Studie zur Identifizierung pH-abhängig regulierter Zellwandkomponenten des humanpathogenen Pilzes Candida albicans

Abstract

Mikroorganismen, die ein breites Spektrum von Habitaten besiedeln, benötigen effektive Mechanismen zur Anpassung an den extrazellulären pH-Wert. Der humanpathogene Pilz Candida albicans besiedelt im menschlichen Körper den Darm und die Vaginalhöhle, in denen jeweils verschiedene pH-Bedingungen herrschen. Die zentrale Rolle der pH-Adaption für das Überleben des Pilzes verleiht der Aufklärung ihrer molekularen Grundlagen besondere Bedeutung im Hinblick auf die Entwicklung neuer Antimykotika. Die vorliegende Arbeit beschäftigt sich mit der pH-abhängigen Genregulation in C. albicans. Der Transkriptionsfaktor Rim101p reguliert die Gene PHR1 und PHR2, welche essentielle Zellwandproteine für das Wachstum von C. albicans in neutraler bzw. saurer Umgebung kodieren. Zur Identifizierung weiterer, RIM101-regulierter Gene in C. albicans wurde für Transkriptionsanalysen ein zellwandspezifischer DNA-Mikroarray eingesetzt. Diese führten zur Identifizierung zum Teil bereits bekannter RIM101-aktivierter Gene (HWP1, RBT1, ALS1). Es wurden aber auch mehrere bisher nicht charakterisierte Gene gefunden, die durch RIM101 reprimiert werden (z.B. RBR1, RBR2, RBR3, für „repressed by Rim101p“). Deletionsstudien für Rbr1p zeigten eine wichtige Funktion dieses Zellwandproteins für die Filamentierung bei saurem pH. Vor allem wies diese Mutante im systemischen Mausmodell eine signifikant verringerte Pathogenität gegenüber dem Wildtyp auf. Da Rbr1p großen Anteil an der RIM101-abhängigen pH-Adaption hat und mit der Virulenz des Pilzes assoziiert ist, stellt es als GPI-Protein eine potentielle Zielstruktur für neue Antimykotika dar. Die RIM101-aktivierten, hyphalen Zellwandproteine Als1p, Hwp1p und Rbt1p werden durch den Transkriptionsfaktor Nrg1p reprimiert. Im Rahmen der vorliegenden Studie konnte gezeigt werden, dass die Expression von Rim101p in C. albicans mit der Herabregulierung des hyphalen Repressors Nrg1p korreliert. Dieses Ergebnis charakterisiert Rim101p als zentralen Mediator an der Schnittstelle zwischen pH-Antwort und der komplexen Regulation der Filamentierung in C. albicans und verdeutlicht die Bedeutung von Rim101p für die Virulenz des Pilzes.

The ascomycetous yeast Candida albicans is the major opportunistic human fungal pathogen. This eukaryotic microorganism is able to inhabit various habitats within the host including acidic environments like the skin or vaginal mucosae and neutral to alkaline environments as encountered in the gut. The ability to sense and adapt to extreme ambient pH-values from 2 to 10 (Odds, 1988) is essential for survival, growth and virulence of the pathogen C. albicans. External pH strongly effects particularly the cell wall and the cell membrane as the outermost barriers of the cell. In the plasma membrane, these effects include changes in protein activity, maintenance of the proton gradient and the availability of nutrients, while the molecular composition of the cell wall is rapidly remodeled due to a shift in external pH. So far, little is known about the molecular mechanisms of this adaptation process of the cell wall. A conserved signal transduction pathway of fungi to respond to external pH is represented by the activity of the zinc finger transcription factor Rim101p/PacC (Su & Mitchell, 1993; Tilburn et al., 1995). At acidic pH, Rim101p/PacC is predominantly found in the full-length form, while at neutral and alkaline pH, the protein is processed by a proteolytic cascade. C. albicans Rim101p has been shown to play a major role in pH-dependent gene regulation. The transcription factor is involved in cell wall biosynthesis as it regulates PHR1 and PHR2 (Porta et al., 1999), two functionally almost redundant cell wall glycosidases important for adaptation to either neutral or acidic habitat (Muhlschlegel & Fonzi, 1997; Saporito-Irwin et al., 1995). To identify additional cell wall components regulated by Rim101p, transcriptional profiling was performed using a cell wall specific DNA-microarray. Experimental data showed that Rim101p contributes to the activation of known hyphal specific genes like HWP1, RBT1 and ALS1 and also is required for repression of several potential cell wall genes. Three yet uncharacterized genes were named RBR1, RBR2 and RBR3 (repressed by Rim101p). Further characterization of RBR1 revealed a small GPI-protein that is expressed under acidic conditions predominantly at lower temperature. Deletion of this gene resulted in a filamentation defect at low pH and furthermore, a significantly reduced virulence of the deletion strain Drbr1 versus the wildtype strain was shown in a systemic mouse model. These data suggest RBR1 as a potential target for new antifungal drugs. Changes in environmental pH also serve as a potent signal for morphological differentiation of C. albicans as it entails the differential expression of several genes affecting morphogenesis. The ability to switch between yeast and filamentous growth form is essential for the virulence of C. albicans and there is great impetus to understand the signals and regulatory pathways that govern this process (Braun & Johnson, 2000; Lo et al., 1997a). One dominant repressor of filamentous growth in C. albicans is the transcription factor Nrg1p. Interestingly, NRG1 is required for the transcription of the acid expressed and RIM101-repressed gene RBR1. This apparently activating effect of NRG1 observed in this study has not been described yet. Transcriptional profiling data give rise to the presumption that this functional property of Nrg1p also affects other genes. In addition, this work shows that expression of NRG1 is not only negatively correlated to temperature, but also pH-dependent. This pH-dependent expression of NRG1 at lower pH seems to be mediated by RIM101, since NRG1-transcription is diminished in the presence of active Rim101p. Summarizing the results presented in this study, the transcription factor Rim101p is regulating the pH-response by activating as well as repressing several cell wall genes of C. albicans and also is part of the signaling pathway regulating hyphal growth of the fungus. Therefore, the function of Rim101p integrates two central pathways of the complex regulatory network required for the ability of C. albicans to be a successful opportunistic pathogen.