A Genomic Safe Haven for Mutant Complementation in Cryptococcus neoformans

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.

A fluorogenic C. neoformans reporter strain with a robust expression of m-cherry expressed from a safe haven site in the genome

“…A previously characterized gene-free region, 1,544 bp in length and located on chromosome 1 between the convergently arranged genes CNAG_00777and CNAG_00778, was suggested to be a good site for gene complementation (Arras et al, 2015). The SH2 locus, located between two tandemly arranged genes on chromosome 11, is 3,278 bp long.…”

“…Both approaches have drawbacks. Inserting the target gene fragment at its original locus requires assembly or cloning of complex and often large DNA constructs; these also typically include selectable markers, which may influence nearby genes (Arras et al, 2015). Random ectopic integration of the target gene fragment is itself mutagenic, potentially interrupting other genes due to the highly compact nature of the C. neoformans genome, and thus confounding interpretation of results (Tenney et al, 2004).…”

“…Random ectopic integration of the target gene fragment is itself mutagenic, potentially interrupting other genes due to the highly compact nature of the C. neoformans genome, and thus confounding interpretation of results (Tenney et al, 2004). These challenges were recently demonstrated with attempts to complement an ade2 Δ mutant, where neither of the above mentioned gene complementation approaches restored wild-type virulence despite complementation of other phenotypes (Arras et al, 2015). As an alternative strategy, the authors identified an intergenic genomic “Safe Haven” region that could serve as a site for DNA insertion without altering the recipient strain phenotypes (Arras et al, 2015).…”

“…These challenges were recently demonstrated with attempts to complement an ade2 Δ mutant, where neither of the above mentioned gene complementation approaches restored wild-type virulence despite complementation of other phenotypes (Arras et al, 2015). As an alternative strategy, the authors identified an intergenic genomic “Safe Haven” region that could serve as a site for DNA insertion without altering the recipient strain phenotypes (Arras et al, 2015). …”

Disruption of de novo ATP biosynthesis abolishes virulence in Cryptococcus neoformans

“…This construct was provided by Andre Koh. APH1 from C. neoformans strain H99 was TOPOcloned as a PCR fragment (primers UQ1750 and UQ1753) into pCR2.1 TOPO, then subcloned as a restriction fragment (SacI and Nhel) into the nourseothricin acetyltransferase (NAT) vector pSDMA25 to give the plasmid pKLB01 (68) . This construct was provided by Kirsten Blake.…”