Construction and Use of a Recyclable Marker To Examine the Role of Major Facilitator Superfamily Protein Members in <named-content content-type="genus-species">Candida glabrata</named-content> Drug Resistance Phenotypes
ABSTRACT Candida glabrata is the second most common species causing candidiasis. C. glabrata can also readily acquire resistance to azole drugs, complicating its treatment. Here we add to the collection of disruption markers to aid in genetic analysis of this yeast. This new construct is marked with...
| Main Authors: | , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2018-04-01
|
| Series: | mSphere |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mSphere.00099-18 |
| _version_ | 1819041648823238656 |
|---|---|
| author | Bao G. Vu W. Scott Moye-Rowley |
| author_facet | Bao G. Vu W. Scott Moye-Rowley |
| author_sort | Bao G. Vu |
| collection | DOAJ |
| description | ABSTRACT Candida glabrata is the second most common species causing candidiasis. C. glabrata can also readily acquire resistance to azole drugs, complicating its treatment. Here we add to the collection of disruption markers to aid in genetic analysis of this yeast. This new construct is marked with a nourseothricin resistance cassette that produces an estrogen-activated form of Cre recombinase in a methionine-regulated manner. This allows eviction and reuse of this cassette in a facile manner. Using this new disruption marker, we have constructed a series of strains lacking different members of the major facilitator superfamily (MFS) of membrane transporter proteins. The presence of 15 MFS proteins that may contribute to drug resistance in C. glabrata placed a premium on development of a marker that could easily be reused to construct multiple gene-disrupted strains. Employing this recyclable marker, we found that loss of the MFS transporter-encoding gene FLR1 caused a dramatic increase in diamide resistance (as seen before), and deletion of two other MFS-encoding genes did not influence this phenotype. Interestingly, loss of FLR1 led to an increase in levels of oxidized glutathione, suggesting a possible molecular explanation for this enhanced oxidant sensitivity. We also found that while overproduction of the transcription factor Yap1 could suppress the fluconazole sensitivity caused by loss of the important ATP-binding cassette transporter protein Cdr1, this required the presence of FLR1. IMPORTANCE Export of drugs is a problem for chemotherapy of infectious organisms. A class of membrane proteins called the major facilitator superfamily contains a large number of proteins that often elevate drug resistance when overproduced but do not impact this phenotype when the gene is removed. We wondered if this absence of a phenotype for a disruption allele might be due to the redundancy of this group of membrane proteins. We describe the production of an easy-to-use recyclable marker cassette that will allow construction of strains lacking multiple members of the MFS family of transporter proteins. |
| first_indexed | 2024-12-21T09:28:20Z |
| format | Article |
| id | doaj.art-3f5ee69e5b1b40e587400858c065a3dc |
| institution | Directory Open Access Journal |
| issn | 2379-5042 |
| language | English |
| last_indexed | 2024-12-21T09:28:20Z |
| publishDate | 2018-04-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mSphere |
| spelling | doaj.art-3f5ee69e5b1b40e587400858c065a3dc2022-12-21T19:08:49ZengAmerican Society for MicrobiologymSphere2379-50422018-04-013210.1128/mSphere.00099-18Construction and Use of a Recyclable Marker To Examine the Role of Major Facilitator Superfamily Protein Members in <named-content content-type="genus-species">Candida glabrata</named-content> Drug Resistance PhenotypesBao G. Vu0W. Scott Moye-Rowley1Department of Molecular Physiology and Biophysics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USADepartment of Molecular Physiology and Biophysics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USAABSTRACT Candida glabrata is the second most common species causing candidiasis. C. glabrata can also readily acquire resistance to azole drugs, complicating its treatment. Here we add to the collection of disruption markers to aid in genetic analysis of this yeast. This new construct is marked with a nourseothricin resistance cassette that produces an estrogen-activated form of Cre recombinase in a methionine-regulated manner. This allows eviction and reuse of this cassette in a facile manner. Using this new disruption marker, we have constructed a series of strains lacking different members of the major facilitator superfamily (MFS) of membrane transporter proteins. The presence of 15 MFS proteins that may contribute to drug resistance in C. glabrata placed a premium on development of a marker that could easily be reused to construct multiple gene-disrupted strains. Employing this recyclable marker, we found that loss of the MFS transporter-encoding gene FLR1 caused a dramatic increase in diamide resistance (as seen before), and deletion of two other MFS-encoding genes did not influence this phenotype. Interestingly, loss of FLR1 led to an increase in levels of oxidized glutathione, suggesting a possible molecular explanation for this enhanced oxidant sensitivity. We also found that while overproduction of the transcription factor Yap1 could suppress the fluconazole sensitivity caused by loss of the important ATP-binding cassette transporter protein Cdr1, this required the presence of FLR1. IMPORTANCE Export of drugs is a problem for chemotherapy of infectious organisms. A class of membrane proteins called the major facilitator superfamily contains a large number of proteins that often elevate drug resistance when overproduced but do not impact this phenotype when the gene is removed. We wondered if this absence of a phenotype for a disruption allele might be due to the redundancy of this group of membrane proteins. We describe the production of an easy-to-use recyclable marker cassette that will allow construction of strains lacking multiple members of the MFS family of transporter proteins.https://journals.asm.org/doi/10.1128/mSphere.00099-18Candida glabratamajor facilitator superfamilyselectable markertranscriptional regulationtransporters |
| spellingShingle | Bao G. Vu W. Scott Moye-Rowley Construction and Use of a Recyclable Marker To Examine the Role of Major Facilitator Superfamily Protein Members in <named-content content-type="genus-species">Candida glabrata</named-content> Drug Resistance Phenotypes mSphere Candida glabrata major facilitator superfamily selectable marker transcriptional regulation transporters |
| title | Construction and Use of a Recyclable Marker To Examine the Role of Major Facilitator Superfamily Protein Members in <named-content content-type="genus-species">Candida glabrata</named-content> Drug Resistance Phenotypes |
| title_full | Construction and Use of a Recyclable Marker To Examine the Role of Major Facilitator Superfamily Protein Members in <named-content content-type="genus-species">Candida glabrata</named-content> Drug Resistance Phenotypes |
| title_fullStr | Construction and Use of a Recyclable Marker To Examine the Role of Major Facilitator Superfamily Protein Members in <named-content content-type="genus-species">Candida glabrata</named-content> Drug Resistance Phenotypes |
| title_full_unstemmed | Construction and Use of a Recyclable Marker To Examine the Role of Major Facilitator Superfamily Protein Members in <named-content content-type="genus-species">Candida glabrata</named-content> Drug Resistance Phenotypes |
| title_short | Construction and Use of a Recyclable Marker To Examine the Role of Major Facilitator Superfamily Protein Members in <named-content content-type="genus-species">Candida glabrata</named-content> Drug Resistance Phenotypes |
| title_sort | construction and use of a recyclable marker to examine the role of major facilitator superfamily protein members in named content content type genus species candida glabrata named content drug resistance phenotypes |
| topic | Candida glabrata major facilitator superfamily selectable marker transcriptional regulation transporters |
| url | https://journals.asm.org/doi/10.1128/mSphere.00099-18 |
| work_keys_str_mv | AT baogvu constructionanduseofarecyclablemarkertoexaminetheroleofmajorfacilitatorsuperfamilyproteinmembersinnamedcontentcontenttypegenusspeciescandidaglabratanamedcontentdrugresistancephenotypes AT wscottmoyerowley constructionanduseofarecyclablemarkertoexaminetheroleofmajorfacilitatorsuperfamilyproteinmembersinnamedcontentcontenttypegenusspeciescandidaglabratanamedcontentdrugresistancephenotypes |