Peraza 1

I assisted with experiments conducted under either Dr. Wozniak or a PhD

students supervision. The research conducted in the Wormley laboratory focused
on the human fungal pathogen Cryptococcus. Cryptococcus is a member of the
Basidiomycota phylum of the fungi, and is more closely related to mushrooms.
The Wormley laboratory uses Cryptococcus neoformans which is serotype
A (as used in older text) to study host-fungal interactions for the purpose of
developing vaccines to treat or prevent invasive fungal infections. However, it is
not limited to only Cryptococcus, other strains of Cryptococcus are also used.
C. neoformans, the causative agent of cryptococcosis, is an opportunistic
fungal pathogen that has the opportunity to cause respiratory tract infections in
severely immune compromised individuals such as AIDS patients. C. neoformans
infects about 5-30% of all AIDS patients worldwide with a mortality rate of 30%.
Cryptococcus is the fourth leading cause of death in AIDS patients in Africa.
These number are unacceptably high and a big reason why it is critical to find
vaccine and better treatment options.
The Wormley lab used a variety of immunological and molecular biology
techniques as well as mouse models to pursue this critical research. The lab has
developed a genetically modified strain of C. neoformans in order to study
protective immunity to cryptococcosis. This strain, designated H99, is
engineered to secrete the interferon- (INF- ). INF- is a cytokine in the pro-

Peraza 2

inflammatory pathway of Th1 cells. Th1 is responsible for the promotion of fungal
clearance and inflammation.
Mouse models infected with H99 demonstrate immunity when challenged
with infections of the highly virulent wild type strain H99. Ongoing projects in the
laboratory utilize this strain (in almost all experiments) to investigate protective
host immunity to cryptococcosis, identify immunodominant C. neoformans
proteins, and evaluate potential vaccine strategies utilizing genetically modified
strains of C. neoformans.
Cryptococcus neoformans comprises four main virulent factors. The
virulent factors are the effects of what makes something infections. The four
classical and prominent virulence factors of C. neoformans include the capsule
formation, melanin pigment production, thermos-tolerance, and intracellular
growth within macrophages.
The prominent antiphagocytic polysaccharide capsule, which is composed
of glucuronoxylomannan (GXM), is unique to Cryptococcus and is considered an
essential virulence factor that has multiple effects on host immunity and can
increase in size with exposure to body tissues and fluids. This was proven when a
mutant strain was produced by removing the gene that accounts for the
polysaccharide capsule. When the mutant was intranasally injected into mice, it
was detected by the immune system and cleared within a few days.

Peraza 3

Normally when a macrophage phagocytes a microbial, the microbial will be

encapsulated and combined with lysosome making a phagolysosome. In the
phagolysosome stage (which is very acidic), the microbial is digested and a piece
of the microbial is bound to a class II receptor on the macrophage surface. The
digested microbial is then spewed out and the piece of microbial bound to a class
II receptor which displays the piece of microbial called antigen presentation.
Antigen presentation induces an immune response, producing antibodies against
it.
However, in the case of Cryptococcus, it does not occur this way. This is
because the capsule contains a number of antiphagocytic proteins on its surface
which detoxifies O2/NO2. Once Cryptococcus is phagocytosed and in the
phagolysosome stage, Cryptococcus breaks down amino acids and used the NH3
group to neutralize the acid produced during digestion. This stops the
phagolysome process and allows the Cryptococcus to live inside the macrophage
cell.
Cryptococcuss polysaccharide capsule has inhibitory effect on
proliferation, survival, and cytokine production of human T-cell. This inhibition is
caused by the GXM ability to cause an increase in the secretion of IL-10. IL-10
activates T-regulatory which suppresses the immune response and Th-17 which
inhibits fungal clearance. This occurs by the alteration of the antigen- presenting
function of macrophages. IL-10 downregulates the expression of MHC class II

Peraza 4

antigen on macrophages, thereby decreasing the MHC complexes available for

interaction and proper stimulation of T cells. Allowing the fungal burden to grow.
Alveolar macrophages and leukocytes are critical during the first stages of
infection. They work directly as antifungals by phagocytosis and intracellular
killing and indirectly by secreting antimicrobial compounds that are proinflammatory mediators. However, the yeast has adapted sophisticated
mechanisms to escape the intracellular environment by modifying the
permeability of the phagosome membrane and via nonlytic, allowing cell-to-cell
transfer of yeast and its virulence factors without damage to the host
macrophages. Through this mechanism, Cryptococcus is able to remain inside
the macrophages and disseminate via bloodstream. Inside the macrophage it can
reproduce
Evidence indicated that Cryptococcus can than cross the blood-brain
barrier in a few different ways. The blood-brain barrier is a barrier that restricts the
passage of solutes and microbes from the capillaries of the central nervous
system into the brain. The way this is done is by direct uptake of the fungal cells
by epithelial cells and transmigration of the fungi through the cytoplasm to reach
the brain. Another theory is the Trojan Horse theory which involves the migration
of phagocytic cells such as macrophages that contain and therefore carry
Cryptococci. Once Cryptococcus has crossed the blood-brain barrier it can invade
the central nervous system causing life-threatening meningoencephalitis.

Peraza 5

Meningoencephalitis is the combination of infection and inflammation of the

meninges (membranes surrounding the central nervous system) and the brain.
The third virulent factor is C. neoformans ability to synthesis melanin. C.
neoformans possesses an enzyme called laccase. Laccase metabolizes a variety
of catechol. Catechol precursors such as L-dopa, dopamine, norepinephrine, and
epinephrine to melanin. When laccase is expressed it may have a biological role
to protect the yeasts from host oxidative stresses, immune defense, and
antifungal drugs.
Finally, the ability to grow at 37C. This is a basic part of the virulence
composite for most pathogenic fungi in humans including Cryptococcus. Without
this virulent factor, as shown with Dr. Wormleys calcenier strain, would not be
able to grow at mammalian body temperature and be cleared within a few days.
The calcenier strain of H99 was a strain that had the gene that accounted for
temperature tolerance. This strain was internasally given to BALB/c mice and
after a few days the mice were sacrificed and their lungs were processed and
then cultured. The culture lacked any growth of Cryptococcus, proving that it was
cleared and had die due to the lack of thermo-tolerance
In recent research in which I had the opportunity to assist in, there was an
experiments that was repeated (since my attendance began at the lab) about
three times to collect data and have it sent an analyzed by one of the postdoctoral students. The experiment began with about forty mice. Dr. Wozniak and

Peraza 6

a fourth year PhD student, intranasally infected the mice with H99, H99 , and
H99HK (heat kill)
I also had the opportunity to work alongside another undergrad and conduct
an experiment under the supervision of Dr. Wormley. The experiment was titled
Cryptococcus neoformans Genomic DNA Isolation. It was basically extracting
DNA from Cryptococcus.
The experiment was tedious and took about a whole work week because of
our contradicting schedules, as well as the times that the protocol required to do
certain things. However, it was a good refresher on our general chemistry and on
getting to know the machines they use in this building. We were asked to make
several stock solution with specific molarities. From the stock solution we then
diluted to obtain the desired molarity. Once the desired molarity was achieved, the
chemicals were added to finally make a buffer solution that was only used once
(10mL) through the whole experiment.
This experiment really tested our abilities to be as careful as we could.
However, we did still unintentionally have some spillages or touched something
with our tips, in which we had to get a new one.
The reason in why this experiment was important was because mice that
had previously been infected with doxycycline, a calcenier mutant, and a nondoxycycline strain. However, the non-doxycycline and the doxycycline mice both
had the same low level lung burden of Cryptococcus. This led the group to believe

Peraza 7

that Cryptococcus had pumped out the modified DNA once it realized it was a
mutant. Besides the calcenier mutant strain, we are also going to take a look at
the other strains. This is critical because as we know organisms become resistant
to certain things with time, if ever.
I also had the opportunity to sit in on the usage of human blood. Working
with human blood is a bit complex. The set up began the day before. The timing
of when the blood would arrive was of great importance because after cell
separation, the experiment would run for about six hours. Human blood cannot be
frozen, therefore if it arrived late then the experiment would begin late, but the
experiment would be conducted either way.
The experiment began by diluting the blood. Then they were separated in to
premade tubes that contained ficol. The ficol worked to separate the red blood
cells from the white blood cells by density. The red blood cell could almost
immediately be seen going to the bottom of the tube. The blood was the spun in a
centrifuge. After they were spun, you could see two distinct layer, one maroon
colored and one yellow colored. The yellow layer contained the white blood cells
that we needed, thus the bottom layer being the red blood cells was disposed of.
The white blood cells were then re-suspended, spun, and washed.
The white blood cells were then marked with CD45- and they process of
cell separation began. The white blood cells were separated according to the
maker. The desired white blood cells such as dendritic cells and macrophages

Peraza 8

remain in the magnetic column and the rest passed straight through. We were
interested in the dendritic cells and the macrophages. The rest were disposed of.
A premade strain of H99 was then pulled out. It was washed and spun, then resuspended. Shortly after, the killing assay began. In a 96 well plate, the
macrophages and dendritic cells were infected with H99 and placed in an
incubator set at 37C. This ran for a total of 6 hours.
An intern in research offers students a taste of the culture of research and
life as a scientist. Allowing students to participate in undergraduate research
engages their intellectual curiosity, satisfies their thirst for discovery, and gives
them an outlet for their creativity.
A lot of learning occurs when undergraduate students do research, learning
that does not happen during traditional coursework. Classroom knowledge is
reinforced and more completely assimilated when students are given the
opportunity to apply that knowledge.
Research always leads to a better understanding of and a deeper
appreciation for the discipline under investigation. Students' career goals are
usually clarified after they participate in research. How do you know you will enjoy
being a biologist without getting a chance to critically think, researching and
writing that a biologist does?
Research is also a significant confidence booster. The more students are
mentally stretched (wrestling with surprising results or unanswered questions or

Peraza 9

pertinence to previous studies), the greater their sense of accomplishment upon

completion of the project. This is especially true when a caring faculty member
guides and encourages the students.
Establishing a relationship with a faculty mentor is another big advantage of
undergraduate participation in research. Students benefit from the wisdom,
knowledge and experience of a mentor, while faculty members benefit from the
questions students ask, the discoveries they make and the energy they bring to
the project.
Scholarly activity also helps make undergraduates' rsums more attractive
to graduate schools and prospective employers, and gives faculty mentors the
ability to write more detailed letters of recommendation.
In other words, its a short-term, risk-free way to investigate whether a
research career or a particular field is a good fit.
The experience I gain in working in the laboratory gave me a different
outlook on where I want to head as far as my career choices. I have found a clear
path to what I would like to continue doing once I graduate. I have decided to
widen my prospective. I will be doing a master at UTSA in the fall on cellular and
molecular biology. However, I will still be pursuing a Doctoral degree in the near
future. At the end my main goal is to continue to help people in any way I can,
whether on the seen or behind the covers.