Accordingly, we aimed to identify a genomic region suitable for acquiring such expression constructs, and provide a set of plasmids that are feasible for these purposes in C. Nonetheless, the goal of this work was to provide a tool for protein tagging in this species and was presented as a proof of principle rather than introducing a general locus for accepting exogenous sequences. albicans enolase 1 was fluorescently labeled in C. Recently a protein tagging method was described by Gonia and coworkers, where one allele of the ortholog of C. This effort, however, was complicated due to the absence of a characterized locus for knock-in approaches and a lack of compatible vectors. For instance, for our virulence studies, it was essential to create an effective pipeline through which we could facilitate the reintegration workflow of our knock-out mutant collection and generate GFP-tagged derivatives of laboratory and also clinical strains to follow their interactions with host immune cells ( Tóth et al., 2018). parapsilosis, our potential to deeply study the fungus remains limited due to the lack a complete set of molecular tools, such as those already available for C. However, while these methods have revealed important characteristics of the physiology and virulence attributes of C. parapsilosis, several molecular tools have been applied that primarily focus on eliminating gene products, such as the caSAT1 flipper, or the double auxotrophic complementation technique, and more recently the CRISPR/Cas9 system ( Ding and Butler, 2007 Gácser et al., 2007 Holland et al., 2014 Lombardi et al., 2017). To better understand the physiology and pathogenicity of C. albicans are not necessarily valid for C. All these observations strengthen the hypothesis that these two pathogens are different by nature, and clinical and basic science findings regarding C. parapsilosis does not ( Nucci and Anaissie, 2001 Miranda et al., 2009 Tóth et al., 2013 Lin et al., 2015 Tóth et al., 2017). albicans requires colonization before infection while C. Notably, they differ significantly in many aspects, including their resistance to antibiotics, recognition by host immune cells and filamentation ability, and C. albicans share some attributes as they are both diploid and both belong to the CUG clade, meaning that during translation the triplet CUG results in the incorporation of serine rather than leucine. parapsilosis as the second most frequently associated Candida species with bloodstream infections, and it has been reported as the major cause of invasive candidiasis in low and very low birth weight neonates ( Pappas, 2006 Pammi et al., 2013 Jayaweera and Reyes, 2018). auris) has occurred ( Yapar, 2014 Cleveland et al., 2015 Cortegiani et al., 2018 Kothalawala et al., 2019). Amongst these opportunistic pathogens, Candida albicans has long been known as the dominant etiologic agent within the genus associated with disease however, according to recent literature, a remarkable shift in the epidemiology toward the so-called non- albicans Candida species ( C. These latter diseases are most commonly associated with Candida species, especially amongst patients with defective immune system due to cancer treatment, HIV infection, or neutropenia, but prolonged hospitalizations and the use of catheters and other medical devices are also established as predisposing factors ( Puzniak et al., 2004 Cheng et al., 2005 Li et al., 2017). parapsilosis.įungal infections represent a global threat with nearly one billion cases per year ranging from mild superficial to invasive infections with high mortality rates ( Pfaller and Diekema, 2007 Bongomin et al., 2017). Hence, our findings demonstrate that N5L is a highly effective neutral locus for knock-in approaches in C. 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. As a proof of principle, we fluorescently tagged laboratory and prototroph strains and established that this locus is also suitable for reintegration purposes. parapsilosis, and introduce an integrative Gateway TM and a classical ligation based replacement plasmid to target it with. Here we report CpNEUT5L (N5L), an intergenic locus in C. To investigate the virulence properties of this pathogen several techniques have been developed for generating knock-out mutants, however, no target locus for knock-in approaches have been published so far. parapsilosis whose incidence is particularly high amongst low birth weight neonates. Besides Candida albicans, other members of the genus have gained relevance in the last two decades, including C. Invasive fungal infections caused by Candida species affect approximately 700,000 people worldwide resulting in 300,000 deaths annually.
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