptides (AMPs) derived from chromogranin A (CgA) [5,6,7,8] and chromogranin B (CgB). [3,9] These peptides are released by stimulated chromaffin cells of adrenal medulla and by activated polymorphonuclear neutrophils (PMNs). [5,7] Sequences of these peptides are highly conserved during evolution, suggesting that they are wellintegrated in innate immune system
As staphylococci colonize skin and epithelia, regardless of the expression of antimicrobial Cgs-derived peptides, [24] we aimed to investigate the antimicrobial effects of CAT and its shorter fragment cateslytin (CTL, CgA344–358) against S. aureus
Taking into account the difference in the activity of these peptides, using HPLC and proteomic analysis, we examined the degradation of these CgA-derived peptides by staphylococcal proteases released into bacterial supernatants
Summary
Chromogranins (Cgs) constitute the predominant family of proteins enclosed in secretory vesicles of chromaffin cells. [1] They are naturally processed to produce numerous peptides with various biological activities. [2,3,4] During the past decade, our group characterized several new antimicrobial peptides (AMPs) derived from chromogranin A (CgA) [5,6,7,8] and chromogranin B (CgB). [3,9] These peptides are released by stimulated chromaffin cells of adrenal medulla and by activated polymorphonuclear neutrophils (PMNs). [5,7] Sequences of these peptides are highly conserved during evolution, suggesting that they are wellintegrated in innate immune system. [10] Among these AMPs: chromofungin (CHR, CgA47–66) and catestatin (CAT, CgA344– 364), derived from bovine CgA, activate PMNs and induce a calcium influx into immune cells. [11].Staphylococcus aureus is the most frequently isolated pathogen in Gram-positive sepsis, often involved in blood clotting disorders and destruction of endocardial tissue. [12] S. aureus has developed several mechanisms to avoid immune response including resistance to AMPs, [13] impairment of phagocyte recruitment, [14] escape from neutrophil extracellular traps, [15] interference with complement, [16] neutrophil lysis, resistance to oxidative burst [17] and non-specific binding and degradation of immunoglobulins. [18] The AMPs evasion mechanisms deployed by S. aureus include proteolytic degradation by extracellular proteases of three major catalytic classes, namely metallo-, serine- and papain-like cysteine proteases. [19] The expression of proteolytic enzymes is controlled directly by global regulators of virulence factors such as agr, sar [20,21] and indirectly by RsbU that controls Sigma(B) activity.[22]. [10] Among these AMPs: chromofungin (CHR, CgA47–66) and catestatin (CAT, CgA344– 364), derived from bovine CgA, activate PMNs and induce a calcium influx into immune cells. [19] The expression of proteolytic enzymes is controlled directly by global regulators of virulence factors such as agr, sar [20,21] and indirectly by RsbU that controls Sigma(B) activity.[22] SarA is a regulator of methicillin resistance factor (fmtA).[23] It has been previously reported that S. aureus metallo-protease aureolysin can cleave and inactivate human cathelicidin LL-37, thereby contributing to bacterial escape from the innate immune system. Taking into account the difference in the activity of these peptides, using HPLC and proteomic analysis (sequencing and MALDITOF mass spectrometry), we examined the degradation of these CgA-derived peptides by staphylococcal proteases released into bacterial supernatants. We report the relationship between peptide sequences and their sensitivity to bacterial proteases, as well as the possibility to use them as new antimicrobial agents in combination with antibiotics
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