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Easy and reliable identification of pathogenic species such as yeasts, emerging as problematic microbes originating from the genus Candida, is a task in the management and treatment of infections, especially in hospitals and other healthcare environments. Aptamers are seizing an already indispensable role in different sensing applications as binding entities with almost arbitrarily tunable specificities and optimizable affinities. Here, we describe a polyclonal SELEX library that not only can specifically recognize and fluorescently label Candida cells, but is also capable to differentiate C. albicans, C. auris and C. parapsilosis cells in flow-cytometry, fluorometric microtiter plate assays and fluorescence microscopy from human cells, exemplified here by human dermal fibroblasts. This offers the opportunity to develop diagnostic tools based on this library. Moreover, these specific and robust affinity molecules could also serve in the future as potent binding entities on biomaterials and as constituents of technical devices and will thus open avenues for the development of cost-effective and easily accessible next generations of electronic biosensors in clinical diagnostics and novel materials for the specific removal of pathogenic cells from human bio-samples., publishedVersion

Tuberculosis is a highly prevalent infectious disease with more than 1.5 million fatalities each year. Antibiotic treatment is available, but intolerable side effects and an increasing rate of drug-resistant strains of Mycobacterium tuberculosis (Mtb) may hamper successful outcomes. Antimicrobial peptides (AMPs) offer an alternative strategy for treatment of infectious diseases in which conventional antibiotic treatment fails. Human serum is a rich resource for endogenous AMPs. Therefore, we screened a library generated from hemofiltrate for activity against Mtb. Taking this unbiased approach, we identified Angiogenin as the single compound in an active fraction. The antimicrobial activity of endogenous Angiogenin against extracellular Mtb could be reproduced by synthetic Angiogenin. Using computational analysis, we identified the hypothetical active site and optimized the lytic activity by amino acid exchanges. The resulting peptide-Angie1-limited the growth of extra‐ and intracellular Mtb and the fast-growing pathogens Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Toward our long-term goal of evaluating Angie1 for therapeutic efficacy in vivo, we demonstrate that the peptide can be efficiently delivered into human macrophages via liposomes and is not toxic for zebrafish embryos. Taken together, we define Angiogenin as a novel endogenous AMP and derive the small, bioactive fragment Angie1, which is ready to be tested for therapeutic activity in animal models of tuberculosis and infections with fast-growing bacterial pathogens., publishedVersion

Antimicrobial peptides (AMPs) are biomolecules with antimicrobial activity against a broad group of pathogens. In the past few decades, AMPs have represented an important alternative for the treatment of infectious diseases. Their isolation from natural sources has been widely investigated. In this sense, mollusks are promising organisms for the identification of AMPs given that their immune system mainly relies on innate response. In this report, we characterized the peptide fraction of the Cuban freshwater snail Pomacea poeyana (Pilsbry, 1927) and identified 37 different peptides by nanoLC-ESI-MS-MS technology. From these peptide sequences, using bioinformatic prediction tools, we discovered two potential antimicrobial peptides named Pom-1 (KCAGSIAWAIGSGLFGGAKLIKIKKYIAELGGLQ) and Pom-2 (KEIERAGQRIRDAIISAAPAVETLAQAQKIIKGG). Database search revealed that Pom-1 is a fragment of Closticin 574 previously isolated from the bacteria Clostridium tyrobutyrium, and Pom-2 is a fragment of cecropin D-like peptide first isolated from Galleria mellonella hemolymph. These sequences were chemically synthesized and evaluated against different human pathogens. Interestingly, structural predictions of both peptides in the presence of micelles showed models that comprise two alpha helices joined by a short loop. The CD spectra analysis of Pom-1 and Pom-2 in water showed for both structures a high random coil content, a certain content of &alpha, helix and a low &beta, sheet content. Like other described AMPs displaying a disordered structure in water, the peptides may adopt a helical conformation in presence of bacterial membranes. In antimicrobial assays, Pom-1 demonstrated high activity against the Gram-negative bacteria Pseudomonas aeruginosa and moderate activity against Klebsiella pneumoniae and Listeria monocytogenes. Neither of the two peptides showed antifungal action. Pom-1 moderately inhibits Zika Virus infection but slightly enhances HIV-1 infectivion in vitro. The evaluation of cell toxicity on primary human macrophages did not show toxicity on THP-1 cells, although slight overall toxicity was observed in high concentrations of Pom-1. We assume that both peptides may play a key role in innate defense of P. poeyana and represent promising antimicrobial candidates for humans.

Streptococcus agalactiae or group B streptococcus (GBS) is a leading cause of serious neonatal infections. GBS is an opportunistic commensal constituting a part of the intestinal and vaginal physiologic flora and maternal colonization is the principal route of GBS transmission. GBS is a pathobiont that converts from the asymptomatic mucosal carriage state to a major bacterial pathogen causing severe invasive infections. At present, as many as 10 serotypes (Ia, Ib, and II–IX) are recognized. The aim of the current review is to shed new light on the latest epidemiological data and clonal distribution of GBS in addition to discussing the most important colonization determinants at a molecular level. The distribution and predominance of certain serotypes is susceptible to variations and can change over time. With the availability of multilocus sequence typing scheme (MLST) data, it became clear that GBS strains of certain clonal complexes possess a higher potential to cause invasive disease, while other harbor mainly colonizing strains. Colonization and persistence in different host niches is dependent on the adherence capacity of GBS to host cells and tissues. Bacterial biofilms represent well-known virulence factors with a vital role in persistence and chronic infections. In addition, GBS colonization, persistence, translocation, and invasion of host barriers are largely dependent on their adherence abilities to host cells and extracellular matrix proteins (ECM). Major adhesins mediating GBS interaction with host cells include the fibrinogen-binding proteins (Fbs), the laminin-binding protein (Lmb), the group B streptococcal C5a peptidase (ScpB), the streptococcal fibronectin binding protein A (SfbA), the GBS immunogenic bacterial adhesin (BibA), and the hypervirulent adhesin (HvgA). These adhesins facilitate persistent and intimate contacts between the bacterial cell and the host, while global virulence regulators play a major role in the transition to invasive infections. This review combines for first time epidemiological data with data on adherence and colonization for GBS. Investigating the epidemiology along with understanding the determinants of mucosal colonization and the development of invasive disease at a molecular level is therefore important for the development of strategies to prevent invasive GBS disease worldwide., publishedVersion

Group B streptococcus (GBS) is a leading cause of neonatal mortality and morbidity in the United States and Europe. It is part of the vaginal microbiota in up to 30% of pregnant women and can be passed on to the newborn through perinatal transmission. GBS has the ability to survive in multiple different host niches. The pathophysiology of this bacterium reveals an outstanding ability to withstand varying pH fluctuations of the surrounding environments inside the human host. GBS host pathogen interations include colonization of the acidic vaginal mucosa, invasion of the neutral human blood or amniotic fluid, breaching of the blood brain barrier as well as survival within the acidic phagolysosomal compartment of macrophages. However, investigations on GBS responses to acid stress are limited. Technologies, such as whole genome sequencing, genome-wide transcription and proteome mapping facilitate large scale identification of genes and proteins. Mechanisms enabling GBS to cope with acid stress have mainly been studied through these techniques and are summarized in the current review

The HCMV protein hIE1 is a multifunctional regulator that can, on one hand, antagonise the immune system in different ways and, on the other hand, promote lytic replication through interaction and stabilisation of the cellular protein hFEN1 or by transactivation of viral promoters. IE1 antagonises PML-NBs, a part of the intrinsic immune system, through interaction with PML followed by it´s deSUMOylation and disruption. Quantitative analysis, using NanoBRET assays, showed that the interaction of PML with IE1 is species-specific. Using further biological methods, it was shown that the deSUMOylation and disruption of PML is also species-specific. The species specificity, similarity of crystal structures and low sequence identity of hIE1 and rIE1 core were used to search for a possible interaction surface of hIE1 with PML. Stably expressed mutants were generated in which helices (H1, H1/2, H4, H5, H8 or H10/11) of hIE1 were exchanged with helices of rIE1. As none of these mutants were able to interact with PML or disperse it, we assume that the interaction surface consists of several protein helices. Furthermore, the functional integrity of the mutants was investigated by analysing additional hIE1 functions. A possible interaction site of hFEN1 was identified in helix 1 of hIE1, while the other helices have no influence on the interaction. Furthermore, since we found that interaction of hIE1 with hFEN1 is not sufficient for protein stabilisation, we assume that recruitment of an additional cellular factor is required for hFEN1 stabilisation. Transactivation of promoters was investigated by Gaussia luciferase assays and showed that only the mutant carrying helix 1 exhibited activity. In summary, these results indicate that the exchange of protein helices affects different functions of hIE1. However, no interaction surface of IE1 and PML could be identified. In addition, various functions of rIE1 were analysed in comparison to hIE1. It could be shown that while hFEN1 interaction and stabilisation is species-specific, the core domain of rIE1 is sufficient to activate viral promoters and antagonise the innate immune system through STAT2 interaction and relocalisation. Furthermore, since rIE1, unlike hIE1, cannot colocalise with mitotic chromatin, RCMV probably encodes a different protein that ensures genome maintenance. These results suggest that specific functions of IE1 have been conserved during evolution, which are probably crucial for maintaining CMV in different species.

Human cytomegalovirus (HCMV) has been known to be a widespread pathogen in the human population. In most cases an infection remains asymptomatic and without consequences for its host. However, immunocompromised patients are at risk of suffering from severe complications like retinitis, colitis or in case of antenatal infections hearing losses or microcephaly. As treatment options are currently limited and associated with high levels of toxicity, new viral targets are needed to treat this growing group of patients. While the crucial role of glycoprotein UL132 (gpUL132), a viral tegument protein, for replication of HCMV has been known for some time, its underlying mechanisms remain elusive. A pronounced morphological and replicative defect in the cytoplasmic viral assembly complex (cVAC) has been described in the absence of gpUL132. This study aimed at better understanding the role of gpUL132 in cVAC biogenesis and viral replication. The emphasis was placed on the interaction between gpUL132 and cellular microtubules. The phenotype was controlled using a circularity assay. The quantitative role of gpUL132 in cVAC biogenesis was investigated with nocodazole washout assays. To control for protein concentrations as possible qualitative differences, Western blots were performed. In this study, it was shown that the cVAC defect observed with gpUL132-deficient viruses does not depend on noncentrosomal nucleation. Furthermore, an SxIP-motif found in gpUL132 did not exhibit any influence on cVAC morphology or nucleation. The protein concentrations did not indicate differences depending on the presence of gpUL132. A hitherto unknown decrease of intracellular calmodulin-regulated spectrin-associated protein 2 levels during late stage infections with gpUL132 competent mutant viruses was observed. Its role remains unclear. More work is needed to reveal the mechanisms of viral modulation of the cellular cytoskeleton.

Severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) emerged in the end of 2019 in Wuhan, China and spread all over the world causing the coronavirus disease 2019 (COVID-19) pandemic. In my thesis, I established methods and protocols to investigate SARS-CoV-2 biology, infection and inhibition. Virus replication was quantified by SARS-CoV-2 genome or titer determination by RT-qPCR, plaque assay and tissue culture infectious dose 50 (TCID50) endpoint titration which also allowed studying cell-to-cell spread and antiviral mechanisms. In order to increase throughput but also specificity and sensitivity of screening assays for the identification and characterization of antiviral compounds, an MTS-based cell death, an enzyme-based immunodetection, and a flow cytometry-based immunodetection assay were developed. All can be performed in 96 well microtiter format and allow quantification of antiviral potency of drugs (50% inhibitory concentrations) after one to three days with good high-throughput performance. However, assay choice depends on cell types, work effort, time expenditure, specificity, sensitivity, equipment and costs. Application of these assays contributed to the identification and characterization of several antivirals, including heparin and carrageenan-containing nasal and oral sprays. The antiviral activity of the sprays was confirmed in physiologically relevant human airway epithelial cells that resemble the entry site for SARS-CoV-2 and was attributed to ι- and κ-carrageenans. Heparin and carrageenan are both sulfated polysaccharides already in clinical use, which may allow fast therapeutic usage. They block viral attachment to cellular receptors preventing virus entry. Heparin has additional anti-inflammatory and anticoagulative properties, making it a unique therapy option for COVID-19, as it covers both the pathogen and the ensuing disease. Oral and nasal administration of the sprays may prevent infection or transmission between individuals. Thus, repurposing heparin and carrageenan-containing nasal and oral sprays may be advisable to treat COVID-19. Studying the virucidal activity of common rapid antigen assay buffers revealed that most commercially available extraction buffers do not inactivate SARS-CoV-2. This may pose a risk of infection during performance of point-of-care tests. Therefore, in cooperation with an industrial partner, we developed a virus-inactivating antigen extraction buffer that allows downstream detection of antigen as well as viral RNA. Thus, ready-to-use extraction buffers with virucidal activity and unchanged analytical performance should replace non-virucidal buffers to allow save handling of the point-of-care tests. Since early in the pandemic, SARS-CoV-2 transmission routes were unclear, human milk was studied to evaluate the potential for spread via breastfeeding. Indeed, we found SARS-CoV-2 RNA in milk for the first time, but infectious virus could not be detected, suggesting a low risk of vertical transmission via breast milk. To assure safety of potentially SARS-CoV-2 containing milk, Holder pasteurization is an effective sterilization measure, as it completely inactivated SARS-CoV-2. It can be used by milk banks or mothers, who might consider discontinuing breastfeeding, but is not suitable as a routine measure. Furthermore, human breast milk has some intrinsic virucidal activity that is generated during milk storage varying with time, temperature and between donors. Studying this antiviral mechanism in detail for Zika virus (ZIKV) revealed that the effect is mediated by the lipid fraction and that lipases are the responsible factor. This suggests that this milk inactivation property on ZIKV and other enveloped viruses such as SARS-CoV-2 is due to membrane-disrupting fatty acids which are generated by lipolysis of triglycerides released from milk fat globules. Probably, this explains why ZIKV transmission via breastfeeding is hardly observed despite the presence of infectious virus in breast milk and why infectious SARS-CoV-2 has never been isolated from human milk despite the presence of viral RNA. Altogether, breast milk seems safe and breastfeeding should be continued in case of ZIKV as well as SARS-CoV-2 infection as it provides many benefits for the newborns. Several non-pulmonary manifestations of COVID-19 have been described, such as new-onset diabetes. Our analysis of its molecular basis showed for the first time that SARS-CoV-2 can directly infect and replicate in ex vivo cultured pancreatic islets, specifically in β-cells. The infection was associated with hormone loss and morphological and functional changes in those cells, supporting a mechanism in which direct infection impairs β-cells leading to decreased insulin production and autoantibody-negative type 1 diabetes. This is in line with histological examinations of patient tissue as well as further laboratory studies, confirming that SARS-CoV-2 infection impairs pancreatic cell survival and/or function. However, the mechanism of β-cells impairment and whether these changes underlie the development of diabetes needs to be further studied. In summary, this work advanced our understanding of the safety of SARS-CoV-2 rapid antigen diagnostics, risk of transmission by breastfeeding, pancreatic tropism and treatment options, thus contributing to the containment of the pandemic.

The respiratory tract is a major entry site for pathogens into the human body. To combat bacterial infections the immune system has a variety of mechanisms of host defence at his disposal. Part of the first line of defence are the so-called antimicrobial peptides (AMPs). To search for novel AMPs from the human respiratory tract, peptide libraries established from bronchoalveolar lavage fluid as well as lung tissue were screened for antimicrobial activity against Gram-positive and Gram-negative bacterial pathogens. After several testing-cycles and subsequent purification of active fractions we identified β-2 microglobulin (B2M) from the BAL library. B2M is a subunit of the major histocompatibility complex class I receptor complex that is present at the surface of every nucleated cell. It is known to express antibacterial activity against Listeria monocytogenes as well as Escherichia coli and to be a precursor of an antimicrobial agent acting against Staphylococcus aureus. Using commercially available B2M we could confirm a dose-dependent inhibition of Pseudomonas aeruginosa as well as Listeria monocytogenes. During investigation of the variables determining the biological effect of B2M a pH-dependent activity of B2M was observed. The strongest biological effect against the two tested bacterial strains was present under acidic conditions and decreased with environmental pH reaching neutral values. Congo Red assays indicate a pH-dependent fibril formation of B2M at acidic conditions suggesting that fibrillogenesis could mediate the observed pH-dependent antibacterial activity. Based on the results of SYTOX Green assays as well as transmission electron microscopy imaging, the inhibition of L. monocytogenes by B2M may be caused by membrane permeabilization. In conclusion B2M as part of a ubiquitous cell surface complex may be a potent inhibitor of bacterial invasion, especially in acidic environments as present in inflamed tissues.

Die durch eine Infektion mit dem Humanen Cytomegalievirus gesteigerte Morbidit��t und Mortalit��t, insbesondere in den klinischen Bereichen P��diatrie und H��matoonkologie, zu verringern, stellt die virologische Forschung noch immer vor eine gro��e Herausforderung. Die Entwicklung antiviraler Therapeutika ist von gro��er Wichtigkeit f��r die Reduzierung HCMV-assoziierter Erkrankungsf��lle und daraus resultierenden Folgesch��den. In der vorliegenden Arbeit wurden potentielle antivirale Zielstrukturen im Hintergrund des klinischen Isolats Merlin auf Ihre Bedeutung f��r das Wachstum dieses Virusstammes hin untersucht. Es wurden hierf��r durch markerlose BAC-Mutagenese gezielt genetische Bereiche modifiziert, die f��r die bereits zuvor im Labor untersuchten Proteine pUL71, pUL103 und gpUL132 kodieren. Die Viren mit Ver��nderungen in diesen Genbereichen wurden dann in Wachstumsanalysen untersucht. Hierbei konnte f��r den pUL71/pUL103-Proteinkomplex gezeigt werden, dass ein Fehlen dieses viralen Genproduktes im klinischen Isolat zu deutlichen Wachstumsdefekten f��hrt. F��r das ebenfalls untersuchte virale Glykoprotein gpUL132 war hingegen in einer gpUL132-defizienten Merlin Virus-Mutante kein eindeutiger ��ber die gesamte Dauer der Analyse bestehender Wachstumsdefekt festzustellen. Zu finden waren im Hintergrund dieses Virus jedoch ein im Durchschnitt weniger rundes vAC, was vermuten l��sst, dass dieses Glykoprotein f��r die korrekte vAC-Biogenese, Bedeutung besitzt. Die genaue Funktion von gpUL132 bleibt jedoch weiterhin unklar und sollte deshalb Gegenstand weiterer Untersuchungen sein.

Streptococcus gallolyticus subspecies gallolyticus (SGG) is an opportunistic pathogen causing invasive infections in the elderly often associated with colon neoplasia. The prevalence of SGG and two other Streptococcus gallolyticus subspecies in the stools of patients with normal colonoscopy (control) was compared with patients with colorectal adenomas (CRA) or with carcinomas (CRC) from stages I to IV.

Die amyotrophe Lateralsklerose ist eine, meist innerhalb weniger Jahre letal endende, neurodegenerative Motoneuronenerkrankung. Eine genaue Ursache ist bislang nicht bekannt. Die Erkrankung wird begleitet von lokalen und peripheren, vermutlich reaktiven, Entzündungsreaktionen und geht mit einem fehlregulierten Energiemetabolismus und Gewichtsverlust einher. Auf neuropathologischer Ebene konnten vermehrt zytoplasmatische Einschlüsse von fehlgefalteten TDP-43-Proteinen beschrieben werden. Ebenso zeigten sich systemische Veränderungen des Immunsystems. Studien haben gezeigt, dass all diese Prozesse durch das intestinale Mikrobiom beeinflusst werden können. Insgesamt wurden 25 ALS-Patienten mit 32 in Alter und Geschlecht gematchten gesunden Personen verglichen und deren fäkales Mikrobiom untersucht. Die Analyse des fäkalen Mikrobioms erfolgte durch 16S-rRNA-Gensequenzierung mittels Next-generation sequencing. Durch eine strikte Auswahl und genaue Ein- und Ausschlusskriterien der Studienpopulation wurden Einflussfaktoren und etwaige Störfaktoren minimiert. Vergleicht man die beiden sorgfältig aufeinander abgestimmten Gruppen, ergaben sich in ihrer Diversität und Häufigkeit von bakteriellen Taxa auf den verschiedenen taxonomischen Ebenen sowie bezüglich ihrer vorhergesagten Metagenome keine signifikanten Unterschiede. Signifikante Unterschiede zwischen ALS-Patienten und gesunden Kontrollpersonen wurden nur in Bezug auf die Gesamtzahl der ermittelten operational taxonomic units (OTUs) und in der Häufigkeit unkultivierter Ruminococcaceae beobachtet. Folglich zeigen ALS-Patienten keine wesentliche Veränderung in der Zusammensetzung des intestinalen Mikrobioms im Vergleich zu Gesunden.

Genomische Rearrangements des Mixed Lineage Leukemia (MLL)-Gens sind assoziiert mit dem Auftreten von therapieassoziierten akuten Leukämien. Hierbei besteht vor allem ein Zusammenhang zur Therapie mit Topoisomerase II-Inhibitoren wie Doxorubicin oder Etoposid. Von derartigen sekundären Leukämien betroffene Patienten weisen eine schlechtere Prognose auf. Um solche Rearrangements zu verhindern, wäre es denkbar Chemotherapien peptidpharmazeutische Zusätze zuzugeben. Im Rahmen eines Projekts der Universität Ulm wurde eine Hämofiltrat-basierte humane Peptidbibliothek erzeugt, aus welcher innerhalb unserer Arbeitsgruppe ein 27 Aminosäuren umfassendes Peptid identifiziert werden konnte. Ziel der vorliegenden Arbeit war es, den Effekt dieses Peptids auf die Rekombinationsfrequenz sowie die Brüchigkeit der MLL-Bruchpunkt-Cluster-Region (MLLbcr) zu untersuchen bei Behandlung mit Doxorubicin oder Etoposid. Außerdem sollte der mögliche Wirkmechanismus des Peptids charakterisiert werden. Dazu wurden verschiedene MLL-gerichtete Schadensantworten wie die Basenexzisionsreparatur (BER), der Nuclear factor κB (NF-κB)-Signalweg und die Endonuklease G untersucht.

Obwohl das Phosphoprotein pp65 ein Hauptbestandteil von humanen Cytomegaloviren (HCMV) und der codierende Leserahmen UL83 zwischen verschiedenen Virusstämmen hochkonserviert ist, ist das Protein für das Wachstum in Fibroblasten verzichtbar. Um pp65 zu untersuchen, wurden bisher Virusstämme verwendet, die teilweise genetische Veränderungen tragen, welche dazu führen, dass sich die Viren im Gegensatz zu klinischen Isolaten unter anderem zellfrei ausbreiten. Möglicherweise verdeckte dies eine Funktion von pp65, da angenommen wird, dass sich frische klinische Isolate zellassoziiert ausbreiten. In dieser Arbeit soll die Hypothese geprüft werden, dass pp65 dazu beiträgt, wie effizient sich HCMV ausbreitet. Es wurde der reparierte Virusstamm Merlin-RL13tetOUL128LtetO als Modell für klinische Isolate verwendet, da gezielt zwei Genregionen reguliert werden können, die für die zellassoziierte Virusausbreitung verantwortlich sind. Eine UL83-Stopmutante wurde durch „en passant“-Mutagenese hergestellt, sodass das Protein pp65 nicht exprimiert wurde. Wildtyp und UL83-Stopmutante wurden in den Versuchen jeweils miteinander verglichen, während die UL83-Revertante als Kontrolle diente. Um festzustellen, wie stark sich Wildtyp und UL83-Stopmutante zellassoziiert ausbreiten, wurde die Herdgröße in Fibroblasten und in Endothelzellen einige Tage nach der Infektion betrachtet. Die UL83-Stopmutante bildete um ein Fünftel kleinere Herde in Fibroblasten und um mehr als ein Drittel kleinere Herde in Endothelzellen. Um einen möglichen Beitrag von pp65 zur zellfreien Virusausbreitung näher zu beleuchten, wurde aus infizierten speziellen Fibroblastenkulturen (HFFFtet-Zellen) zu verschiedenen Zeitpunkten der Virustiter von Wildtyp und UL83-Stopmutante untersucht. Die UL83-Stopmutante setzte aus HFFFtet-Zellen nur halb so viel Virus frei wie der Wildtyp. Da sich die UL83-Revertante nicht wie die UL83-Stopmutante verhielt, sondern wie der Wildtyp, sind die UL83-Stopmutationen für den Wachstumsdefekt der Mutante verantwortlich zu machen. Dass pp65 zur Virusausbreitung in Fibroblasten und in Endothelzellen beiträgt, wurde hier erstmals als statistisch signifikant nachgewiesen. Zusammenfassend lässt sich sagen, dass das virale Protein pp65 die Ausbreitung eines Isolat-ähnlichen HCMV-Stamms unabhängig vom Ausbreitungsmodus und vom Zelltyp fördert und demzufolge bedeutender für das Viruswachstum ist, als bisher angenommen.

Streptococcus anginosus is considered a human commensal of mucosal membranes. However, increasing number of reports showed their isolation from invasive infections highlighting the clinical relevance of this species. We aimed to establish a fast and reliable genetic manipulation technique to be able to investigate S. anginosus on the genetic level which would help to increase our scarce knowledge about the pathogenicity mechanisms in this species. Therefore, we combined the competence system of S. anginosus and the Cre-recombinase system which allows the generation of markerless gene deletions. With the help of the established genetic manipulation technique we were able to investigate the role of the Catabolite control protein A (CcpA) in the regulation of the Streptolysin S (SLS) expression. We were able to demonstrate that different sugars had a different influence on the SLS promoter activity in an EGFP based reporter system, indicating a Carbon Catabolite Repression mechanism mediated by CcpA and we further confirmed this by the generation of a ccpA deletion mutant. The findings could be reproduced using a functional hemolytic assay and we verified the effect of CcpA on the SLS expression by constructing a ccpA complementation strain. Analysis of the SLS promoter finally led to the identification of a CcpA binding site that was verified by the promoter reporter assay using mutated promoter sequences as well as electrophoretic mobility shift assays. Thus, we were able to demonstrate for the first time the regulation of a potential virulence factor in the emerging pathogen S. anginosus and we established a Cre-lox based genetic engineering technique that will facilitate the investigation of pathogenicity mechanisms in this species.

Streptococcus agalactiae (Gruppe B Streptokokken, GBS) gilt als der häufigste Erreger von Neugeboreneninfektionen in industrialisierten Ländern und wird auch bei Erwachsenen immer häufiger als Erreger invasiver Infektionen isoliert. Als Kommensale kommen Gruppe B Streptokokken im Urogenitaltrakt bei etwa jedem dritten Erwachsenen vor; um eine schwerwiegende Infektion zu verursachen, muss S. agalactiae demnach der Immunantwort des Wirts entgehen. Die so genannten Neutrophil Extracellular Traps (NETs), ein Abwehrmechanismus des Immunsystems, können durch extrazelluläre DNasen von Streptokokken abgebaut werden. Bislang wurden bei S. agalactiae drei extrazelluläre Nukleasen beschrieben, zu deren genetischer Grundlage es bislang nur wenige Arbeiten in der Literatur gibt. Mithilfe einer Insertionsmutantenbank wurde nach GBS-Mutanten gescreent, die eine verminderte DNase-Aktivität auf Methylgrünagar zeigten. Bei 22 Isolaten mit phänotypisch eingeschränkter DNase-Aktivität wurden die Insertionsstellen untersucht. Dies führte zur Identifizierung von drei mutmaßlich DNase-kodierenden Genen, die bezüglich des Molekulargewichtes zu den 18,6 kDa, 33 kDa und 26,5 kDa großen Nukleasen I, II und III von S. agalactiae passen. Um den enzymatischen DNA-Abbau durch die mutmaßlichen Nukleasen nachzuweisen, wurden die Gene in den Vektor pET21a kloniert und als rekombinante His-Tag-Proteine in Escherichia coli exprimiert. Die nativen Proteine zeigten in vitro einen zeit- und konzentrationsabhängigen Abbau von DNA, wobei das rekombinant exprimierte SAK_0814 den stärksten DNA-Abbau zeigte. In Genexpressionsanalysen wurde die Transkription von der Nuklease III durch Zugabe von DNA unterdrückt. Die rekombinanten Proteine wurden mittels SDS-Polyacrylamidgelelektrophorese und Western Blot auf ihre tatsächliche Größe untersucht. Dadurch ergeben sich folgende Zuordnungen zwischen Gen und der jeweiligen bekannten Nuklease: SAK_0271 (SAK_RS01335) – Nuklease I SAK_0220 (SAK_RS01090) – Nuklease II SAK_0814 (SAK_RS04075) – Nuklease III Mithilfe des oben gewählten Ansatzes konnte ein Zusammenhang zwischen SAK_0814 und Nuklease III-Produktion bestätigt werden, der bereits von anderen Arbeitsgruppen postuliert worden war. Zudem gelang es mit SAK_0271 und SAK_0220 zwei weitere Kandidatengene zu identifizieren, die direkt mit der DNase-Aktivität von Gruppe B Streptokokken zusammenhängen. Da Nukleasen wichtige Pathogenitätsfaktoren bei der Entstehung und Etablierung einer GBS-Infektion darstellen, liefert die Kenntnis der genetischen Basis und der pathophysiologischen Zusammenhänge einen wichtigen Beitrag, um zukünftig gezielt Maßnahmen gegen GBS-Infektionen bei Erwachsenen und Neugeborenen zu entwickeln.

Low transduction efficiencies are a common problem in retro- and lentiviral gene transfer. Here, I have presented a new approach to boost retroviral gene transfer using self-assembled nanofibrils formed by the 12-amino acid peptide termed EF-C (Enhancement Fragment C). This peptide was derived from the HIV-1 external envelope glycoprotein gp120. The fibrils form instantaneously upon dilution of peptide in aqueous media and adopt a typical amyloid structure that was observed due to Thioflavin T and Congo Red staining and atomic force microscopy. Measurement of ζ-potential charge of aqueous solution of EF-C revealed that the nanofibrils have positively charged surface, supposed that their enhance infection by neutralizing the repulsion between the negatively charged viral and cellular membranes. My data obtained using confocal fluorescence microscopy shown that fibrils bind virions and increase their adsorption to cells and subsequent infection. Addition of EF-C to virus stocks or target cells boosted gene delivery into multiple cell types, including primary T cells, macrophages and CD34+ stem cells, and was substantially more effective than other reagents commonly used to increase transduction. Moreover, like RetroNection, the most commonly used reagent to increase retroviral transduction, EF-C potently boosts transduction also when immobilized on cell culture dishes. This effect was confirmed with retro- and lentiviral particles carrying various viral envelope proteins frequently used for gene transfer and therapy. The enhancing effect was independent of the viral glycoprotein but still required the respective cellular receptor(s). Furthermore, the fibrils allow a fast and convenient concentration of virions without the need of ultracentrifugation. The result presented in this thesis demonstrate that EF-C nanofibrils provide a convenient, flexible and effective means to increase retro- and lentiviral gene transfer in basic research and clinical applications.