| Abstract: | Dermcidin (DCD) is an antimicrobial peptide which is constitutively expressed in eccrine sweat glands. By postsecretory proteolytic processing in sweat, the DCD protein gives rise to anionic and cationic DCD peptides with a broad spectrum of antimicrobial activity. Many antimicrobial peptides induce membrane permeabilization as part of their killing mechanism, which is accompanied by a loss of the bacterial membrane potential. In this study we show that there is a time-dependent bactericidal activity of anionic and cationic DCD-derived peptides which is followed by bacterial membrane depolarization. However, DCD-derived peptides do not induce pore formation in the membranes of gram-negative and gram-positive bacteria. This is in contrast to the mode of action of the cathelicidin LL-37. Interestingly, LL-37 as well as DCD-derived peptides inhibit bacterial macromolecular synthesis, especially RNA and protein synthesis, without binding to microbial DNA or RNA. Binding studies with components of the cell envelope of gram-positive and gram-negative bacteria and with model membranes indicated that DCD-derived peptides bind to the bacterial envelope but show only a weak binding to lipopolysaccharide (LPS) from gram-negative bacteria or to peptidoglycan, lipoteichoic acid, and wall teichoic acid, isolated from Staphylococcus aureus. In contrast, LL-37 binds strongly in a dose-dependent fashion to these components. Altogether, these data indicate that the mode of action of DCD-derived peptides is different from that of the cathelicidin LL-37 and that components of the bacterial cell envelope play a role in the antimicrobial activity of DCD.Antimicrobial peptides (AMPs) serve as a first line of innate host defense in many species such as plants, amphibians, insects, and mammals. AMPs show a broad-spectrum antimicrobial activity against a wide range of pathogens including bacteria, fungi, and enveloped viruses (51). Most gene-encoded AMPs are synthesized as proforms, which are subsequently processed into mature peptides of various lengths. A common feature of most of these peptides is that they are cationic and form amphipathic structures (3). The mode of action of most AMPs is incompletely understood. It is believed that most AMPs kill microorganisms by membrane permeation either via pore formation or via membrane disintegration like that induced by the human cathelicidin LL-37. However, membrane disruption may not reflect the complex processes involved in the killing of microorganisms (5, 48). In addition, several AMPs clearly act differently and intracellular target sites have been identified (15). The mode of action has been unraveled for only a few AMPs which act via defined targets, such as the lantibiotic nisin, which specifically binds to bacterial lipid II, a membrane-bound component involved in peptidoglycan (PG) synthesis (7, 46). Similarly, the lantibiotic mersacidin interferes with transglycosylation and PG synthesis in gram-positive bacteria by direct targeting of lipid II (6). Furthermore, buforin II kills microorganisms by disruption of critical intracellular processes such as the inhibition of macromolecular biosynthesis or by interacting with nucleic acids inside the microorganisms (33). For several AMPs it has been demonstrated that the charge and the composition of the bacterial cell envelope determine sensitivity to AMPs (37). Staphylococcus aureus mutants lacking specific modifications in the bacterial envelope are highly susceptible to a variety of cationic AMPs. For example, incorporation of d-alanine into S. aureus teichoic acids by the dltA enzymes or the lysinylation of phosphatidylglycerol by mprF confers resistance to defensins, protegrins, and other AMPs by repulsion of the cationic peptides (36).Dermcidin (DCD) was identified by our group as a human AMP which is constitutively expressed in eccrine sweat glands and secreted into sweat (40). By postsecretory proteolytic processing in human sweat, the precursor protein gives rise to several truncated DCD peptides varying in length from 25 to 48 amino acids and with net charges between −2 and +2 (2, 10, 38). Several DCD peptides show antimicrobial activity against pathogenic microorganisms such as S. aureus, Escherichia coli, Enterococcus faecalis, Candida albicans, Staphylococcus epidermidis, Pseudomonas putida, and methicillin-resistant S. aureus as well as rifampin- and isoniazid-resistant Mycobacterium tuberculosis (9, 25, 40, 41, 45). We were able to show that DCD-derived peptides are also active under high-salt conditions and in a buffer resembling human sweat (40). Antimicrobially active DCD peptides, namely, the anionic peptides DCD-1L (48-mer) and DCD-1 (47-mer) and the cationic peptides SSL-25 (25-mer) and SSL-23 (23-mer), are derived from the C-terminal region of the precursor protein. Interestingly, these peptides have diverse and overlapping spectra of activity which are independent of the net peptide charge (41). In previous studies we showed that DCD peptides interact with the bacterial cell envelope and kill gram-negative bacteria without forming pores in membranes (41). In this study we investigated the mode of antimicrobial activity of DCD-derived peptides in more detail and studied bacterial factors that govern sensitivity or tolerance to DCD in the model microorganism S. aureus. In our first approach, we tried to identify the bacterial surface molecules to which DCD peptides bind. Second, we analyzed the bacterial response to DCD peptide challenge. Finally, we analyzed bacterial mutants to elucidate the mechanisms determining DCD sensitivity. |
