Project title : Creating an overexpression library and developing an inducible gene expression system in Candida parapsilosis
To study the interaction between mammalian macrophages and Candida parapsilosis an in vitro model system was set up and optimised involving J774.2 murine cells. We established that these phagocytes were capable of uptake and eliminate the yeast cells. To characterise transcriptional changes of the host upon interaction microarray analysis and quantitative real-time PCR were performed. The C. parapsilosis – host interaction was examined by using THP-1 human monocytes as well. Up- and downregualted genes of both the host and the pathogen were identified by RNAseq analysis at four different time-points. Our major goal is to create a BAR-coded overexpression library in C. parpasilosis based on these data to study virulence in vitro and in vivo. To create mutants in such a large scale LifeTechnolgies Gateway system is applied. This approach is also used for creating reintegrant mutants for C. parapsilosis knock-out library and to adopt TETON-TETOFF system optimised for C. albicans. This approach enables us to regulate the expression (overexpress or silence) of any genes under in vitro or in vivo circumstances at any timeponts of the interaction.
Metal ions are essential nutrients for both the host and the fungus. The way how pathogens can obtain specific ions upon infection, when accessible metal ions are limited, is still not known in every details. Previous data revealed that C. albicans possessed a “siderophore-like” scavenger-transporter system that was responsible for uptake zinc ions upon in vitro infection. This so-called “zincophore system” consists of two proteins in C. albicans, however C. parapsilosis lacks the scavenger, suggesting a different mechanism for zinc-uptake in this species. Based on homology analysis a total of six potential zinc-transporters were identified in C. parapsilosis. Our aim is to knock these genes out to reveal how this pathogen is capable of acquiring zinc upon interaction with the host.