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Zerebrale kavernöse Malformationen (CCM) sind maulbeerartige Gefäßfehlbildungen, die sich klinisch in Form von rezidivierenden, migräneartigen Kopfschmerzen, epileptischen Anfällen oder hämorrhagischen Schlaganfällen äußern können. CCMs treten sowohl sporadisch als auch in einer autosomal-dominant erblichen Form auf. Die Prävalenz der symptomatischen erblichen Kavernome liegt bei 1:5400 bis 1:6200. Somit zählen sie zu den seltenen Erkrankungen. Pathogene Varianten in den Genen CCM1, CCM2 und CCM3 sind mit der Entstehung der Malformationen assoziiert. Speziell Träger einer pathogenen CCM3-Variante zeigen meist ein frühes Manifestationsalter und einen schwerwiegenderen klinischen Krankheitsverlauf.
Durch in der Greifswalder Arbeitsgruppe durchgeführte Transkriptomanalysen von CCM3-/- CI-huVECs konnten die im Vergleich zu den CCM3+/+ Zellen neben FN1 (Fibronektin-1-Gen) am stärksten herunterregulierten Gene FBLN5 und POSTN identifiziert werden. Sie kodieren für Proteine der EZM. In der vorliegenden Arbeit wurden die Auswirkungen der matrizellulären Proteine FBLN5 und POSTN erstmals im Rahmen der CCM3-Pathogenese untersucht. Weder die akute Herunterregulation von FBLN5 noch die von POSTN hatte einen Einfluss auf die Wachstumsmorphologie oder die Organisation des Aktinzytoskeletts. In Bezug auf die Angiogenese führten die akuten Herunterregulationen von FBLN5 und POSTN zu einer verminderten Ausbildung gefäßähnlicher Strukturen. Im nächsten Schritt wurde eine mögliche Rettung des Phänotyps der CCM3-/- Endothelzellen durch die Hinzugabe der rekombinanten Proteine FBLN5 und POSTN untersucht. Die Addition von rFBLN5 veränderte die Morphologie der Endothelzellen maßgeblich und führte zu einer gezielten Reorganisation des Aktinzytoskeletts. Die Supplementierung von rPOSTN wirkte im Zeitverlauf stabilitätssteigernd auf die gefäßähnlichen Strukturen und hatte einen rettenden Einfluss auf die angiogenetischen Eigenschaften der CCM3-/- Endothelzellen. In Zusammenschau mit den von Schwefel et al. (2020) publizierten Daten über Fibronektin ist davon auszugehen, dass Bestandteile der EZM eine modulatorische Rolle in der Entstehung von CCMs einnehmen. Allerdings bleibt bisher ungeklärt welche Mechanismen hierbei von Bedeutung sind.
In vitro and in vivo analyses of mono- and mixed-species biofilms formed by microbial pathogens
(2022)
Microbial biofilms can be defined as multicellular clusters of microorganisms embedded in a self-produced extracellular matrix (ECM), which is primarily composed of polymeric biomolecules. Biofilms represent one of the most severe burdens in both industry and healthcare worldwide, causing billions of dollars of treatment costs annually because biofilms are inherently difficult to prevent, treat, and eradicate. In health care settings, patients suffering from cystic fibrosis, or patients with medical implants are highly susceptible to biofilm infections. Once a biofilm is formed, it is almost impossible to quantitatively eradicate it by mechanical, enzymatical, chemical, or antimicrobial treatment. Often the only remaining option to fully eradicate the biofilm is removing of the infected implant or body part. The primary reasons for the inherent resistance of biofilms against all forms of antimicrobial treatment are (I) a reduced metabolic activity of biofilm-embedded cells climaxing in the presence of metabolic inactive persister cells, as well as (II) the protective nature of the biofilm matrix acting as a (diffusion) barrier against antimicrobials and the host immune system. Consequently, there is an urgent need to better understand microbial biofilms from a structural and (patho-) physiological point of view in order to be able to develop new treatment strategies.
Therefore, the aims of this study were to investigate fundamental physiological properties of different clinically relevant single and multi-species biofilms, both in vitro and in vivo. Furthermore, the effectiveness of a novel treatment strategy using cold atmospheric pressure plasma was evaluated in vitro to treat biofilms of the pathogenic fungus C. albicans.
In article I, the intracellular and ECM protein inventory of Staphylococcus aureus during in vitro biofilm growth in a flow reactor was analyzed by liquid-chromatography coupled to tandem mass-spectrometry (LC-MS/MS) analysis combined with metabolic footprint analysis. This analysis showed that anaerobiosis within biofilms releases organic acids lowering the ECM pH. This, in turn, leads to protonation of alkaline proteins – mostly ribosomal proteins originating from cell lysis as well as actively secreted virulence factors – resulting in a positive net charge of these proteins. As a consequence, these proteins accumulate within the ECM and form an electrostatic network with negatively charged cell surfaces, eDNA, and metabolites contributing to the overall biofilm stability.
In article II, the in vivo metaproteome of the multi-species biofilm community in cystic fibrosis sputum was investigated. To this end, an innovative protocol was developed allowing the enrichment of microbial cells, the extraction of proteins from a small amount of cystic fibrosis sputum, and subsequent metaproteome analysis. This protocol also allows 16S sequencing, metabolic footprint analysis, and microscopy of the same sample to complement the metaproteome data. Applying this protocol, we were able to significantly enhance microbial protein coverage providing first insights into important physiological pathways during CF lung infection. A key finding was that the arginine deaminase pathway as well as microbial proteases play a so far underappreciated role in CF pathophysiology.
In articles III and IV, a novel treatment strategy for biofilms formed by the important fungal pathogen Candida albicans was evaluated in vitro. Biofilms were treated with two different sources of nonthermal plasma (with the Nonthermal Plasma Jet “kINPen09” as well as with the Microwave-induced plasma torch “MiniMIP”) and the effect on growth, survival, and viability was assessed by counting colony-forming units (CFU), by cell proliferation assays, as well as by live/dead staining combined with fluorescence microscopy, confocal laser scanning microscopy, (CLSM) and atomic force microscopy (AFM). These tests revealed that biofilms were effectively inactivated mostly on the bottom side of biofilms, indicating a great potential of these two plasma sources to fight biofilms.