Transcript Introduction of Point Mutations through Sequential PCR Steps

Hydrophobic interactions in the N-terminal of Alsn
protein of Canidada albicans are responsible for
epithelial adhesion events when the protein is
expressed in Saccromyces cerevisiae
By Augusto Cigliano: Undergraduate Research Assistant
for Dr. Zhang at California State University Long Beach
Lecture Outline
• General background information
• Talk about motivations for this experiment
• The experimental design
– Specific methods and their results
– Problems with testing hydrophobicity
• Conclusions
• Future experiments
Background information
• Candida albicans is an opportunistic pathogen that has a diverse range
of associations with its host. (yeast infection?)
• If adhesion to host cells is the first step on the road to C.albicans
pathogenesis, blocking this first step could stop its pathogenesis, and
save lives. This would be simple, accept evolution has given this little
guy lots of different proteins to use to adhere to its host.
• Numerous studies were done on the sexual agglutinin proteins of
Saccromyces cerevisiae, a yeast that does not adhere to human cells.
• Scientists blasted the agglutinin gene sequence into the C. albicans
known geneome and came up with numerous close matches, now
called agglutinin-like sequences (Als).
• The different Als proteins are coded for on different loci, but they all
similar 3-domain structure with a highly conserved N-terminus
• The N-terminal is believed to give the protein its binding properties
This experiment
• Recent studies have uncovered yet another Als protein: Alsn. What
role does it play in cell adhesion? What causes it to adhere? How is it
different from the other Als proteins?
• What is known is that it binds weakly to endothelial cells and is the
least conserved of all the proteins in the Als family. It also has fewer
hydrophobic sequences than all the other Als proteins.
• Could this decrease in hydrophobicity be the cause in its weak
adherence to endothelial cells?
• This experiment shows that hydrophobicity could be the main cause
for the aherence of Alsn protein in vitro.
• Studying the mechanisms by which this protein adheres might give a
clue to how the other proteins in this same family adhere.
The experimental design
Using PCR technology, I mutated the areas of the Alsn C. albicans gene involved
in hydrophobic interactions with host cells.
Then, I expressed the gene into a normally nonadherent S. cerevisiae
yeast using a shuttle vector.
Using immunostaining, I made sure the protein was being expressed on
the surface of the yeast cell.
I assayed for adherence to epithelial cells
Finally I used a hydrophobic microsphere assay to double checked to
make sure the mutations of the hydrophobic regions resulted in actual
decrease in the cells hydrophobic character.
Expected areas involved in
hydrophobic interactions.
N-terminus of Alsn protein
Amino acids
350-365
and
690-706
Three mutations were constructed. M1 mutated regions 350
to 365, M2 mutated regions 690 to 706, and M3 combined
both mutations.
Introduction of Point Mutations
through Sequential PCR Steps
SacII
Xho1
The plasmid is made by
cutting out
the N-terminus
with Xho1 and
SacII restriction
sites, and subcloning
into a high copy number
vector.
N-terminus of
ALSn
Xho1
F=forward primer
R=reverse primer
M= mutated primers
SacII
Xho1
SacII
From here, the Nterminus was
amplified for 25
cycles with the two
sets of primers.
Primers 5 and 6 were synthesized with mutations, while F and R
were synthesized for the end sequences to include restriction
sites Xho 1 and SacII respectively.
Example of a synthesized primer
PRIMER 5
Hydrophobic
coding region
5’ TATCATCAGCAGGATAATAACAGTCAACAGAGCTTTTCCCTTTATCATCAGETC 3’
5’ CCCCGACTACTAGATGCTCCCT TTAT CATCAGCAGGATATTATCGGTCTACTGGGCTTTTCCCTTTATCATCAGCAG 3’
3’ GGGGCTGATGATCTACGAGGGAAATAGTAGTCGTCCTA TAATAGCCAGATGACCCGAAAAGGGAAATAGTAGTCGTC 5’
3’ ETCTCTACGAGGGAAATAGTAGTCGTCCTATTATT GTCA GTTGTCTCGAAAAGGGAAATAGTAG 5’
PRIMER 6
The specific sequences for hydrophobic amino acids found
on the surface of the Alsn protein were manipulated to code
for polar amino acids.
~Ile-Ile-Gly-Leu-Leu-Gly~ now reads ~Asn-Asn-Ser-Gln-Gln-Ser~
The Amplified DNA was obtained and put through one cycle
with no Forward and reverse primers.
Mutated Primers
Xho1
SacII
SacII
Xho1
The N-terminal with
SacII restriction site
specific for its own gene
(that has the Nterminus missing) and
XhoI site for the
lambda-YES shuttle
vector, should insert
itself into the Alsn gene
and the vector.
Run on a gel to ensure
the gene has been
inserted.
.
This vector is a shuttle
vector that passes freely
from E.coli into S.
cerevisie
ALSn
Alsn expression in S. cerevisiae
• S. cerevisiae ura- yeast were transformed with
lambda-YES Alsn mutants and controls.
• The Ura+ mutants were selected for as being
transformed and were colony-purified.
• The mutant yeast were grown in a galactose
containing minimal medium to induce expression
of the Alsn protein. The lambda-YES vector’s
promoter region is galactose induced.
Preventing false negatives
• Did the Alsn proteins even
make it to the yeast’s surface.
If they didn’t, I would be
getting false negatives!
• Surface proteins were detected
with immunostaining:
polyclonal antibodies were
added, then washed.
• Goat-anti-rabbit-FITC was
added, incubated, then washed.
• Any polyclonal antibodies that
initially adhered would attach
the labeled secondary
antibodies.
• If fluorescence is visualized, the
Alsn protein is being expressed
on the membrane’s surface.
Assay for Adherence to epithelial cells
• Each strain was tested for adherence to
epithelial cells using a standard adherence
assay and the percent adherence was
calculated by actually counting the adhered
cells microscopically and using the formula
(CFUs per well/total input cells)*100.
This graph depicts the % binding of C. albicans ALSn protein expressed S. cervisiae yeast to
epithelial cells. The positive control is S. cervisiae with both surface N-terminal hydrophobic
sections intact. Negative control shows yeast with an empty vector. The mutant one (M1) has
one hydrophobic section mutated. Mutant two (M2) has the other hydrophobic section mutated.
Mutant three (M3) has both sections mutated. The yellow arms represent how the hydrophobic
sections grab onto the red epithelial cells. The sad M3 mutant has no arms and has a hard time
adhering.
E
E
E
E
Determining Hydrophobicity
• A hydrophobic microsphere test was done in addition to
the epithelial adhesion assay to assess whether the mutant
Alsn proteins are less hydrophobic than the normal Alsn
proteins.
• Microspheres are very small polystyrene hydrophobic
beads. In essence, they are mixed in with the cells and
bind to its hydrophobic parts.
• The % of cells which are hydrophobic was determined
from bright-field microscopy (a method for illuminating
the beads). A count of 100 cells containing 3 or more
attached microspheres determined the hydrophobicity of
each population. The difference of the cell surface
adhesion (CSH) value from 100% determines the
hydrophobic level of each population.
Things get complicated! Selection of optimal microsphere size
100
Alsn (+)
control
% CSH adhesion
80
Alsn (-)
control
60
Mutant 1
40
20
Mutant 2
0
Mutant 3
0.8um
0.9um
1.0um
1.5um
As can be seen in the graph, the smaller the hydrophobic pellet, the more
hydrophobicity
is expressed
in both
the
- controls.
Thisthe
is for
because
the
small
Of course
The
question
another
is are
separate
the protein’s
test
needs
hydrophobic
to+beand
made
sections
to validate
available
statement
a yeast-host
that
the
cell
pellets
get into
grooves
of all
foldedtest
proteins
adhere.
thewill
pellets
are
tooThe
Alsn’s hydrophobicity
interaction?
A the
simple
hydrophobicity
is even
affected
by
the
formutations
theand
isolated
to protein
theIfhydrophobic
not work.
region.
large,
there
adhesion.
If we
assume
that
the
protein
in such
The level
protein
needs
of will
hydrophobicity
to be
be no
properly
expressed
folded,
processed
by
a protein
and glycosilated,
andAlsn
the number
andfolds
of
expressed
hydrophobic
onathe
way
as
to offer
up its
N-terminus
so
that
the and
hydrophobic
region is positioned
for
amino
cells
surface.
acids
itEven
has
do
though
not goS.hand
cerevisiae
and
hand
cells
do not
so hydrophobicity
show adherence,
can
they
notdobeexpress
adhesion,
we
can on
find
a bead that is big enough not to adhere proteins native to S.
calculatedhydrophobicity.
“hidden”
based
structure.
cerevisiae, yet will still adhere to the Alsn protein. This bead is 1.0um in diameter.
Using this size bead, we can now say that proteins hydrophobicity was altered by the
mutations.
So what do we gather from this experiment?
• N-terminal hydrophobic residues 350 to 365
and 690 to 706 are mainly responsible for
Alsn adhesion.
• Mutations of these hydrophobic residues
that made them expessess polar residues
decreased cellular adhesion in vitro and also
showed a decrease in hydrophobicity.
• We can conclude that nonspecific
hydrophobic interactions are probably the
main cause of the Alsn binding character.
Looking at my results and
drawing conclusions
•What about the possibility that there is another
mechanism that was affected by the mutations?
•This is probably unlikely because the loss of
hydrophobicity was proportional to the loss of
adhesion events.
•If hydrophobicity was decreased and adhesion
events remained the same, then there would
probably be another mechanism associated.
•If hydrophobicity wasn’t affected by the
mutation, then something went wrong in the
design of my experiment, or my detection of
hydrophobicity was not accurate.
In conclusion
•Whether or not these nonspecific interactions are the cause of
cellular adhesion events in other proteins of the Als family is still
an area of research.
•A more broad question is why do C. albicans have such diversity?
If the majority of cellular adhesion events are believed to be caused
by other surface proteins such as mannoproteins and C3d-Binding
proteins, why do the Alsn proteins exist? (Survival?)
•The main goal of this and similar research is the identification and
characterization of any and all proteins believed to be involved in
adhesion. Continue research along these lines.
•Who knows to what extent this knowledge will be useful in
combating future diseases.
Future experiments
•Because the Alsn protein is so new, we need more basic
experiments to characterize its functional domains (this is being
done right now at UCLA.)
•What causes the C. albicans cells to express any one of the C.
albicans Als proteins in humans? Set up experiments that mimic
diferent enviornmental conditions found in the the human body.
•See if adhesion of this protein is linked to pathogenesis of the C.
albicans Alsn protein. This is a hard experiment because
adherence and pathogenesis is already known to be mediated by
many other adhesion proteins. But to what extent does Alsn
participate C. albicans adhesion? Determining its roll in
pathogenesis is tricky because you have to work with animals.
Acknowledgments
• The Howard Hughes institute
• Dr. Zhang
– Department of Microbiology CSULB
• Natalie Lucindo
• Dr. Mason and Dr. Archie
– my biology 490 instructors at CSULB.