Examination of the Role of Alkaline pH Regulator RIM101 in Filamentation in Clinical Strains of Candida albicans
Presenter Type
UNO Undergraduate Student
Major/Field of Study
Biology
Advisor Information
Associate Professor and Chair of Biology
Location
MBSC Ballroom Poster # 206 - U
Presentation Type
Poster
Start Date
24-3-2023 1:00 PM
End Date
24-3-2023 2:15 PM
Abstract
pH is an important environmental factor in the ability of Candida albicans to grow and differentiate in the human microbiome. As such, changes in pH can cause the disruption of its stable commensal status within our microbiome and lead to infection. Currently, more than 25,000 systemic infections are reported yearly in the US across Candida species with high mortality rates of 25-40% despite treatment. Key to its ability to cause these infections is the process of filamentation, with alkaline pH changes being a known trigger for filamentation in C. albicans. In our study, we examine the role of neutral-alkaline pH response regulator Rim101 in filamentation across several clinical strains. This gene plays a significant role in filamentation in the C. albicans type strain SC5314, but this type strain has clear filamentation differences compared to clinical strains. Thus, it is unclear if the filamentation-defective phenotype for the rim101 mutant strain was specific to the type strain or if it represents a species-wide role in filamentation. To test this, initial clinical isolates P87 and P76067 were mutated using CRISPR-Cas9 to generate novel homozygous deletion mutants of RIM101. Knockout constructs were synthesized from pJK1354 using cloning techniques to introduce complementary RIM101 gene sequences to both ends of the knockout construct for proper homologous recombination upon CRISPR-Cas9 cutting. A Cas9 single guide RNA encoding the desired cut site within RIM101 was synthesized through overlap PCR. The knockout construct, guide RNA construct, and CAS9 DNA were transformed into the clinical strains and cells were grown on selective media. We have putative deletion strains for both P87 and P76067. Confirmation of true homozygous deletion of RIM101 through colony PCR will be performed before subsequent phenotypic analysis in classical filamentation assays across liquid and solid media.
Scheduling
9:15-10:30 a.m., 10:45 a.m.-Noon, 1-2:15 p.m., 2:30 -3:45 p.m.
Examination of the Role of Alkaline pH Regulator RIM101 in Filamentation in Clinical Strains of Candida albicans
MBSC Ballroom Poster # 206 - U
pH is an important environmental factor in the ability of Candida albicans to grow and differentiate in the human microbiome. As such, changes in pH can cause the disruption of its stable commensal status within our microbiome and lead to infection. Currently, more than 25,000 systemic infections are reported yearly in the US across Candida species with high mortality rates of 25-40% despite treatment. Key to its ability to cause these infections is the process of filamentation, with alkaline pH changes being a known trigger for filamentation in C. albicans. In our study, we examine the role of neutral-alkaline pH response regulator Rim101 in filamentation across several clinical strains. This gene plays a significant role in filamentation in the C. albicans type strain SC5314, but this type strain has clear filamentation differences compared to clinical strains. Thus, it is unclear if the filamentation-defective phenotype for the rim101 mutant strain was specific to the type strain or if it represents a species-wide role in filamentation. To test this, initial clinical isolates P87 and P76067 were mutated using CRISPR-Cas9 to generate novel homozygous deletion mutants of RIM101. Knockout constructs were synthesized from pJK1354 using cloning techniques to introduce complementary RIM101 gene sequences to both ends of the knockout construct for proper homologous recombination upon CRISPR-Cas9 cutting. A Cas9 single guide RNA encoding the desired cut site within RIM101 was synthesized through overlap PCR. The knockout construct, guide RNA construct, and CAS9 DNA were transformed into the clinical strains and cells were grown on selective media. We have putative deletion strains for both P87 and P76067. Confirmation of true homozygous deletion of RIM101 through colony PCR will be performed before subsequent phenotypic analysis in classical filamentation assays across liquid and solid media.