The vast majority of systemic bacterial infections are caused by facultative, often antibiotic-resistant, pathogens colonizing human body surfaces. Nasal carriage of Staphylococcus aureus predisposes to invasive infection, but the mechanisms that permit or interfere with pathogen colonization are largely unknown. Whereas soil microbes are known to compete by production of antibiotics, such processes have rarely been reported for human microbiota. We show that nasal Staphylococcus lugdunensis strains produce lugdunin, a novel thiazolidine-containing cyclic peptide antibiotic that prohibits colonization by S. aureus, and a rare example of a non-ribosomally synthesized bioactive compound from human-associated bacteria. Lugdunin is bactericidal against major pathogens, effective in animal models, and not prone to causing development of resistance in S. aureus. Notably, human nasal colonization by S. lugdunensis was associated with a significantly reduced S. aureus carriage rate, suggesting that lugdunin or lugdunin-producing commensal bacteria could be valuable for preventing staphylococcal infections. Moreover, human microbiota should be considered as a source for new antibiotics.

Ever since the discovery of endogenous host defense antimicrobial peptides it has been discussed how these evolutionary conserved molecules avoid to induce resistance and to remain effective. Human ß-defensin 1 (hBD1) is an ubiquitously expressed endogenous antimicrobial peptide that exhibits qualitatively distinct activities between its oxidized and reduced forms. Here, we explore these antimicrobial mechanisms. Surprisingly, using electron microscopy we detected a so far unknown net-like structure surrounding bacteria, which were treated with the reduced but not the oxidized form of hBD1. A transmigration assay demonstrated that hBD1-derived nets capture bacteria and inhibit bacterial transmigration independent of bacterial killing. The presence of nets could completely prevent migration of hBD1 resistant pathogens and are stable in the presence of human duodenal secretion with a high amount of proteases. In contrast to HD6, cysteins are necessary for net formation. This redox-dependent function serves as an additional mechanism of action for hBD1 and differs from net formation by other defensins such as Paneth cell-derived human α-defensin 6 (HD6). While hBD1red and hBD1ox have distinct antimicrobial profiles and functions, only the reduced form provides additional host protection by entrapping bacteria in extracellular net structures preventing bacterial invasion. Better understanding of the modes of action of endogenous host peptides will help to find new antimicrobial strategies.

Lugdunin, a novel thiazolidine cyclopeptide, exhibits micromolar activity against methicillin‐resistant Staphylococcus aureus (MRSA). For structure–activity relationship (SAR) studies, synthetic analogues obtained from alanine and stereo scanning as well as peptides with modified thiazolidine rings were tested for antimicrobial activity. The thiazolidine ring and the alternating d ‐ and l ‐amino acid backbone are essential. Notably, the non‐natural enantiomer displays equal activity, thus indicating the absence of a chiral target. The antibacterial activity strongly correlates with dissipation of the membrane potential in S. aureus. Lugdunin equalizes pH gradients in artificial membrane vesicles, thereby maintaining membrane integrity, which demonstrates that proton translocation is the mode of action (MoA). The incorporation of extra tryptophan or propargyl moieties further expands the diversity of this class of thiazolidine cyclopeptides.

‡ Equally contributedThe complex cell envelope of Gram-negative bacteria comprises two membranes: the outer membrane (OM) and the cytoplasmic membrane. The two membranes delimit the periplasmic space of the bacterial cell and prevent the accumulation of toxic agents in the cytosol while regulating the access of nutrients vital for growth and cell function. The OM is the first barrier during compound uptake. It is composed of an asymmetric bilayer: an inner leaflet of phospholipids and an outer leaflet of lipopolysaccharides (LPS). Both OM leaflets combined prevent the efficient diffusion of hydrophilic as well as hydrophobic molecules. Porins, waterfilled channels spanning across the OM, enable passive diffusion of small, hydrophilic molecules into the periplasm. Substrate specificity is mainly defined by the constriction zone within the barrel structure of these porins, determining entry of molecules by factors such as size, shape, electric multipoles, and rigidity. 1,2,3 E. coli encodes multiple porins. The major porins OmpF and OmpC are highly abundant and both cation-selective, and it has been thought that they restrict the passage to compounds with a size-exclusion limit of about 600 Da. 4 However, it has recently been suggested that this limit should be redefined using other parameters. 5,6 The translocation of several classes of antibiotics, e.g. β-lactams and fluoroquinolones, through porins has been investigated extensively. Also, porin modification emerged as antibiotic resistance mechanism in clinical isolates, 7 based on specific changes in amino acid residues or decreased expression of wild-type porins. 8 Aminoglycosides (AGs) target the ribosome in the cytoplasm, thus they have to overcome both membranes in Gram-negative bacteria. Despite their frequent use as therapeutic agents, the mechanisms of their OM translocation remain incompletely understood. The self-promoted pathway is a proposed uptake mechanism. Here, divalent cations between LPS molecules are displaced by AGs, which leads to brief OM destabilization, thereby enabling OM translocation. 9