1. 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.usas
k.ca
www.usas
k.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