Molekulare Charakterisierung von antimikrobiellen und cytolytischen Polypeptiden von Acanthamoeba culbertsoni, einem freilebenden und potenziell humanpathogenen Protozoon

Culbicin ist ein neuartiges Peptid, welches aufgrund seiner porenbildenden Aktivität aus humanpathogenen Acanthamoeba culbertsoni isoliert wurde. Die Aufklärung der Nukleotidsequenz von Culbicin zeigte, dass das translatierte Culbicin in einem Precursor mit aminoterminalem Signalpeptid organisiert ist. Es wird postuliert, dass die Nukleotidsequenz zwischen dem 5'-Ende der Culbicinsequenz und der Signalpeptidcodierenden Sequenz ein weiteres Peptid codiert, welches KURZ genannt wurde. KURZ weist weder strukturelle Ähnlichkeit zu bekannten Proteinen noch zu Culbicin auf. Für beide Peptide wurde jeweils ein heterologes Expressionssystem etabliert, mit dem ausreichende Mengen der rekombinanten Peptide für die Antikörpergenerierung, Funktions- und Strukturanalysen synthetisiert wurden. Die prozentuale Verteilung der Sekundärstrukturelemente wurde untersucht und rCulbicin als ein vorwiegend α-helikales Peptid identifiziert. Die Tertiärstruktur von rCulbicin wird von fünf Disulfidbrücken stabilisiert, die dem Peptid eine enorme Stabilität unter Hitze und Säureeinwirkung verleiht. KURZ hingegen wurde als vorwiegend β-Faltblattstrukturiertes Peptid identifiziert, dessen Tertiärstruktur ebenfalls durch fünf Disulfidbrücken stabilisiert wird. Ferner wurde festgestellt, dass rCulbicin ein breites Wirkspektrum aufweist: es permeabilisiert die Membranen Gram-positiver und Gram-negativer Bakterien, bildet Poren in artifiziellen Liposomen, lysiert Human- und Schaf-Erythrocyten und wirkt cytotoxisch auf Candida albicans sowie zwei verschiedene humane Tumorzelllinien. Bei KURZ wurde ebenfalls antibakterielle, porenbildende und hämolytische Aktivität nachgewiesen. Mit den rekombinanten Peptiden Culbicin und KURZ wurden spezifische Antikörper generiert, die zur Immunolokalisation der natürlichen Peptide in Acanthamoeba culbertsoni-Trophozoiten verwendet wurden. Natürliches Culbicin wurde in cytosolischen Granula der Trophozoiten nachgewiesen, KURZ hingegen konnte nicht lokalisiert werden. Mit dem spezifischen Anti-Culbicin-Antikörper wurde im Immunoblot ein Protein von Acanthamoeba castellanii erkannt, bei dem es sich vermutlich um ein Culbicin-Ortholog handelt. Die Analyse einer online-cDNA-Bank von A. castellanii untermauert dieses Postulat, da eine translatierte cDNA-Sequenz 93 % Ähnlichkeit zur Culbicinsequenzaufweist. Dieses Culbicin-Ortholog, welches in dieser Arbeit den Arbeitstitel Castellanicin erhielt, ist gleichfalls in einem Precursor organisiert. Die Aminosäuresequenz des gesamten Castellanicin-Precursors ist der des Culbicin-Precursors zu 71 % ähnlich. Aufgrund dessen wird postuliert, dass mit Culbicin und KURZ sowie deren Homologen von Acanthamoeba castellanii in dieser Arbeit zwei gänzlich neue Proteinfamilien entdeckt wurden, die keine strukturellen Ähnlichkeiten zu bekannten Proteinfamilien aufweisen

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 an

RNase 7 in Cutaneous Defense

RNase 7 belongs to the RNase A superfamily and exhibits a broad spectrum of antimicrobial activity against various microorganisms. RNase 7 is expressed in human skin, and expression in keratinocytes can be induced by cytokines and microbes. These properties suggest that RNase 7 participates in innate cutaneous defense. In this review, we provide an overview about the role of RNase 7 in cutaneous defense with focus on the molecular mechanism of the antimicrobial activity of RNase 7, the regulation of RNase 7 expression, and the role of RNase 7 in skin diseases

The Inflammasome and the Epidermal Growth Factor Receptor (EGFR) Are Involved in the Staphylococcus aureus-Mediated Induction of IL-1alpha and IL-1beta in Human Keratinocytes.

Staphylococcus (S.) aureus is an important pathogen causing various infections including those of the skin. Keratinocytes are able to sense invading S. aureus and to initiate a fast defense reaction by the rapid release of innate defense mediators such as antimicrobial peptides and cytokines. There is increasing evidence that the cytokines IL-1alpha and IL-1beta, which both signal through the IL-1 receptor, play an important role in cutaneous defense against S. aureus. The aim of this study was to gain more insight into the underlying mechanisms leading to the S. aureus-induced IL-1alpha and IL-1beta expression in keratinocytes. Infection of human primary keratinocytes with S. aureus led to the induction of gene expression and protein secretion of IL-1alpha and IL-1beta. Full S. aureus-induced IL-1 protein release required the inflammasome components caspase-1 and ASC (apoptosis-associated speck-like protein containing a CARD) whereas gene induction of IL-1alpha and IL-beta by S. aureus was not dependent on caspase-1 and ASC. Since patients receiving anti-cancer therapy by inhibition of the epidermal growth factor receptor (EGFR) often suffer from skin infections caused by S. aureus we additionally evaluated whether the EGFR pathway may be involved in the IL-1alpha and IL-1beta induction by S. aureus. Inactivation of the EGFR with a blocking antibody decreased the S. aureus-mediated IL-1alpha and IL-1beta induction in primary keratinocytes. Moreover, the use of siRNA experiments revealed that ADAM17 (A Disintegrin and A Metalloprotease 17), a metalloproteinase known to mediate the shedding and release of EGFR ligands, was required for full induction of IL-1alpha and IL-1beta in keratinocytes infected with S. aureus. A failure of keratinocytes to adequately upregulate IL-1alpha and IL-1beta may promote S. aureus skin infections

IL-17A and IFN-γ synergistically induce RNase 7 expression via STAT3 in primary keratinocytes.

Human keratinocytes produce several antimicrobial peptides and proteins (AMP) which contribute to the protection of human skin against infection. RNase 7 is a major AMP involved in cutaneous defense with a high expression in keratinocytes and a broad spectrum of antimicrobial activity. The cytokine IL-17A has been recently identified as a potent inducer of several AMP in keratinocytes. Since the role of IL-17A to induce RNase 7 expression is unknown we analyzed IL-17A alone and in combination with other cytokines to induce RNase 7 expression in keratinocytes. Whereas IL-17A alone only weakly induced RNase 7 expression, the synergistic combination of IL-17A and IFN-γ (IL-17A/IFN-γ) was identified as a potent inducer of RNase 7 expression. This combination was more effective in inducing RNase 7 than the combination of IL-17A/TNF-α, a combination previously identified as a strong inducer of psoriasis-related immune response genes including several AMP. IFN-γ and IL-17A both have been reported to activate the transcription factor STAT3 (Signal transducer and activator of transcription 3). Therefore we investigated the influence of STAT3 on the IL-17A/IFN-γ -mediated RNase 7 induction. The use of a STAT3 inhibitor as well as siRNA-mediated downregulation of STAT3 resulted in a diminished IL-17A/IFN-γ -mediated RNase 7 induction in keratinocytes indicating that STAT3 is involved in this process. Similarly as seen with RNase 7, treatment of keratinocytes with IL-17A/IFN-γ revealed also a synergistic induction of gene expression of the AMP human beta-defensin (hBD)-2 and -3 as well as the S100 protein psoriasin (S100A7) indicating that the combination of IL-17A/IFN-γ is a potent inducer of various AMP classes in general. This was also reflected by an increase of the Staphylococcus aureus-killing activity of IL-17A/IFN-γ -treated keratinocytes

Infection of keratinocytes with Trichophytum rubrum induces epidermal growth factor-dependent RNase 7 and human beta-defensin-3 expression.

Human keratinocytes are able to express various antimicrobial peptides (AMP) to protect the skin from exaggerated microbial colonization and infection. Recently, in vitro growth-inhibiting activity of the skin-derived AMP psoriasin, RNase 7 and human beta-defensin (hBD)-2 against dermatophytes such as Trichophyton (T.) rubrum have been reported. To evaluate whether keratinocytes are able to respond to T. rubrum infection by an induced expression of AMP we exposed primary keratinocytes to living conidia of T. rubrum. This led to conidia germination and mycelial growth which was paralleled by a strong gene induction of the skin-derived AMP RNase 7 and hBD-3. Gene expression of the AMP psoriasin (S100A7) and hBD-2 were only slightly induced. The T. rubrum-mediated RNase 7 gene induction was accompanied by increased secretion of RNase 7. Parallel treatment of the keratinocytes with T. rubrum and the cytokine combination IL-17A/IFN-γ resulted in synergistic induction of RNase 7 and hBD-3 expression. Since patients receiving therapy by inhibition of the epidermal growth factor receptor (EGFR) more often suffer from dermatophytoses we investigated whether EGFR may be involved in the T. rubrum-mediated RNase 7 and hBD-3 induction. Primary keratinocytes incubated with an EGFR blocking antibody as well as with the EGFR antagonist AG1478 showed a significantly diminished RNase 7 and hBD-3 induction upon exposure of the keratinocytes to T. rubrum indicating that EGFR is involved in the T. rubrum-mediated induction of RNase 7 and hBD-3. The growth of T. rubrum in vitro was inhibited by hBD-3 in a dose-dependent manner suggesting that hBD-3 may contribute to cutaneous innate defense against T. rubrum. Taken together our data indicate that keratinocytes are able to initiate a fast defense response towards T. rubrum by the increased expression of AMP active against T. rubrum. A dysregulation of AMP may contribute to chronic and recurring dermatophytoses

Evaluation of the impact of technical and technological adjustments to the water treatment plant on the quality of treated water

The subject of this bachelor thesis is the evaluation of sequential technical and technological changes of water collection and modification at a specific water treatment facility – the water treatment facility in Ivančice. The first part of the thesis explains the basic terminology such as technological processes of the water treatment plant, types of water treatment facilities, the range of different types of water based on its quality and technologies that can be found at the water treatment plant in Ivančice. The next two chapters describe the water treatment process before and after intensification. The primary focus is on raw water collection, the quality of both raw and treated water, and the water treatment technology. The last part of the paper offers multiple comparisons of the original and renovated facility and recommendations for improving the operations of the renovated water treatment facility. The final evaluation was carried out based on the information collected mainly by visiting the facility in person, and other sources