Antimicrobial Host Defensins – Specific Antibiotic Activities and Innate Defense Modulation

infections heavily relies on strategies which aim to inhibit and kill pathogens with high specificity. These strategies are very successful and antibiotics have contributed to increasing human life expectancy more than any other class of therapeutic drugs. However, antibiotics are losing efficacy as a result of high selection pressure and rapid resistance development. Thus, strategies that rely on boosting natural host defenses are gaining more attention, since compounds targeting host mechanisms should control infections regardless of the antibiotic resistance levels of pathogens. Antimicrobial peptides (AMPs) are considered as ideal candidates for such novel anti-infective strategies since they combine direct antibiotic activities with modulation of immune responses (Figure 1). However, AMPs frequently lack specific molecular targets and tend to have membrane disruptive activities, bearing risks of cytotoxicity. For antiinfective drug development, AMPs should ideally inhibit specific microbial targets without impacting on membranes; peptides with such properties were recently identified in a large subfamily of AMPs, the defensins. All multicellular organisms produce AMPs to protect surfaces and tissues from invading pathogens. These peptides have been referred to as AMPs and more recently as host defense peptides (HDPs). HDPs are ancient effector molecules of innate immunity with multiple functions. They do not share specific sequence similarities, but can be generally defined as amphiphatic cationic peptides consisting of 12–50 amino acids. They are either linear (e.g., LL-37, magainin, and indolicidin) or have tertiary structures stabilized by disulfide bonds (Hancock and Lehrer, 1998; Shai, 2002; Zasloff, 2002). Defensins sensu stricto belong to the latter class and were first isolated from mammals, and subsequently also found in invertebrates and plants. Plant, fungal, and invertebrate defensins share a common structural motif, the cysteine-stabilized αβ-motif that is composed of an α-helix linked to an antiparallel β-sheet with three or four disulfide bonds; they display either antifungal or antibacterial activity. Recently, it has been demonstrated that antibacterial defensins of fungi and invertebrates bind with high affinity to the bacterial cell wall precursor lipid II. They form an equimolar stoichiometric complex with lipid II, thereby inhibiting the incorporation of the cell wall building-block into the nascent peptidoglycan network (Schmitt et al., 2010; Schneider et al., 2010). NMRbased modeling of the plectasin-lipid II complex indicated that the fungal defensin interacts with the pyrophosphate moiety of lipid II by forming four hydrogen bonds (involving residues F2, G3, C4, and C37). Additionally, a salt bridge between the N-terminus (His18) and the d-glutamic acid in position 2 of the lipid II stem peptide is important for binding (Schneider et al., 2010). Interestingly, the amino acid residues involved in the lipid II binding of plectasin are also present in many other fungal and invertebrate defensins, suggesting a conserved lipid II binding motif. Cell wall biosynthesis is a prominent target of clinically used antibiotics. For example, the glycopeptide vancomycin, a last-resort antibiotic for treatment of infections with multi-resistant Gram-positive bacteria, binds to the D-alanyl-D-alanine terminus of the lipid II pentapeptide. However, cross-resistance between vancomycin and plectasin could not be observed and also the presence of D-alanine-D-lactate found in vancomycin-resistant bacteria did not affect the activity of plectasin (Schneider et al., 2010). In general, only modest resistance development toward HDPs has been observed under in vitro selection pressure (Zhang et al., 2005). The lipid II isoprenoid anchor (C 55 P) is also involved in the biosynthesis of other major cell envelope polymers (e.g., wall teichoic acid, capsules). Synthesis of C 55 P-anchored molecules always starts with the transfer of a sugar moiety to the lipid carrier, forming a pyrophosphate linkage. This structural motif is highly conserved, as it is part of several essential building blocks and therefore cannot be easily modified to confer resistance. The antifungal action of plant and invertebrate defensins also appears to be highly specific and is based on interaction with particular sphingolipids in membranes and cell walls of susceptible fungi. For example, the interaction of RsAFP2 (from radish seeds) with fungal glucosylceramides causes the production of radical oxygen species and apoptosis as well as cell wall stress, septin delocalization, and ceramide accumulation (Thevissen et al., 2012). Other plant defensins such as DmaMp1 (from Dahlia merckii) bind specifically to inositol phosphoryl-containing sphingolipids leading to membrane permeabilization and ion efflux (Thevissen et al., 1996, 2003). In contrast, the activity of vertebrate defensins may be of intermediate specificity for microbial targets with a broader activity spectrum. Vertebrate defensins comprise three subfamilies, α-, β-, and θ-defensins, which differ in their pairing of Antimicrobial host defensins – specific antibiotic activities and innate defense modulation