Rainer Podschun

Uncovering the evolutionary history of innate immunity: The simple metazoan Hydra uses epithelial cells for host defence

Although many properties of the innate immune system are shared among multicellular animals, the evolutionary origin remains poorly understood. Here we characterize the innate immune system in Hydra, one of the simplest multicellular animals known. In the complete absence of both protective mechanical barriers and mobile phagocytes, Hydras epithelium is remarkably well equipped with potent antimicrobial peptides to prevent pathogen infection. Induction of antimicrobial peptide production is mediated by the interaction of a leucine-rich repeats (LRRs) domain containing protein with a TIR-domain containing protein lacking LRRs. Conventional Toll-like receptors (TLRs) are absent in the Hydra genome. Our findings support the hypothesis that the epithelium represents the ancient system of host defence.

Physico-chemical and antimicrobial properties of co-sputtered Ag?Au/PTFE nanocomposite coatings

In this work, we used co-sputtering of noble metals together with polytetrafluorethylene (PTFE) as a method for producing antibacterial metal/polymer nanocomposite coatings, where the precious metals are only incorporated in a thin surface layer. Moreover, they are finely dispersed as nanoparticles, thus saving additional material and providing a very large effective surface for metal ion release. Nanocomposite films with thickness between 100 and 300 nm were prepared with a wide range of metal filling between 10 and 40%. The antimicrobial effect of the nanocomposite coatings was evaluated by means of two different assays. The bactericidal activity due to silver release from the surface was determined by a modification of conventional disc diffusion methods. Inhibition of bacterial growth on the coated surface was investigated through a modified proliferation assay. Staphylococcus aureus and S. epidermidis were used as test bacteria, as these species commonly cause infections associated with medical polymer devices. The antibacterial efficiency of the coatings against different bacteria was demonstrated at extremely small noble metal consumption: Au: ~1 mg m−2 and Ag: ~0.1 g m−2. The maximum ability for having an antibacterial effect was shown by the Ag–Au/PTFE nanocomposite, followed by the Ag/PTFE nanocomposite.

Hydramacin-1, Structure and Antibacterial Activity of a Protein from the Basal Metazoan Hydra

Hydramacin-1 is a novel antimicrobial protein recently discovered during investigations of the epithelial defense of the ancient metazoan Hydra. The amino acid sequence of hydramacin-1 shows no sequence homology to any known antimicrobial proteins. Determination of the solution structure revealed that hydramacin-1 possesses a disulfide bridge-stabilized αβ motif. This motif is the common scaffold of the knottin protein fold. The structurally closest relatives are the scorpion oxin-like superfamily. Within this superfamily hydramacin-1 establishes a new family of proteins that all share antimicrobial activity. Hydramacin-1 is potently active against Gram-positive and Gram-negative bacteria including multi-resistant human pathogenic strains. It leads to aggregation of bacteria as an initial step of its bactericidal mechanism. Aggregated cells are connected via electron-dense contacts and adopt a thorn apple-like morphology. Analysis of the hydramacin-1 structure revealed an unusual distribution of amino acid side chains on the surface. A belt of positively charged residues is sandwiched by two hydrophobic areas. Based on this characteristic surface feature and on biophysical analysis of protein-membrane interactions, we propose a model that describes the aggregation effect exhibited by hydramacin-1.

The battle against multi-resistant strains: Renaissance of antimicrobial essential oils as a promising force to fight hospital-acquired infections

Hospital-acquired infections and antibiotic-resistant bacteria continue to be major health concerns worldwide. Particularly problematic is methicillin-resistant Staphylococcus aureus (MRSA) and its ability to cause severe soft tissue, bone or implant infections. First used by the Australian Aborigines, Tea tree oil and Eucalyptus oil (and several other essential oils) have each demonstrated promising efficacy against several bacteria and have been used clinically against multi-resistant strains. Several common and hospital-acquired bacterial and yeast isolates (6 Staphylococcus strains including MRSA, 4 Streptococcus strains and 3 Candida strains including Candida krusei) were tested for their susceptibility for Eucalyptus, Tea tree, Thyme white, Lavender, Lemon, Lemongrass, Cinnamon, Grapefruit, Clove Bud, Sandalwood, Peppermint, Kunzea and Sage oil with the agar diffusion test. Olive oil, Paraffin oil, Ethanol (70%), Povidone iodine, Chlorhexidine and hydrogen peroxide (H(2)O(2)) served as controls. Large prevailing effective zones of inhibition were observed for Thyme white, Lemon, Lemongrass and Cinnamon oil. The other oils also showed considerable efficacy. Remarkably, almost all tested oils demonstrated efficacy against hospital-acquired isolates and reference strains, whereas Olive and Paraffin oil from the control group produced no inhibition. As proven in vitro, essential oils represent a cheap and effective antiseptic topical treatment option even for antibiotic-resistant strains as MRSA and antimycotic-resistant Candida species.

Capsule impedes adhesion to and invasion of epithelial cells by Klebsiella pneumoniae.

ABSTRACT The adhesion of K21a, K26, K36, and K50 capsulatedKlebsiella strains to ileocecal (HCT-8) and bladder (T24) epithelial cell lines was significantly lower than that of their corresponding spontaneous noncapsulated variants K21a/3, K26/1, K36/3, and K50/3, respectively. Internalization of the bacteria by both epithelial cell lines was also significantly reduced. Similarly, a capsule-switched derivative, K2(K36), that exhibited a morphologically larger K36 capsule and formed more capsular material invaded the ileocecal epithelial cell line poorly compared to the corresponding K2 parent strain. None of the capsulated strains exhibited significant mannose-sensitive type 1 fimbriae, whereas two of the noncapsulated variants K21a/3 and K50/3 exhibited potent mannose-sensitive hemagglutinating activity. Although hemagglutinating activity that could be attributed to mannose-resistantKlebsiella type 3 fimbriae was weak in all strains, in several cases the encapsulated parent strains exhibited lower titers than their corresponding noncapsulated variants. Although the level of adhesion to the ileocecal cells is not different from adhesion to bladder cells, bacterial internalization by bladder cells was significantly lower than internalization by ileocecal cells, suggesting that bladder cells lack components required for the internalization ofKlebsiella.

RNase 7 Contributes to the Cutaneous Defense against Enterococcus faecium

Background Human skin is able to mount a fast response against invading microorganisms by the release of antimicrobial proteins such as the ribonuclease RNase 7. Because RNase 7 exhibits high activity against Enterococcus faecium the aim of this study was to further explore the role of RNase 7 in the cutaneous innate defense system against E. faecium. Methodology/Principal Findings Absolute quantification using real-time PCR and ELISA revealed that primary keratinocytes expressed high levels of RNase 7. Immunohistochemistry showed RNase 7 expression in all epidermal layers of the skin with an intensification in the upper more differentiated layers. Furthermore, RNase 7 was secreted by keratinocytes in vitro and in vivo in a site-dependent way. RNase 7 was still active against E. faecium at low pH (5.5) or high NaCl (150 mM) concentration and the bactericidal activity of RNase 7 against E. faecium required no ribonuclease activity as shown by recombinant RNase 7 lacking enzymatic activity. To further explore the role of RNase 7 in cutaneous defense against E. faecium, we investigated whether RNase 7 contributes to the E. faecium killing activity of skin extracts derived from stratum corneum. Treatment of the skin extract with an RNase 7 specific antibody, which neutralizes the antimicrobial activity of RNase 7, diminished its E. faecium killing activity. Conclusions/Significance Our data indicate that RNase 7 contributes to the E. faecium-killing activity of skin extracts and suggest an important role for RNase 7 in the protection of human skin against E. faecium colonization.

Recommended Test Panel for Differentiation of Klebsiella Species on the Basis of a Trilateral Interlaboratory Evaluation of 18 Biochemical Tests

ABSTRACT Klebsiella pneumoniae and Klebsiella oxytoca are the two most frequently encountered Klebsiella species giving rise to infections in humans, but other Klebsiella species can also be found in clinical specimens: Klebsiella ozaenae, Klebsiella rhinoscleromatis, Klebsiella terrigena, Klebsiella planticola, Klebsiella ornithinolytica, and Enterobacter aerogenes (Klebsiella mobilis). However, many of these species are indistinguishable by the conventional methods employed routinely in the clinical microbiological laboratory. Several investigators have suggested various additional tests, but as yet there is no standardized test panel for identifying all Klebsiella species and subspecies. In the present study, performed in three national Klebsiella reference laboratories, we have evaluated a test panel consisting of 18 biochemical tests on 242 strains comprising all Klebsiella species and subspecies. The test panel was designed to identify organisms preliminarily identified as belonging to the genus Klebsiella on the basis of conventional methods or automated identification systems. With the described test panel it is possible to find one or more positive test results differentiating any Klebsiella species, except Klebsiella rhinoscleromatis, from its closest relative.

Burkholderia Is Highly Resistant to Human Beta-Defensin 3

ABSTRACT The bactericidal activity of the novel beta-defensin hBD-3 against 28 species and 55 strains of gram-positive cocci and gram-negative fermentative and nonfermentative rods was tested. All strains proved to be highly or intermediately susceptible to hBD-3 (minimal bactericidal concentration [MBC], ≤50 μg/ml), except for Burkholderia cepacia, for all 23 tested strains of which MBCs were >100 μg/ml.

Impaired Phagosomal Maturation in Neutrophils Leads to Periodontitis in Lysosomal-Associated Membrane Protein-2 Knockout Mice

Inflammatory periodontal diseases constitute one of the most common infections in humans, resulting in the destruction of the supporting structures of the dentition. Circulating neutrophils are an essential component of the human innate immune system. We observed that mice deficient for the major lysosomal-associated membrane protein-2 (LAMP-2) developed severe periodontitis early in life. This development was accompanied by a massive accumulation of bacterial plaque along the tooth surfaces, gingival inflammation, alveolar bone resorption, loss of connective tissue fiber attachment, apical migration of junctional epithelium, and pathological movement of the molars. The inflammatory lesions were dominated by polymorphonuclear leukocytes (PMNs) apparently being unable to efficiently clear bacterial pathogens. Systemic treatment of LAMP-2-deficient mice with antibiotics prevented the periodontal pathology. Isolated PMNs from LAMP-2-deficient mice showed an accumulation of autophagic vacuoles and a reduced bacterial killing capacity. Oxidative burst response was not altered in these cells. Latex bead and bacterial feeding experiments showed a reduced ability of the phagosomes to acquire an acidic pH and late endocytic markers, suggesting an impaired fusion of late endosomes-lysosomes with phagosomes. This study underlines the importance of LAMP-2 for the maturation of phagosomes in PMNs. It also underscores the requirement of lysosomal fusion events to provide sufficient antimicrobial activity in PMNs, which is needed to prevent periodontal disease.

Activity of the Novel Peptide Arminin against Multiresistant Human Pathogens Shows the Considerable Potential of Phylogenetically Ancient Organisms as Drug Sources

ABSTRACT The emergence of multidrug-resistant bacteria highlights the need for new antibacterial agents. Arminin 1a is a novel antimicrobial peptide discovered during investigations of the epithelial defense of the ancient metazoan Hydra. Following proteolytic processing, the 31-amino-acid-long positively charged C-terminal part of arminin 1a exhibits potent and broad-spectrum activity against bacteria, including multiresistant human pathogenic strains, such as methicillin-resistant Staphylococcus aureus (MRSA) strains (minimal bactericidal concentration, 0.4 μM to 0.8 μM). Ultrastructural observations indicate that bacteria are killed by disruption of the bacterial cell wall. Remarkably, the antibacterial activity of arminin 1a is not affected under the physiological salt conditions of human blood. In addition, arminin 1a is a selective antibacterial agent that does not affect human erythrocyte membranes. Arminin 1a shows no sequence homology to any known antimicrobial peptide. Because of its high level of activity against multiresistant bacterial strains pathogenic for humans, the peptide arminin 1a is a promising template for a new class of antibiotics. Our data suggest that ancient metazoan organisms such as Hydra hold promise for the detection of novel antimicrobial molecules and the treatment of infections caused by multiresistant bacteria.