Skin Sentinels: Defensive Effector Molecules Produced by the Skin Microbiome

Organisms which rely solely on innate defense systems must combat a large number of antagonists with a comparatively low number of defense effector molecules. As one solution of this problem, these organisms have evolved effector molecules targeting epitopes that are conserved between different antagonists of a specific taxon or, if possible, even of different taxa. In order to restrict the activity of the defense effector molecules to physiologically relevant taxa, these target epitopes should, on the other hand, be taxon-specific and easily accessible. Glycans fulfill all these requirements and are therefore a preferred target of defense effector molecules, in particular defense proteins. Here, we review this defense strategy using the example of the defense system of multicellular (filamentous) fungi against microbial competitors and animal predators.

Preformed mediators of defense-Gatekeepers enter the spotlight.

Effects of Candida albicans infection on defense effector secretion by human oral mucosal epithelial cells

Host defense effector molecules in mucosal secretions

The Fc portion of immunoglobulin G (IgG) promotes defensive effector functions in the immune system by interacting with Fcγ receptors and complement component C1q. These interactions critically depend on N-glycosylation at Asn297 of each CH2 domain, where biantennary complex-type oligosaccharides contain microheterogeneities resulting primarily from the presence or absence of non-reducing terminal galactose residues. Crystal structures of Fc have shown that a pair of N-glycans is located between the two CH2 domains. Here we applied our metabolic isotope labeling technique using mammalian cells for in-solution structural characterization of mouse IgG2b-Fc glycoforms with a molecular mass of 54kDa. Based on spectral assignments of the N-glycans as well as polypeptide backbones of Fc, we probed conformational perturbations of Fc induced by N-glycan trimming, especially enzymatic degalactosylation. The results indicated that degalactosylation structurally perturbed the Fc region through rearrangement of glycan-protein interactions. The spectral assignments of IgG2b-Fc glycoprotein will provide the basis for NMR investigation of its dynamic conformations and interactions with effector molecules in solution.

Conventional analyses distinguish between antimicrobial peptides by differences in amino acid sequence. Yet structural paradigms common to broader classes of these molecules have not been established. The current analyses examined the potential conservation of structural themes in antimicrobial peptides from evolutionarily diverse organisms. Using proteomics, an antimicrobial peptide signature was discovered to integrate stereospecific sequence patterns and a hallmark three-dimensional motif. This striking multidimensional signature is conserved among disulfide-containing antimicrobial peptides spanning biological kingdoms, and it transcends motifs previously limited to defined peptide subclasses. Experimental data validating this model enabled the identification of previously unrecognized antimicrobial activity in peptides of known identity. The multidimensional signature model provides a unifying structural theme in broad classes of antimicrobial peptides, will facilitate discovery of antimicrobial peptides as yet unknown, and offers insights into the evolution of molecular determinants in these and related host defense effector molecules.

The natural antimicrobial peptides are ancient host defense effector molecules, present in organisms across the evolutionary spectrum. Several properties of α-melanocyte stimulating hormone (α-MSH) suggested that it could be a natural antimicrobial peptide. α-MSH is a primordial peptide that appeared during the Paleozoic era, long before adaptive immunity developed and, like natural antimicrobial molecules, is produced by barrier epithelia, immunocytes, and within the central nervous system. α-MSH was discovered to have antimicrobial activity against two representative pathogens, Staphylococcus aureus and Candida albicans. The candidacidal influences of α-MSH appeared to be mediated by increases in cell cyclic adenosine monophosphate (cAMP). The cAMP-inducing capacity of α-MSH likely interferes with the yeast's own regulatory mechanisms of this essential signaling pathway. It is remarkable that this mechanism of action in yeast mimics the influences of α-MSH in mammalian cells in which the peptide binds to G-protein-linked melanocortin receptors, activates adenylyl cyclase, and increases cAMP. When considering that most of the natural antimicrobial peptides enhance the local inflammatory reaction, the anti-inflammatory and antipyretic effects of α-MSH confer unique properties to this molecule relative to other natural antimicrobial molecules. Synthetic derivatives, chemically stable and resistant to enzymatic degradation, could form the basis for novel therapies that combine anti-inflammatory and antimicrobial properties.

Virulence is thought to coevolve as a result of reciprocal selection between pathogens and their hosts. This paper focuses on coevolution between microbial proteinases operating as virulence factors and host defense molecules of insects. Owing to shorter generation times and smaller genomes, microbes exhibit a high evolutionary adaptability in comparison with their hosts. Indeed, the latter can only compete with pathogens if they evolve mechanisms providing a comparable genetic plasticity. Gene or domain duplication and shuffling by recombination is the driving force behind the countermeasures in host defense effectors. Recent literature provides evidence for both diversifications of fungal proteinases involved in pathogenesis and expansion host proteinase inhibitors subsets contributing to insect innate immunity. For example, the pathogen-associated spectrum of proteolytic enzymes encompasses thermolysin-like metalloproteinases that putatively promoted the evolution of corresponding host inhibitors of these virulence factors which complement the insect repertoire of antimicrobial defense molecules. Beyond mutual diversification of effector molecules coevolution resulted also in sophisticated molecular adaptations of host insects such as sensing and feedback-loop regulation of microbial metalloproteinases and corresponding countermeasures of pathogens providing evasion of host immunity induced by these virulence factors.

Antimicrobial peptides represent ancient host defense effector molecules present in organisms across the evolutionary spectrum. Lots of antimicrobial peptides were synthesized based on well-known structural motif widely existed in a variety of lives. Leucine-rich repeats (LRRs) are sequence motifs present in over 60,000 proteins identified from viruses, bacteria, and eukaryotes. To elucidate if LRR motif possesses antimicrobial potency, two peptides containing one or two LRRs were designed. The biological activity and membrane-peptide interactions of the peptides were analyzed. The results showed that the tandem of two LRRs exhibited similar antibacterial activity and significantly weaker hemolytic activity against hRBCs than the well-known membrane active peptide melittin. The peptide with one LRR was defective at antimicrobial and hemolytic activity. The peptide containing two LRRs formed α-helical structure, respectively, in the presence of membrane-mimicking environment. LRR-2 retained strong resistance to cations, heat, and some proteolytic enzymes. The blue shifts of the peptides in two lipid systems correlated positively with their biological activities. Other membrane-peptide experiments further provide the evidence that the peptide with two LRRs kills bacteria via membrane-involving mechanism. The present study increases our new understanding of well-known LRR motif in antimicrobial potency and presents a potential strategy to develop novel antibacterial agents.

Over the last decade, decreasing effectiveness of conventional antimicrobial-drugs has caused serious problems due to the rapid emergence of multidrug-resistant pathogens. Furthermore, biofilms, which are microbial communities that cause serious chronic infections and dental plaque, form environments that enhance antimicrobial resistance. As a result, there is a continuous search to overcome or control such problems, which has resulted in antimicrobial peptides being considered as an alternative to conventional drugs. Antimicrobial peptides are ancient host defense effector molecules in living organisms. These peptides have been identified in diverse organisms and synthetically developed by using peptidomimic techniques. This review was conducted to demonstrate the mode of action by which antimicrobial peptides combat multidrug-resistant bacteria and prevent biofilm formation and to introduce clinical uses of these compounds for chronic disease, medical devices, and oral health. In addition, combinations of antimicrobial peptides and conventional drugs were considered due to their synergetic effects and low cost for therapeutic treatment.

It is said that nature is the greatest innovator, yet molecular conservation can be equally powerful. One key requirement for the survival of any host is its ability to defend against infection, predation and competition. Recent discoveries, including the presence of a multidimensional structural signature, have revealed a previously unforeseen structural and functional congruence among host defence effector molecules spanning all kingdoms of life. Antimicrobial peptides, kinocidins, polypeptide venoms and other molecules that were once thought to be distinct in form and function now appear to be members of an ancient family of host defence effectors. These molecules probably descended from archetype predecessors that emerged during the beginning of life on earth. Understanding how nature has sustained these host defence molecules with a potent efficacy in the face of dynamic microbial evolution should provide new opportunities to prevent or treat life-threatening infections.

P149 Role of the humoral pattern recognition molecule PTX3 in defence against urinary tract infections

Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a staphylocidal peptide released by activated platelets. This peptide initiates its microbicidal activity by membrane permeabilization, with ensuing inhibition of intracellular macromolecular synthesis. RP-1 is a synthetic congener modeled on the C-terminal microbicidal alpha-helix of tPMP-1. This study compared the staphylocidal mechanisms of RP-1 with those of tPMP-1, focusing on isogenic tPMP-1-susceptible (ISP479C) and -resistant (ISP479R) Staphylococcus aureus strains for the following quantitative evaluations: staphylocidal efficacy; comparative MIC; membrane permeabilization (MP) and depolarization; and DNA, RNA, and protein synthesis. Although the proteins had similar MICs, RP-1 caused significant killing of ISP479C (<50% survival), correlating with extensive MP (>95%) and inhibition of DNA and RNA synthesis (>90%), versus substantially reduced killing of ISP479R (>80% survival), with less MP (55%) and less inhibition of DNA or RNA synthesis (70 to 80%). Interestingly, RP-1-induced protein synthesis inhibition was equivalent in both strains. RP-1 did not depolarize the cell membrane and caused a relatively short postexposure growth inhibition. These data closely parallel those previously reported for tPMP-1 against this strain set and exemplify how synthetic molecules can be engineered to reflect structure-activity relationships of functional domains in native host defense effector molecules.

Lymphotoxin β receptor (LTβR) signaling plays an important role in efficient initiation of host responses to a variety of pathogens, encompassing viruses, bacteria, and protozoans via induction of the type I interferon response. The present study reveals that after Toxoplasma gondii infection, LTβR−/− mice show a substantially reduced survival rate when compared to wild-type mice. LTβR−/− mice exhibit an increased parasite load and a more pronounced organ pathology. Also, a delayed increase of serum IL-12p40 and a failure of the protective IFNγ response in LTβR−/− mice were observed. Serum NO levels in LTβR−/− animals rose later and were markedly decreased compared to wild-type animals. At the transcriptional level, LTβR−/− animals exhibited a deregulated expression profile of several cytokines known to play a role in activation of innate immunity in T. gondii infection. Importantly, expression of the IFNγ-regulated murine guanylate-binding protein (mGBP) genes was virtually absent in the lungs of LTβR−/− mice. This demonstrates clearly that the LTβR is essential for the induction of a type II IFN-mediated immune response against T. gondii. The pronounced inability to effectively upregulate host defense effector molecules such as GBPs explains the high mortality rates of LTβR−/− animals after T. gondii infection.

Antibiotic peptides are host defense effector molecules broadly distributed throughout the animal kingdom. Many different families of peptides can be identified based on mature peptide structure or, in some cases, by similar propeptide structure (1). These peptides have been identified in a variety of different cell and tissue types. In several instances, members of a single family of antibiotic peptides are distributed in multiple tissues in a single species. In some species, such as human, more than one family of antibiotic peptide has been identified (2,3). These features afford opportunities to better understand the principles underlying the evolution of this system of host defense.