Elucidating the role of MBP1 in the pathogenesis of Candida albicans
Gregory J. Fischer Julie Anderson Daniel Herman
Department of Biology
University of Wisconsin–Eau Claire
Plasmid Construction and Isolation
Abstract
The yeast species Candida albicans is the most commonly-isolated yeast in human disease.
Systemic infections of C. albicans have emerged as important causes of mortality in premature
infants and in the immunocompromised, with the number of cases on the rise. To infect host
tissue, the usual unicellular form of C. albicans switches into an invasive, multicellular filamentous
form. This morphogenesis or conversion to the filamentous state has been shown to contribute
significantly to C. albicans’ pathogenicity. We have been investigating the role of the MBP1 gene
in this process. The MBP1 homolog in the nonpathogenic yeast Saccharomyces cerevisiae has
been well studied and plays a role in the G1-S transition within the cell cycle. To further our
understanding of the function of MBP1 in C. albicans, we are expressing C. albicans’ MBP1 within
a S. cerevisiae MBP1 knockout strain to assess whether the MBP1 protein carries out similar
functions in both organisms. Experimental results will help elucidate the role MBP1 plays in
morphogenesis which could lead to novel therapies for these types of infections.
Experimental Design
Acquiring Double Mutant
MBP1
Mating
YJJ1068
MBP1
MBP1
n
Figure 4: Following transformation, plasmid DNA was isolated and
cut to determine whether the MBP1 insert was present. The results
indicate that homologous recombination was successful in DNA of
colonies 2 and 5.
Figure 5: The use of homologous recombination for the creation of
a vector construct. C. albicans MBP1 was placed downstream of a
Gal promoter, allowing for conditional expression of MBP1 in the
presence of galactose.
Meiotic Product Characteristics
MBP1 Protein Expression
B)
Mouse anti-myc
Figure 2: Prevalence of Candida species in fungal infections for ICU and
cancer patients. Recent studies continue to identify C. albicans as the
predominant yeast species isolated in fungal infections.
mbp1Δ/swi4Δ
[pLEU-MBP1-1]
Figure 6: A) Immunoblot using chemiluminescence to detect C. albicans MBP1 protein
under the control of a GAL promoter. Primary and secondary antibodies were used at
concentrations of 1:1000 and 1:5000, respectively. These results confirmed that C.
albicans MBP1 expression is induced by galactose. B) Schematic of a typical
immunoblot using chemiluminescence. A myc-epitope was added to MBP1 in the
expression system and an anti-myc antibody was used to detect the protein.
MBP1 myc-fusion protein
93.9 kDa
B)
Swi6
MBF
Complex
mbp1Δ/swi4Δ
[pLEU-MBP1-2]
MBP1
Vector Only
Molecular Genetics of Yeast
Master Plate
Swi6
Jorgenson, et al., 2002
Experimental Results
MBP1
Role of MBP1 in Cell Cycle
SBF/MBF
Leu+/Ura-/G418S
Leu+/Ura+/G418S
Leu+/Ura-/G418R
Leu+/Ura+/G418R
Donkey anti-mouse
Light
~94kDa
Bud emergence
(CLA4, BEM1,
BEM2, BEM4)
DNA replication
SPB duplication
MBP1+/Swi4+
MBP1+/swi4Δ
mbp1Δ/Swi4+
mbp1Δ/swi4Δ (LETHAL)
mbp1Δ/swi4Δ
Candida Candida Candida Candida
albicans tropicalis glabrata krusei
Enzyme
Phenotype
Leu-MBP1
0%
Leu
Shoham, et al., 2009
Lalla, et al., 2010
5%
HRP
Genotype
Vector Only
10%
Luminol &
H2O2
Ura-MBP1
15%
Ura
Cancer Patients
Leu
20%
Galactose
Ura-MBP1
ICU
Ura
30%
Leu-MBP1
Glucose
35%
Leu-MBP1
A)
25%
Sporulation
 Separate spores by micro-dissection.
 Identify genotype via replica plating.
(JA2011)
45%
40%
GJF2011
leu2∆1 his3∆200 ura3-52 swi4∆::URA3
mbp1 ∆::KNMX4 [pLEU-MBP1]
YSMG, Duke Univ.
50%
CLN1/2
n
2n
YJJ1000 (Betz, et al., 2002)
Mat a leu2 ∆1 his3∆200 ura3-52 swi4∆::URA3
JA2011
Mat a/α leu-/leu- ura+/ura- mbp1+/mbp1∆ swi4+/swi4∆
[pLEU-MBP1]
Prevalence of Candida Species in Various Medical
Settings
A)
YJJ1000
YJJ1068 (Betz, et al., 2002)
Mat α leu2∆1 his3∆200 ura3-52 mbp1∆::KNMX4
[pLEU-MBP1]
pESC-Leu
 Cases of fungal infections, such as oropharyngeal candidiasis (OPC) continue to
be problematic, especially in neonatal intensive care units (Dotis, et al., 2010).
 Recent studies of ICU and cancer patients consistently find C. albicans to be the
predominant yeast species isolated (Figure 2) (Shoham, et al., 2009 & Lalla, et
al., 2010).
 There is evidence that MBP1 plays a role in the filamentous growth of C.
albicans and is similar in sequence to the S. cerevisiae gene (Herman, et al., in
progress).
Figure 1: Fungal infections, such as OPC,
continue to be problematic, especially within
NICUs, with antifungal resistant strains
emerging.
JA2011
MBP1 insert
~2.5kb
Pathogenesis
Image Courtesy C. Halde
Genotype of Yeast Strains Used in Study
SBF
Complex
Swi4
A)
Gene Disruptions
Swi4
MBP1
Swi4
KNMX4
MBP1
Swi4
Alberts B., D. Gray, J. Lewis, et al., (1994). Molecular Biology of the Cell 3rd Edition. New York: Garland Science.
MBP1
Betz, J.L., M. Chang, T.M. Washburn. S.E. Porter, C.L. Mueller, & J.A. Jaehning (2002). Phenotypic analysis of Paf1/RNA
polymerase II mutations reveals connections to cell cycle regulation, protein synthesis, and lipid and nucleic acid
metabolism. Mol Genet Genomics, 268:272-285.
Dotis, J. & E. Roilides (2010). Candidemia in the pediatric intensive care unit: What’s different from candidemia in adults?
Current Fungal Infection Reports, 5(1): 49-55.
Jorgenson, P., J.L. Nishikawa, B. Breitkreutz, & M. Tyers (2002). Systemic Identification of pathways that couple cell growth
and division in yeast. Science, 297:395-400.
Shoham, S. & S. Marwaha (2009). Invasive fungal infections in the ICU. Journal of Intensive Care Medicine, 25(2):78-92.
Immunoblot
Conclusions
References
Lalla, R.V. M.C. Latortue, C.H. Hong, et al., (2010). A systemic review of oral fungal infection in patients receiving cancer
therapy. Support Care Cancer, 18:985-992.
Ura3
Glucose
Figure 8: Replica plating revealed that a double mutant yeast strain grew on galactose media but not glucose. Yeast with functional S.
cerevisiae MBP1 transformed with vector only was used as a positive control. Immunoblot of the double mutant strain confirmed C.
albicans MBP1 protein expression. Therefore, C. albicans MBP1 is able to functionally replace the MBP1 ortholog in S. cerevisiae.
B)
Figure 3: A) Schematic of the activation or inhibition of SBF and MBF by various proteins within S. cerevisiae. During growth phases, in
which the size of the cell increases, transcription factor complexes like SBF and MPF are inhibited so that cell division does not occur. Once
conditions merit division, SBF and MBF are directly involved with the activation of genes responsible for DNA replication and bud
emergence. B) MBP1 and Swi4 interact with the adapter protein Swi6 to form the MBF and SBF complexes respectively.
Galactose
Alberts, et al., 1994
Figure 7: A) In the budding yeast lifecycle, haploid yeast cells of opposite mating types undergo
conjugation to produce a diploid yeast strain. Mitosis of this diploid strain continues until meiosis
is triggered by lack of essential nutrients. Sporulation of the diploid cell results in four haploid
meiotic products known as spores. The genotypes of these spores can be determined by
separating and growing them on selective media. A mbp1/swi4 double mutant was isolated using
this technique in order to assess the activity of C. albicans MBP1. B) Gene disruptions of MBP1 via
Geneticin (G418) resistance cassette (KNMX4) in YJJ1000 strain and Swi4 disruption using a URA3
cassette in YJJ1068 strain. Growth of these strains on media containing Geneticin (G418) or
lacking uracil indicates that genomic MBP1 and Swi4 are disrupted.
 Candida albicans MBP1 is functionally equivalent to Saccharomyces cerevisiae MBP1
and is able to rescue the lethal phenotype of a mbp1/swi4 double mutant, thus
playing a role in the cell cycle.
 Suggests that C. albicans MBP1 activates genes in the G1/S transition that are
orthologous to those targeted by S. cerevisiae MBP1.
 Is evidence that the MBP1 protein could potentially serve as a drug target for the next
generation of antifungal agents.
This faculty/student research collaboration was made possible through Differential
Tuition and a grant from the Office of Research and Sponsored Programs.