Investigation of the localization and phenotypic effects of the

mRNA transport protein She3 in Candida albicans Amanda Estes- SMCC

Advisor: Dr. Anne McBride- Bowdoin College

Goals

Determine localization of She3 protein in C. albicans cells • Hypothesis 1: When linked with GFP, She3 protein will be

visible localizing in the cytoplasm of the tips of yeast buds and hyphae.

Determine if presence of She3 affects phenotypes of cell colonies under different conditions • Hypothesis 2: Deletion of SHE3 affects filamentation of C. albicans in various environmental conditions.

• Integrate GFP::URA3 PCR product into genome of she3/SHE3∆ ura3∆/ura3∆ strain of C. albicans through transformation

• Use PCR to create DNA that will combine SHE3with GFP and a selectable marker (URA3)

• Determine expression of GFP::URA3 with SHE3 • Epifluorescence microscopy • Anti-GFP immunoblot

Methods & Results

• Select for transformants on Ura- medium

Create strains of C. albicans with GFP linked to 3’ end of She3

Acknowledgements: Thank you: Dr. Anne McBride for her guidance and encouragement Judi Medlin & MDIB Frank Pellerin Bowdoin College SMCC Dr. Elizabeth Ehrenfeld Research reported in this project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103423

Absence of She3 affects filamentation but does not affect lipase secretion.

SHE3/she 3∆ she3∆/she∆

RPMI agar

SHE3/she 3∆ she3∆/she 3∆

Spider agar

Conclusions

• She3 likely localizes in the nucleus of C. albicans cells • She3-GFP is expressed in cells transformed with GFP linked to SHE3 • Filamentation is affected by the absence of She3 while lipase

activity is not

Future Directions

• PCR confirmation of GFP integration of into SHE3 locus • DAPI staining to confirm nuclear localization of She3-GFP • Immunoblot with higher protein concentrations • Use epifluorescent microscopy to visualize She3-GFP in

hyphal cells • Link GFP to N-terminus of She3 to determine if site of GFP tag

affects She3 localization • Test effect of GFP tag on She3 function on filamentation in

media assays

She3-GFP may localize to the nucleus in budding yeast cells

Methods & Results Grow single colonies of C. albicans on plates with different environmental conditions.

• Filamentation assays • 5µl of 107 cells of each strain spotted on

medium • Colonies grown on plates for 6-10 days at

37⁰C

• Lipase assay • 3µl of cultures diluted to OD600~ 0.5 • Colonies grown for 2 days in 37⁰C and

observed every 24 hours.

SHE3/she3∆ colonies develop higher ratio of filamentous periphery to central region in SHE3/she3∆ colonies .

SHE3/she3∆ she3∆/she3∆

Egg yolk agar

No difference in lipase activity was detected.

Budding Yeast

Hyphae

www.usask.ca

www.usask.ca

Introduction Candida albicans • Commensal fungus in normal microflora of

mammals • Opportunistic pathogen in certain

conditions • Potential for fatal infections in

immunocompromised individuals • Rapid morphological changes in response

to environment contribute to virulence4 • Optimal hyphal growth and host invasion

require proteins to be located to the hypha or its surface by:

• protein transport • localized translation of

transported mRNA3.

She3: mRNA transport protein • Part of well-established mRNA transport

model in Saccharomyces cerevisiae1

• C. albicans protein She3 homologous to S. cerevisiae She3

• Important for proper

filamentation2

• Part of less-understood transport

system of mRNAs translated at C.albicans hyphal tip

Anti-GFP immunoblot

• Log-phase yeast cells collected and lysed

• Proteins resolved by SDS-PAGE • GFP detected by immunoblot • She3-GFP predicted size=83 kDa

Detection of putative She3-GFP protein in yeast lysate

Weak band detected between 80-100 kDa in SHE3-GFP::URA3-transformed strains

SHE3/she3∆ SHE3/she3∆ she3∆/she3∆

References 1. Bohl F, Kruse C, Frank A, Ferring D, Jansen RP She2p, a novel RNA binding protein tethers ASH1 mRNA to the Myo4p myosin motor via She3p. EMBO J 2000, 19: 5514–5524 2. Elson SL, Noble SM, Solis NV, Filler SG, Johnson AD: An RNA transport system in Candida albicans regulates hyphal morphology and invasive growth. PLoS genetics 2009, 5(9):e1000664. 3. Mayer, F.L., Wilson, D., and Hube, B. (2013). Candida albicans pathogenicity mechanisms. Virulence 4, 119-128 4. Sudbery, P.E.. Growth of Candida albicans hyphae. Nat. Rev. Microbiol 2011, 9, 737-748.

She3-GFP No GFP