A Genomic Safe Haven for Mutant Complementation in Cryptococcus neoformans.

Arras, Samantha D. M., Chitty, Jessica L., Blake, Kirsten L., Schulz, Benjamin L. and Fraser, James A. (2015) A Genomic Safe Haven for Mutant Complementation in Cryptococcus neoformans.. PLoS One, 10 4: e0122916-e0122916. doi:10.1371/journal.pone.0122916

Author Arras, Samantha D. M.
Chitty, Jessica L.
Blake, Kirsten L.
Schulz, Benjamin L.
Fraser, James A.
Title A Genomic Safe Haven for Mutant Complementation in Cryptococcus neoformans.
Journal name PLoS One   Check publisher's open access policy
ISSN 1932-6203
Publication date 2015-04
Year available 2015
Sub-type Article (original research)
DOI 10.1371/journal.pone.0122916
Open Access Status DOI
Volume 10
Issue 4
Start page e0122916
End page e0122916
Total pages 16
Place of publication San Francisco, CA United States
Publisher Public Library of Science
Collection year 2016
Language eng
Formatted abstract
Just as Koch's postulates formed the foundation of early infectious disease study, Stanley Falkow's molecular Koch's postulates define best practice in determining whether a specific gene contributes to virulence of a pathogen. Fundamentally, these molecular postulates state that if a gene is involved in virulence, its removal will compromise virulence. Likewise, its reintroduction should restore virulence to the mutant. These approaches are widely employed in Cryptococcus neoformans, where gene deletion via biolistic transformation is a well-established technique. However, the complementation of these mutants is less straightforward. Currently, one of three approaches will be taken: the gene is reintroduced at the original locus, the gene is reintroduced into a random site in the genome, or the mutant is not complemented at all. Depending on which approach is utilized, the mutant may be complemented but other genes are potentially disrupted in the process. To counter the drawbacks of the current approaches to complementation we have created a new tool to assist in this key step in the study of a gene's role in virulence. We have identified and characterized a small gene-free region in the C. neoformans genome dubbed the "safe haven", and constructed a plasmid vector that targets DNA constructs to this preselected site. The plasmid vector integrates with high frequency, effectively complementing a mutant strain without disrupting adjacent genes. qRT-PCR of the flanking genes on either side of the safe haven site following integration of the targeting vector revealed no changes in their expression, and no secondary phenotypes were observed in a range of phenotypic assays including an intranasal murine infection model. Combined, these data confirm that we have successfully created a much-needed molecular resource for the Cryptococcus community, enabling the reliable fulfillment of the molecular Koch's postulates.
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2016 Collection
School of Chemistry and Molecular Biosciences
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Citation counts: TR Web of Science Citation Count  Cited 3 times in Thomson Reuters Web of Science Article | Citations
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Created: Fri, 17 Apr 2015, 13:57:11 EST by Mrs Louise Nimwegen on behalf of School of Chemistry & Molecular Biosciences