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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

Streptococcus anginosus produces the novel antimicrobial peptide Angicin, which inhibits Gram positive microorganisms and is classified as a group IId bacteriocin. Production of Angicin is regulated by the quorum sensing system Sil (Streptococcus invasion locus), which is located adjacent to the bacteriocin gene cluster. Within this genetic region a typical CAAX protease is encoded, which was designated SilX. Nelfinavir, a HIV protease inhibitor, led to a concentration dependent reduction in antimicrobial activity, presumably through the inhibition of SilX. Concentrations exceeding 25 μM Nelfinavir caused a complete abolishment of bacteriocin activity against Listeria monocytogenes. These results are supported by the observation, that a SilX deletion mutant of S. anginosus strain BSU 1211 no longer inhibits the growth of L. monocytogenes. Antimicrobial activity could be restored by addition of synthetically synthesized mature SilCR, implying that SilX may be involved in the export and processing of the signal peptide SilCR. Some CAAX proteases have been reported to provide immunity against bacteriocins. However, in a radial diffusion assay the deletion mutant S. anginosus BSU 1211ΔSilX showed no sensitivity toward Angicin arguing against a role of SilX in the immunity of S. anginosus. The putative processing of the signal peptide SilCR indicates a novel function of the CAAX protease SilX, in the context of S. anginosus bacteriocin production.

Streptococcus agalactiae or group B streptococcus (GBS) is a commensal of the gastrointestinal and genitourinary tracts of healthy women and an important cause of neonatal invasive infections worldwide. Transmission of bacteria to the newborn occurs at birth and can be prevented by intrapartum antibiotic prophylaxis. However, this not available in resource limited settings in Africa, which carries a particular high burden of disease. Serotype based vaccines are in development and present a suitable alternative to prevent neonatal infections. To be able to assess vaccine efficacy, knowledge and surveillance of GBS epidemiological data are required. This review summarizes investigations about the serotype distribution and the multi-locus sequence types (MLST) found in different African countries. While most serotypes and MLST data are comparable to findings from other continents, some specific differences exist. Serotype V is predominant among colonizing maternal strains in many different African countries. Serotypes that are rarely detected in western industrialized nations, such as serotypes VI, VII and IX, are prevalent in studies from Ghana and Egypt. Moreover, some specific MLST sequence types that seem to be more or less unique to Africa have been detected. However, overall, the data confirm that a hexavalent vaccine can provide broad coverage for the African continent and that a protein vaccine could represent a promising alternative., publishedVersion

Protein hydrogels represent ideal materials for advanced cell culture applications, including 3D-cultivation of even fastidious cells. Key properties of fully functional and, at the same time, economically successful cell culture materials are excellent biocompatibility and advanced fabrication processes allowing their easy production even on a large scale based on affordable compounds. Chemical crosslinking of bovine serum albumin (BSA) with N-(3-dimethylaminopropyl)-N���-ethylcarbodiimide hydrochloride (EDC) in a water-in-oil emulsion with isoparaffinic oil as the continuous phase and sorbitan monooleate as surfactant generates micro-meter-scale spherical particles. They allow a significant simplification of an indispensable and laborious step in traditional cell culture workflows. This cell passaging (or splitting) to fresh culture vessels/flasks conventionally requires harsh trypsinization, which can be omitted by using the ���trans-ferry-beads��� presented here. When added to different pre-cultivated adherent cell lines, the beads are efficiently boarded by cells as passengers and can be easily transferred afterward for the embarkment of novel flasks. After this procedure, cells are perfectly viable and show normal growth behavior. Thus, the trans-ferry-beads not only may become extremely affordable as a final product but also may generally replace trypsinization in conventional cell culture, thereby opening new routes for the establishment of optimized and resource-efficient workflows in biological and medical cell culture laboratories., publishedVersion

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.