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Tools for high efficiency genetic manipulation of the human pathogen Penicillium marneffei
Hayley E Bugeja1, Kylie J Boyce, Harshini Weerasinghe
1Department of Genetics, The University of Melbourne, Parkville, Victoria 3010, Australia.
Abstract:
Penicillium marneffei is an opportunistic pathogen of humans and displays a temperature dependent dimorphic transition. Like many fungi, exogenous DNA introduced by DNA mediated transformation is integrated randomly into the genome resulting in inefficient gene deletion and position-specific effects. To enhance successful gene targeting, the consequences of perturbing components of the non-homologous end joining recombination pathway have been examined. The deletion of the KU70 and LIG4 orthologs, pkuA and ligD, respectively, dramatically enhanced the observed homologous recombination frequency leading to efficient gene deletion. While ΔpkuA was associated with reduced genetic stability over-time, ΔligD represents a suitable recipient strain for downstream applications and combined with a modified Gateway™ system for the rapid generation of gene deletion constructs, this represents an efficient pipeline for characterizing gene function in P. marneffei.
Insights
Researchers improved gene deletion efficiency in Penicillium marneffei by targeting the non-homologous end joining pathway. Deleting the LIG4 ortholog (ligD) creates a suitable strain for fungal gene function characterization.
Area of Science:
- Mycology
- Molecular Biology
- Medical Mycology
Background:
- Penicillium marneffei is an opportunistic fungal pathogen causing human infections.
- Random DNA integration limits gene deletion efficiency and introduces position-specific effects in P. marneffei.
- Targeting the non-homologous end joining (NHEJ) pathway is a strategy to enhance homologous recombination.
Purpose of the Study:
- To improve gene targeting and deletion efficiency in Penicillium marneffei.
- To investigate the role of NHEJ pathway components (pkuA and ligD) in homologous recombination.
- To develop an efficient pipeline for characterizing gene function in P. marneffei.
Main Methods:
- Deletion of the KU70 ortholog (pkuA) and LIG4 ortholog (ligD) in P. marneffei.
- Assessment of homologous recombination frequency in the generated deletion strains.
- Utilizing a modified Gateway™ system for rapid construction of gene deletion cassettes.
Main Results:
- Deletion of pkuA and ligD significantly enhanced homologous recombination frequency.
- Efficient gene deletion was achieved in the modified strains.
- The ΔligD strain showed suitability for downstream applications, unlike the ΔpkuA strain which had reduced genetic stability.
Conclusions:
- Perturbing the NHEJ pathway, specifically ligD deletion, enhances homologous recombination and enables efficient gene deletion in P. marneffei.
- The ΔligD strain provides a stable and efficient recipient for genetic manipulation.
- This study establishes a robust pipeline for functional genomics in P. marneffei, aiding in understanding this important fungal pathogen.
