Thioredoxin Peroxidase des Humanpathogens Candida albicans : ein differenziell lokalisiertes und multifunktionelles Enzym

Abstract

Die Zellwand und im Besonderen die Zellwand-Proteine nehmen bei der Etablierung von Infektionen der humanpathogenen Hefe Candida albicans eine entscheidende Stellung ein, da sie z. B. für die Adhäsion und Penetration des Wirtsgewebes essenziell sind. Folglich stellen Zellwand-Proteine geeignete Zielstrukturen für die Entwick-lung neuer Antimykotika dar. Die vorliegende Arbeit beschäftigt sich mit der Dynamik zellwand-assoziierter Proteine in C. albicans. Zellwand-Proteine sind entweder kovalent oder nicht-kovalent an die unlöslichen Zuckerpolymere (beta-1,3- und beta-1,6-Glukane, sowie Chitin) der Zellwandstruktur gebunden. Nicht-kovalent gebundene Zellwand-Proteine werden daher als lösliche Zellwand-Proteine bezeichnet. Im Rahmen meiner Diplomarbeit konnten durch Markierung mittels eines membranimpermeablen Biotinderivats und anschließender Aufreinigung mittels Affinitäts-Chromatographie insgesamt 29 unterschiedliche lösliche Zellwand-Proteine in C. albicans identifiziert werden. Neben der Analyse der löslichen Zellwand-Proteine aus Blastosporen wurde auch der Vergleich zu filamentösen Hyphen gezogen, da die Filamentierung für die Invasivität des Pathogens essenziell ist. Unter den identifizierten Proteinen von C. albicans befand sich ein Homolog der Thioredoxin Peroxidase 1 (Tsa1p) aus S. cerevisiae, das nur aus der Zellwand von Hyphen isoliert wurde. In Stämmen, die Gfp-markiertes Tsa1p exprimierten, wurde Tsa1p ebenfalls ausschließlich in der Hyphenzellwand lokalisiert. Darüber hinaus wurde Tsa1p sowohl in Hyphen, als auch in Blastosporen im Zellkern und im Zytoplasma detektiert. Daher handelt es sich bei Tsa1p eher um ein differenziell lokalisiertes als um ein differenziell exprimiertes Protein. Hyphenbildung diente dabei als spezifisches Signal für die Translokation von Tsa1p an die Zellwand. Anhand von Deletionsstudien wurde gezeigt, dass Tsa1p Funktionen als Peroxidase übernimmt, die Stabilität des Candida-Genoms erhöht und für die transkriptionelle Induktion hyphen-spezifischer Zellwand-Gene notwendig ist. Um den Einfluss von Tsa1p auf Zellwand-Gene im Detail zu untersuchen, wurden in Co-immunpräzipitationen mittels Anti-Tsa1p-Antikörper mit Tsa1p interagierende Zellwand-Proteine analysiert. Die Ergebnisse dieser Arbeit deuten darauf hin, dass in C. albicans die unterschiedlichen Funktionen von Tsa1p durch differenzielle Lokalisa-tion ermöglicht werden.

During the past years there has been an increase of fungal infections in humans predominantly caused by Candida albicans. This opportunistic pathogen coexists in the majority of the population as a commensal. But particularly in immunocompromised patients, the sensitive relationship of the host and the fungus is out of balance. This may result in life-threatening, systemic infections. Major virulence traits of C. albicans are the ability to switch from growth of singular budding cells (blastospores) to a filamentous growth form (hyphae), called the dimorphic transition, as well as adhesion to host tissues mediated by cell wall components. The composition of fungal cell walls is highly dynamic. The cell wall has been implicated in several physiological processes, including the maintenance of cellular morphology and osmotic protection of the cell. In addition to these essential functions, the cell wall of fungal pathogens is crucial for virulence. Antigenic determinants which are involved in adhesion and therefore colonization and modulation of the immunological response by the host contribute to pathogenesis. Moreover, the absence of the cell wall in mammalian cells makes it an attractive target for the development of novel antifungal substances. In general, the cell wall is composed of complex polymers of glucose (beta-1,3- and beta-1,6-glucan), chitin and mannoproteins. These components interact to give rise to the overall architecture. Proteins either are covalently linked to the glucan network or are non-covalently incorporated into the cell wall. Covalently linked cell wall proteins can be released from walls by treatment with specific hydrolases. Non-covalently linked or SDS-extractable cell sur-face proteins can be released by using detergents. Only little is known about the structure of non-covalent attachments of proteins to the cell wall.

In the course of my diploma thesis an approach using Sulfo-NHS-LC-Biotin has been established to specifically label these cell surface proteins of intact cells. Non-covalent cell wall proteins were extracted by detergent and purified by affinity chromatography using immobilized neutravidin. In total 29 different cell surface proteins from C. albicans under two distinct conditions have been identified. Among these proteins was a homolog of Thiol-specific antioxidant like protein 1 (Tsa1p) which has been studied in more detail during this work. Tsa1p localized to the cell wall depending on the growth conditions as it was exclusively isolated from hyphally grown cells. A Tsa1p-GFP fusion revealed that depending on the conditions applied the protein in blastospores is localized to the nucleus and the cytoplasm and can be found at the cell surface only in hyphal cells. Thus, Tsa1p is differentially localized rather than being differentially expressed depending on the growth conditions. Although Tsa1p has no aparent secretion signal sequence and is abundant, it could be demonstrated in binding experiments that Tsa1p did not stick unspecifically to the cell surface of growing cells as it might be artificially released from the cytoplasm for example by lysis of cells. Deletion of four copies of Tsa1p in the C. albicans genome indicated a function of Tsa1p in maintenance of genetic stability and resistance to oxidative stress, which both could be shown phenotypically. The latter was confirmed by the findings that oxidative substances like hydrogen peroxid induce TSA1 as has been shown in northern and western analysis. Moreover, transcriptional analysis revealed that genes implicated in oxidative stress response are upregulated in the TSA1 deletion strain even under mild oxidative conditions. The decreased genetic stability was in good agreement with the nuclear localization of Tsa1p that was found under all conditions applied. Furthermore, genes encoding for hyphal specific covalently linked cell wall proteins were found to be significantly repressed in a TSA1 mutant strain exclusively under hyphae inducing conditions, indicating that Tsa1p might have other functions in the hyphal cell wall as it has in the cytoplasm and in the nucleus. Additionally, the translocation of Tsa1p to the cell wall was not mediated by hydrogen peroxid but in contrast by formation of filaments under two distinct hyphae inducing conditions. To address how Tsa1p is involved in cell wall biosynthesis potential interaction partners of Tsa1p were analysed using co-immune precipitation. The presented data indicate that different functions of Tsa1p in C. albicans might be regulated by differential localization.