Identification and Characterization of a Neutral Locus for Knock-in Purposes in C. parapsilosis

Abstract

Invasive fungal infections caused by Candida species affect approximately 700,000 people worldwide resulting in 300,000 deaths annually. Besides Candida albicans, other members of the genus have gained relevance in the last two decades, including C. parapsilosis whose incidence is particularly high amongst low birth weight neonates. To investigate the virulence properties of this pathogen several techniques have been developed for generating knockout mutants, however, no target locus for knock-in approaches have been published so far. Here we report CpNEUT5L (N5L), an intergenic locus in C. parapsilosis, and introduce an integrative Gateway TM and a classical ligation based replacement plasmid to target it with. As a proof of principle, we fluorescently tagged laboratory and prototroph strains and established that this locus is also suitable for reintegration purposes. We concluded that GFP-expressing constructs integrated into this region provide strong, homogenous fluorescent signals while alteration of this locus affects neither the growth of the mutants in liquid or on solid media, even in the presence of different stressors, nor their basic virulence properties. Hence, our findings demonstrate that N5L is a highly effective neutral locus for knock-in approaches in C. parapsilosis.

A Candida parapsilosis Overexpression Collection Reveals Genes Required for Pathogenesis

“…To generate overexpression mutants, the Gateway TM system was used. Our work is based on the previous work of Chauvel and colleagues with C. albicans [ 50 ], which we modified and optimized for C. parapsilosis [ 53 ]. Selected ORFs were amplified along with a 20 nt long bar code (See Table S1 TAG sequences) in the non-transcribed region to enable subsequent identification by molecular methods.…”

“…The linearized plasmid (or integrative cassette) was then integrated into the CpNEUT5L (CpN5L) intergenic region of C. parapsilosis ( Figure S2 ). We targeted the CpN5L region because, in contrast to the ortholog of RPS1 , this locus not only provides a strong homogenous expression for exogenous constructs, but it does not affect the fitness of the transformed/modified strain [ 53 , 56 ]. The expression vector utilizes the C. maltosa LEU2 marker for selection that is compatible with the leucine auxotroph laboratory strain of C. parapsilosis CPL2 [ 28 ].…”

“…We also included an mCherry fluorescent protein-expressing strain as a control OE strain (mCherry OE ), which was generated using the same procedure. We found this pertinent because expressing an irrelevant gene (coding a “neutral” protein) in terms of virulence would imitate the same load on the expression machinery, which itself might have an effect on the physiological and virulence properties of the fungus [ 53 ].…”

“…Overexpression constructs were generated using Gateway TM cloning method with pDONR TM 221 for BP and pDCpOE-L-N5L for LR cloning [ 53 ]. Target ORFs were amplified with an attB1 site at the 5′ end, and an attB2 site at the 3′ end.…”

“…Although extensive studies in gene OE in Saccharomyces cerevisiae and C. albicans have enriched our knowledge on their fitness and virulence [ 49 , 50 , 51 , 52 ], no such an effort has yet been achieved in the case of the neonatal pathogen C. parapsilosis . Therefore, we have generated and characterized an OE collection in C. parapsilosis using a recently adapted system by Németh and co-workers [ 53 ] and explored the collection in order to reveal novel virulence traits.…”