Construction of Candida albicans Strains with ATP-Analog-Sensitive Protein Kinase A and Hog1
ABSTRACT Candida albicans is an opportunistic human fungal pathogen and a member of the mucosal microbiota. To survive in the host and cause disease, C. albicans utilizes several virulence traits, including the ability to respond and adapt to diverse stressors, as well as the morphogenetic switch be...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2023-06-01
|
| Series: | mSphere |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/msphere.00095-23 |
| _version_ | 1827919025660755968 |
|---|---|
| author | Zhongle Liu Jessie MacAlpine Nicole Robbins Leah E. Cowen |
| author_facet | Zhongle Liu Jessie MacAlpine Nicole Robbins Leah E. Cowen |
| author_sort | Zhongle Liu |
| collection | DOAJ |
| description | ABSTRACT Candida albicans is an opportunistic human fungal pathogen and a member of the mucosal microbiota. To survive in the host and cause disease, C. albicans utilizes several virulence traits, including the ability to respond and adapt to diverse stressors, as well as the morphogenetic switch between yeast and filamentous morphologies. While complex cellular circuitry governs these virulence attributes, the following two kinase-mediated signaling pathways play particularly critical roles in controlling these processes: the Hog1 mitogen-activated protein kinase (MAPK) cascade and the protein kinase A (PKA) pathway. Here, we describe the construction of C. albicans strains harboring substitutions in the ATP-binding pockets of Hog1 and the catalytic subunits of PKA, Tpk1, and Tpk2 to render their activities sensitive to the addition of bulky ATP analogs. Specifically, inhibition by the ATP analog 1NM-PP1 resulted in phenotypes characteristic of the corresponding homozygous deletion mutants for each kinase gene. These strains represent a toolset for the rapid and specific inhibition of PKA and Hog1 kinase activity to further understand their roles in regulating C. albicans morphogenesis and stress responses. IMPORTANCE As an opportunistic pathogen in humans, the fungus Candida albicans relies on virulence traits to cause disease. They include the ability to transition from yeast to filamentous morphologies and the ability to grow in diverse environmental stress conditions, including nutrient limitation, as well as osmotic and heat shock. Previous work identified the following two kinases that play a critical role in regulating these responses: Hog1 and PKA. Here, we generated versions of each kinase that are sensitive to inhibition by a bulky ATP analog, 1NM-PP1. In the presence of the analog, kinase activity is inhibited rapidly and specifically, facilitating the analysis of both kinases in regulating C. albicans morphogenesis and stress responses. Together, these strains represent an important toolset to further our understanding of C. albicans biology and virulence. |
| first_indexed | 2024-03-13T03:51:56Z |
| format | Article |
| id | doaj.art-909882a2fce04f71862340b827526dd6 |
| institution | Directory Open Access Journal |
| issn | 2379-5042 |
| language | English |
| last_indexed | 2024-03-13T03:51:56Z |
| publishDate | 2023-06-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mSphere |
| spelling | doaj.art-909882a2fce04f71862340b827526dd62023-06-22T13:00:23ZengAmerican Society for MicrobiologymSphere2379-50422023-06-018310.1128/msphere.00095-23Construction of Candida albicans Strains with ATP-Analog-Sensitive Protein Kinase A and Hog1Zhongle Liu0Jessie MacAlpine1Nicole Robbins2Leah E. Cowen3Department of Molecular Genetics, University of Toronto, Toronto, Ontario, CanadaDepartment of Molecular Genetics, University of Toronto, Toronto, Ontario, CanadaDepartment of Molecular Genetics, University of Toronto, Toronto, Ontario, CanadaDepartment of Molecular Genetics, University of Toronto, Toronto, Ontario, CanadaABSTRACT Candida albicans is an opportunistic human fungal pathogen and a member of the mucosal microbiota. To survive in the host and cause disease, C. albicans utilizes several virulence traits, including the ability to respond and adapt to diverse stressors, as well as the morphogenetic switch between yeast and filamentous morphologies. While complex cellular circuitry governs these virulence attributes, the following two kinase-mediated signaling pathways play particularly critical roles in controlling these processes: the Hog1 mitogen-activated protein kinase (MAPK) cascade and the protein kinase A (PKA) pathway. Here, we describe the construction of C. albicans strains harboring substitutions in the ATP-binding pockets of Hog1 and the catalytic subunits of PKA, Tpk1, and Tpk2 to render their activities sensitive to the addition of bulky ATP analogs. Specifically, inhibition by the ATP analog 1NM-PP1 resulted in phenotypes characteristic of the corresponding homozygous deletion mutants for each kinase gene. These strains represent a toolset for the rapid and specific inhibition of PKA and Hog1 kinase activity to further understand their roles in regulating C. albicans morphogenesis and stress responses. IMPORTANCE As an opportunistic pathogen in humans, the fungus Candida albicans relies on virulence traits to cause disease. They include the ability to transition from yeast to filamentous morphologies and the ability to grow in diverse environmental stress conditions, including nutrient limitation, as well as osmotic and heat shock. Previous work identified the following two kinases that play a critical role in regulating these responses: Hog1 and PKA. Here, we generated versions of each kinase that are sensitive to inhibition by a bulky ATP analog, 1NM-PP1. In the presence of the analog, kinase activity is inhibited rapidly and specifically, facilitating the analysis of both kinases in regulating C. albicans morphogenesis and stress responses. Together, these strains represent an important toolset to further our understanding of C. albicans biology and virulence.https://journals.asm.org/doi/10.1128/msphere.00095-231NM-PP1ATPCandida albicansHog1protein kinase Agenetic resource |
| spellingShingle | Zhongle Liu Jessie MacAlpine Nicole Robbins Leah E. Cowen Construction of Candida albicans Strains with ATP-Analog-Sensitive Protein Kinase A and Hog1 mSphere 1NM-PP1 ATP Candida albicans Hog1 protein kinase A genetic resource |
| title | Construction of Candida albicans Strains with ATP-Analog-Sensitive Protein Kinase A and Hog1 |
| title_full | Construction of Candida albicans Strains with ATP-Analog-Sensitive Protein Kinase A and Hog1 |
| title_fullStr | Construction of Candida albicans Strains with ATP-Analog-Sensitive Protein Kinase A and Hog1 |
| title_full_unstemmed | Construction of Candida albicans Strains with ATP-Analog-Sensitive Protein Kinase A and Hog1 |
| title_short | Construction of Candida albicans Strains with ATP-Analog-Sensitive Protein Kinase A and Hog1 |
| title_sort | construction of candida albicans strains with atp analog sensitive protein kinase a and hog1 |
| topic | 1NM-PP1 ATP Candida albicans Hog1 protein kinase A genetic resource |
| url | https://journals.asm.org/doi/10.1128/msphere.00095-23 |
| work_keys_str_mv | AT zhongleliu constructionofcandidaalbicansstrainswithatpanalogsensitiveproteinkinaseaandhog1 AT jessiemacalpine constructionofcandidaalbicansstrainswithatpanalogsensitiveproteinkinaseaandhog1 AT nicolerobbins constructionofcandidaalbicansstrainswithatpanalogsensitiveproteinkinaseaandhog1 AT leahecowen constructionofcandidaalbicansstrainswithatpanalogsensitiveproteinkinaseaandhog1 |