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Cytolytic proteins and peptide toxins are classical virulence factors of several bacterial pathogens which disrupt epithelial barrier function, damage cells and activate or modulate host immune responses. Such toxins have not been identified previously in human pathogenic fungi. Here we identify the first, to our knowledge, fungal cytolytic peptide toxin in the opportunistic pathogen Candida albicans . This secreted toxin directly damages epithelial membranes, triggers a danger response signalling pathway and activates epithelial immunity. Membrane permeabilization is enhanced by a positive charge at the carboxy terminus of the peptide, which triggers an inward current concomitant with calcium influx. C. albicans strains lacking this toxin do not activate or damage epithelial cells and are avirulent in animal models of mucosal infection. We propose the name ‘Candidalysin’ for this cytolytic peptide toxin; a newly identified, critical molecular determinant of epithelial damage and host recognition of the clinically important fungus, C. albicans . This study identifies a cytolytic peptide toxin in the opportunistic human fungal pathogen Candida albicans —the peptide is both a crucial virulence factor that permeabilizes the host cell plasma membrane and a key signal that triggers a host danger response pathway. A toxin identified in a human fungal pathogen This study identifies and characterizes a cytolytic peptide toxin in the opportunistic human fungal pathogen Candida albicans . The peptide, termed Candidalysin, acts both as a crucial virulence factor that permeabilizes the host cell plasma membrane and as a key signal that triggers a host danger-response pathway.

The methanoarchaea Methanosphaera stadtmanae and Methanobrevibacter smithii are known to be part of the indigenous human gut microbiota. Although the immunomodulatory effects of bacterial gut commensals have been studied extensively in the last decade, the impact of methanoarchaea in human's health and disease was rarely examined. Consequently, we studied and report here on the effects of M. stadtmanae and M. smithii on human immune cells. Whereas exposure to M. stadtmanae leads to substantial release of proinflammatory cytokines in monocyte-derived dendritic cells (moDCs), only weak activation was detected after incubation with M. smithii. Phagocytosis of M. stadtmanae by moDCs was demonstrated by confocal microscopy as well as transmission electronic microscopy (TEM) and shown to be crucial for cellular activation by using specific inhibitors. Both strains, albeit to different extents, initiate a maturation program in moDCs as revealed by up-regulation of the cell-surface receptors CD86 and CD197 suggesting additional activation of adaptive immune responses. Furthermore, M. stadtmanae and M. smithii were capable to alter the gene expression of antimicrobial peptides in moDCs to different extents. Taken together, our findings strongly argue that the archaeal gut inhabitants M. stadtmanae and M. smithii are specifically recognized by the human innate immune system. Moreover, both strains are capable of inducing an inflammatory cytokine response to different extents arguing that they might have diverse immunomodulatory functions. In conclusion, we propose that the impact of intestinal methanoarchaea on pathological conditions involving the gut microbiota has been underestimated until now.

More than two million people worldwide are affected by life-threatening, invasive fungal infections annually. Candida species are the most common cause of nosocomial, invasive fungal infections and are associated with mortality rates above 40%. Despite the increasing incidence of drug-resistance, the development of novel antifungal formulations has been limited. Here we investigate the antifungal mode of action and therapeutic potential of positively charged, synthetic peptide mimics to combat Candida albicans infections. Our data indicates that these synthetic polymers cause endoplasmic reticulum stress and affect protein glycosylation, a mode of action distinct from currently approved antifungal drugs. The most promising polymer composition damaged the mannan layer of the cell wall, with additional membrane-disrupting activity. The synergistic combination of the polymer with caspofungin prevented infection of human epithelial cells in vitro, improved fungal clearance by human macrophages, and significantly increased host survival in a Galleria mellonella model of systemic candidiasis. Additionally, prolonged exposure of C.   albicans to the synergistic combination of polymer and caspofungin did not lead to the evolution of tolerant strains in vitro. Together, this work highlights the enormous potential of these synthetic peptide mimics to be used as novel antifungal formulations as well as adjunctive antifungal therapy. Fungal infections are severely underestimated as a cause of mortality, and alternative drugs are urgently needed. Here, Schaefer et al. show that a synthetic polymer mimicking defensins shows different, but synergistic activity with known antifungals.

ADAM17, a prominent member of the ‘Disintegrin and Metalloproteinase’ (ADAM) family, controls vital cellular functions through cleavage of transmembrane substrates. Here we present evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. PS exposure is tightly coupled to substrate shedding provoked by diverse ADAM17 activators. PS dependency is demonstrated in the following: (a) in Raji cells undergoing apoptosis; (b) in mutant PSA-3 cells with manipulatable PS content; and (c) in Scott syndrome lymphocytes genetically defunct in their capacity to externalize PS in response to intracellular Ca 2+ elevation. Soluble phosphorylserine but not phosphorylcholine inhibits substrate cleavage. The isolated membrane proximal domain (MPD) of ADAM17 binds to PS but not to phosphatidylcholine liposomes. A cationic PS-binding motif is identified in this domain, replacement of which abrogates liposome-binding and renders the protease incapable of cleaving its substrates in cells. We speculate that surface-exposed PS directs the protease to its targets where it then executes its shedding function. ADAM17 is a member of the ‘Disintegrin and Metalloproteinase’ family of proteases, that cleaves transmembrane substrates from the surfaces of cells. Here the authors show that surface exposure of phosphatidylserine is required for ADAM17 sheddase activity, possibly by directing the protease to its substrates.

The methyltransferase FliB posttranslationally modifies surface-exposed ɛ-N-lysine residues of flagellin, the protomer of the flagellar filament in Salmonella enterica (S . enterica) . Flagellin methylation, reported originally in 1959, was recently shown to enhance host cell adhesion and invasion by increasing the flagellar hydrophobicity. The role of FliB in this process, however, remained enigmatic. In this study, we investigated the properties and mechanisms of FliB from S . enterica in vivo and in vitro . We show that FliB is an S-adenosylmethionine (SAM) dependent methyltransferase, forming a membrane associated oligomer that modifies flagellin in the bacterial cytosol. Using X-band electron paramagnetic resonance (EPR) spectroscopy, zero-field 57 Fe Mössbauer spectroscopy, methylation assays and chromatography coupled mass spectrometry (MS) analysis, we further found that FliB contains an oxygen sensitive [4Fe-4S] cluster that is essential for the methyl transfer reaction and might mediate a radical mechanism. Our data indicate that the [4Fe-4S] cluster is coordinated by a cysteine rich motif in FliB that is highly conserved among multiple genera of the Enterobacteriaceae family.

A multitude of membrane active peptides exists that divides into subclasses, such as cell penetrating peptides (CPPs) capable to enter eukaryotic cells or antimicrobial peptides (AMPs) able to interact with prokaryotic cell envelops. Peptide membrane interactions arise from unique sequence motifs of the peptides that account for particular physicochemical properties. Membrane active peptides are mainly cationic, often primary or secondary amphipathic, and they interact with membranes depending on the composition of the bilayer lipids. Sequences of these peptides consist of short 5–30 amino acid sections derived from natural proteins or synthetic sources. Membrane active peptides can be designed using computational methods or can be identified in screenings of combinatorial libraries. This review focuses on strategies that were successfully applied to the design and optimization of membrane active peptides with respect to the fact that diverse features of successful peptide candidates are prerequisites for biomedical application. Not only membrane activity but also degradation stability in biological environments, propensity to induce resistances, and advantageous toxicological properties are crucial parameters that have to be considered in attempts to design useful membrane active peptides. Reliable assay systems to access the different biological characteristics of numerous membrane active peptides are essential tools for multi-objective peptide optimization.

The methanogenic archaeon Methanomassiliicoccus luminyensis strain B10T was isolated from human feces just a few years ago. Due to its remarkable metabolic properties, particularly the degradation of trimethylamines, this strain was supposed to be used as \"Archaebiotic\" during metabolic disorders of the human intestine. However, there is still no data published regarding adaptations to the natural habitat of M. luminyensis as it has been shown for the other two reported mucosa-associated methanoarchaea. This study aimed at unraveling susceptibility of M. luminyensis to antimicrobial peptides as well as its immunogenicity. By using the established microtiter plate assay adapted to the anaerobic growth requirements of methanogenic archaea, we demonstrated that M. luminyensis is highly sensitive against LL32, a derivative of human cathelicidin (MIC = 2 μM). However, the strain was highly resistant against the porcine lysin NK-2 (MIC = 10 μM) and the synthetic antilipopolysaccharide peptide (Lpep) (MIC>10 μM) and overall differed from the two other methanoarchaea, Methanobrevibacter smithii and Methanosphaera stadtmanae in respect to AMP sensitivity. Moreover, only weak immunogenic potential of M. luminyensis was demonstrated using peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (moDCs) by determining release of pro-inflammatory cytokines. Overall, our findings clearly demonstrate that the archaeal gut inhabitant M. luminyensis is susceptible to the release of human-derived antimicrobial peptides and exhibits low immunogenicity towards human immune cells in vitro-revealing characteristics of a typical commensal gut microbe.

The effect of asymmetric membranes containing lipopolysaccharides (LPS) on the outer membrane protein F (OmpF) reconstitution, channel orientation, and antibiotic permeation across the outer membrane was investigated. After forming an asymmetric planar lipid bilayer composed of LPS on one and phospholipids on the other side, the membrane channel OmpF was added. The ion current recordings demonstrate that LPS has a strong influence on the OmpF membrane insertion, orientation, and gating. Enrofloxacin was used as an example of an antibiotic interacting with the asymmetric membrane and with OmpF. The enrofloxacin caused the blockage of the ion current through the OmpF, depending on the side of addition, the transmembrane voltage applied, and the composition of the buffer. Furthermore, the enrofloxacin changed the phase behavior of the LPS-containing membranes, demonstrating that its membrane activity influences the function of OmpF and potentially the membrane permeability.

The most potent cell wall-derived inflammatory toxins (\"pathogenicity factors\") of Gram-negative and -positive bacteria are lipopolysaccharides (LPS) (endotoxins) and lipoproteins (LP), respectively. Despite the fact that the former signals toll-like receptor 4 (TLR4) and the latter TLR2, the physico-chemistry of these compounds exhibits considerable similarity, an amphiphilic molecule with a polar and charged backbone and a lipid moiety. While the exterior portion of the LPS (i.e., the O-chain) represents the serologically relevant structure, the inner part, the lipid A, is responsible for one of the strongest inflammatory activities known. In the last years, we have demonstrated that antimicrobial peptides from the Pep19-2.5 family, which were designed to bind to LPS and LP, act as anti-inflammatory agents against sepsis and endotoxic shock caused by severe bacterial infections. We also showed that this anti-inflammatory activity requires specific interactions of the peptides with LPS and LP leading to exothermic reactions with saturation characteristics in calorimetry assays. Parallel to this, peptide-mediated neutralization of LPS and LP involves changes in various physical parameters, including both the gel to liquid crystalline phase transition of the acyl chains and the three-dimensional aggregate structures of the toxins. Furthermore, the effectivity of neutralization of pathogenicity factors by peptides was demonstrated in several models together with the finding that a peptide-based therapy sensitizes bacteria (also antimicrobial resistant) to antibiotics. Finally, a significant step in the understanding of the broad anti-inflammatory function of Pep19-2.5 was the demonstration that this compound is able to block the intracellular endotoxin signaling cascade.

Candida albicans produces an important virulence factor, the hypha-associated Ece1-derived secreted peptide toxin candidalysin, which is crucial for the establishment of mucosal and systemic infections. C. albicans has also long been known to be hemolytic, yet the hemolytic factor has not been clearly identified. Here, we show that candidalysin is the hemolytic factor of C. albicans. Its hemolytic activity is modulated by fragments of another Ece1 peptide, P7. Hemolysis by candidalysin can be neutralized by the purinergic receptor antagonist pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS). PPADS also affects candidalysin’s ability to intercalate into synthetic membranes. We also describe the neutralization potential of two anti-candidalysin nanobodies, which are promising candidates for future anti-Candida therapy. This work provides evidence that the historically proposed hemolytic factor of C. albicans is in fact candidalysin and sheds more light on the complex roles of this toxin in C. albicans biology and pathogenicity.