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

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...

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Autores principales: Nemeth, Tibor, Papp, Csaba, Vagvolgyi, Csaba, Chakraborty, Tanmoy, Gacser, Attila
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289963/
https://www.ncbi.nlm.nih.gov/pubmed/32582114
http://dx.doi.org/10.3389/fmicb.2020.01194
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author Nemeth, Tibor
Papp, Csaba
Vagvolgyi, Csaba
Chakraborty, Tanmoy
Gacser, Attila
author_facet Nemeth, Tibor
Papp, Csaba
Vagvolgyi, Csaba
Chakraborty, Tanmoy
Gacser, Attila
author_sort Nemeth, Tibor
collection PubMed
description 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 knock-out 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.
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spelling pubmed-72899632020-06-23 Identification and Characterization of a Neutral Locus for Knock-in Purposes in C. parapsilosis Nemeth, Tibor Papp, Csaba Vagvolgyi, Csaba Chakraborty, Tanmoy Gacser, Attila Front Microbiol Microbiology 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 knock-out 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. Frontiers Media S.A. 2020-06-05 /pmc/articles/PMC7289963/ /pubmed/32582114 http://dx.doi.org/10.3389/fmicb.2020.01194 Text en Copyright © 2020 Nemeth, Papp, Vagvolgyi, Chakraborty and Gacser. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Nemeth, Tibor
Papp, Csaba
Vagvolgyi, Csaba
Chakraborty, Tanmoy
Gacser, Attila
Identification and Characterization of a Neutral Locus for Knock-in Purposes in C. parapsilosis
title Identification and Characterization of a Neutral Locus for Knock-in Purposes in C. parapsilosis
title_full Identification and Characterization of a Neutral Locus for Knock-in Purposes in C. parapsilosis
title_fullStr Identification and Characterization of a Neutral Locus for Knock-in Purposes in C. parapsilosis
title_full_unstemmed Identification and Characterization of a Neutral Locus for Knock-in Purposes in C. parapsilosis
title_short Identification and Characterization of a Neutral Locus for Knock-in Purposes in C. parapsilosis
title_sort identification and characterization of a neutral locus for knock-in purposes in c. parapsilosis
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289963/
https://www.ncbi.nlm.nih.gov/pubmed/32582114
http://dx.doi.org/10.3389/fmicb.2020.01194
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