Candida parapsilosis is an opportunistic human fungal pathogen, threatening a wide range of patient group ranging from low birth-weight neonates to the critically ill and severely immune suppressed patients treated at intensive care units. Furthermore, C. parapsilosis infection rates can even exceed the candidaemia episodes caused by C. albicans at certain geographical regions. Therefore, excessive research is now focused on the investigation of C. parapsilosis’ pathogenesis. In order to search for genes potentially influencing the virulence of C. parapsilosis, we aimed to generate and characterize over-expression mutant strains of ORFs that, according to our preliminary experiments, are thought to influence fungal pathogenesis.
To identify such set of genes, transcriptional analysis of fungi co-incubated with THP-1 human monocytes was performed. Differentially expressed kinases and transcriptional factors and additional orthologous genes of C. albicans virulence regulatory ORFs (adherence, biofilm formation, antifungal resistance) were sentenced to over-expression. We used the Thermo Fisher Scientific Gateway cloning technology to create over-expression mutant strains. Transformants were validated by colony-PCR, Southern blot and real-time PCR. The fitness of the verified mutants was monitored in vitro at various temperatures and under conditions that are thought to influence the virulence of C. parapsilosis. These included different pH conditions, minimal and complex media and also solid media supplemented with osmotic, oxidative or cell wall stressors. Fitness under metal iron limiting conditions was also examined.
During our work, we successfully generated 36 over-expression mutant strains with 2 parallels and added unique 20-mer nucleotide barcode sequences to each in order to be able to identify them during later co-infection studies. Integration of the over-expression constructs was confirmed by both colony PCR and real-time PCR. To exclude the possibility of ectopic integrations, Southern-blot analysis was also applied. We confirmed that all mutant strains contained only one copy of the integrating constitutive promoter containing over-expression cassette. Thereafter, all available mutant strains’ viability was examined under the aforementioned conditions. During the characterization, altered phenotypes were detected mainly in the presence of SDS, Calcofluor white and Congo Red. Under these conditions, mutants showed growth disabilities when compared to the parental strain. 
In this study, we successfully generated 36 C. parapsilosis over-expression mutant strains. According to our results, several of these showed a phenotype different from the wild type under conditions that are thought to impact the pathogenicity of this species. In order to supplement this hypothesis we further aim to perform virulence studies including both in vitro and in vivo infection models.
This project was funded by NKFIH K123952 and GINOP-2.3.2-15-2016-00015.

Generation and characterization of Candida parapsilosis over-expression mutant strains