Download Mucins expression in intestinal epithelial cells infected with

Mucin expression in intestinal epithelial cells infected with Toxoplasma gondii
Joshua A. Mayoral
11/10/11
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Mucin glycoproteins, which make up mucus, are created and discharged as a barrier to
pathogenic organisms (Linden et al., 2008). Mucin up-regulation is a crucial and innate host
immune response in mammals, to a wide variety of pathogens that invade the respiratory,
reproductive, urinary, and particularly the intestinal tracts (Linden et al., 2008). Considering
how crucial the various functions of the mucus layer are in the host’s intestine (McGuckin et al.,
2011), understanding the conditions in which a parasite successfully infects the host intestine can
lead to important knowledge in developing new therapeutic remedies to such infections.
Currently, no studies have researched the consequences of an intestinal infection by the
protozoan parasite Toxoplasma gondii on epithelial cells. The purpose of this study is to fill these
research gaps, investigating whether a pathogenic protozoan, T. gondii, induces or reduces gene
expression of mucins in intestinal epithelial cells and to determine which pathways are involved
in mucin regulation during infection.
Mucins are glycoproteins that mainly compose the highly hydrated gel known as mucus
present in airways and the gastrointestinal cavity (Thornton and Sheehan, 2004). Three different
categories of mucins exist, which are: secreted gel forming, secreted non-gel forming, and cell
surface mucins (Linden et al., 2008). Gel-forming mucins are the most studied of the mucins,
and are the primary proteins that make up mucus in the airways and intestine (Linden et al.,
2008). In order for microbes to cause infection, they must adhere to the epithelial cells by
disrupting the mucin coating either through direct penetration or by toxin secretion (Linden et al.,
2008). However, mucin hypersecretion can be dangerous to host airways and lead to increased
mortality and morbidity (Thornton and Sheehan, 2004). Mucin hypersecretion is typically
pathogen induced, one example being the up-regulation of pro-inflammatory cytokines (such as
IL-13) and neutrophils, which eventually lead to acute asthma (Shim et al., 2001). Reactive
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oxidative species and oxidative stress are also causes of mucin hypersecretion in airways through
neutrophil pathways, indicating complex mechanisms in the production of mucins (Takeyama et
al., 2000; Shao and Nadel, 2005).
Toxoplasma gondii is an obligate intracellular protozoan parasite that is prevalent in
many humans and animals around the world. As of 2011, over six billion people have been
infected with T. gondii, with South America and Africa having the most seropositivity (Furtado
et al., 2011). Infection occurs in humans and animals when a contaminated substance containing
T. gondii cysts is ingested (Furtado et al., 2011). The prevalence of T. gondii worldwide
illustrates the necessity to further understand the role intestinal mucins play during infection with
this protozoan parasite. Additionally, by obtaining this information, other worldwide infections
involving parasite-mucin interactions can be studied using knowledge acquired through this
study.
A wide variety of parasitic infections are able to induce mucin expression, or penetrate
the mucus barrier, within hosts. One study demonstrated the ability of Naegleria fowleri
trophozoites to induce expression of the Muc5AC gene, among other cytokines and chemokines,
in a human airway epithelial cell line and in a mouse model (Cervantes-Sandoval et al., 2009).
Another study investigated the manner in which the protozoan parasite Entamoeba histolytica
cleaves through the mucus barrier (Lidell et al., 2006). E. histolytica was shown to release
cysteine proteases that degrade the peptide bonds of Muc2 in a human intestinal epithelial cell
line (Lidell et al., 2006). In vivo mucin-parasite interactions provide key insights to the
vulnerabilities and strengths of mucins composing the mucus layer. By observing strategies
employed by other protozoan parasites, predictions can be made as to how T. gondii penetrates
the mucosal barrier.
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Mucin deficiencies and Toll-like receptors (TLRs) play crucial roles in a host immune
response to protozoan parasites (Hasnain et al., 2010) (Moncada et al., 2011). The correlation of
TLR and mucin deficiencies to mucin expression and pathogenicity of a parasite may yield vital
knowledge in an in vivo model of T. gondii infection. A deficiency in Muc2 expression was
shown to lead to delayed worm expulsion of the nematode Trichurus muris in Muc2 knockout
mice when compared to wild-type mice (Hasnain et al., 2010). Toll-like receptors have been
described as a second line of defense to parasitic infections of the gastrointestinal epithelium
when compared to mucins (Moncada et al., 2011). TLRs activate signaling pathways that
produce pro-inflammatory cytokines and other innate responses, which may prove harmful if not
modulated due to their destructive inflammatory nature (Moncada et al., 2011). The antigens of
the intracellular parasite Gymnophalloides seoi were shown to stimulate mRNA expression of
TLR2, TLR4, as well as Muc2, Muc3, and Muc4 in a human epithelial cell line (Lee et al.,
2010). Currently, Muc1, Muc2, Muc4, Muc5B and Muc13 have been identified to be expressed
in mouse intestines, all of which are cell surface mucins with the exception of Muc2 and Muc5B,
which are secreted gel-forming mucins (Lindén et al., 2008). Understanding the pathways of
mucin activation and their relationship to immune responses through the afore-mentioned studies
help in providing likely outcomes of our specific parasite-mucin study when moved to an in vivo
study.
Little information is known concerning Toxoplasma gondii and mucin expression
compared to other protozoan parasites. However, knowledge obtained from mucin expression
during other protozoan parasitic infections has allowed us to make inferences concerning T.
gondii and mucin expression. It was hypothesized that Toxoplasma gondii infection in mouse
intestinal epithelial cells induces mucin expression. In this preliminary study, we expect to
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characterize mucin expression in a mouse intestinal epithelial cell line. If adequate results are
achieved indicating mucin expression by infection, a mouse model will be used to further
investigate mucin expression and function in vivo. It is known that T. gondii infection interferes
with host immune responses to facilitate its own survival (Peng et al., 2011); this is why downregulation of mucin gene expression by infection would not be unexpected. A second part of this
proposal waiting to be answered is the time-course of mucin expression in a mouse model of
infection. The relevance of this research applies mainly to the medical and biology field. By
obtaining a better understanding of how T. gondii infection regulates mucin expression, more
information can become available to researchers investigating other parasite-mucin interactions.
In addition, by finding the specific methods T. gondii penetrates the mucosal barrier, new
therapeutic methods can be developed for this prevalent infection worldwide.
Materials and Methods
To test the hypothesis, mouse intestinal epithelial (Mode-K) cells will be maintained in a
T-25 flask using DMEM cell media. The cells will be trypsinized to detach and remove the
majority of cells forming a monolayer on the bottom of the flask. This will be done once a week
while changing media to prevent overgrowth of cells in the flask. The cells will be counted using
a hemocytometer, and then transferred to six different petri dishes. 10,000 cells will be
transferred to each petri dish by diluting cells with DMEM media with an appropriate ratio of
media to cells.
Type II T. gondii trophozoites will be used to infect the cells in the petri dishes. It is
expected that the T. gondii trophozoites will induce or reduce mucin expression in the cells
between 3-6 and 12-24 hour timepoints. These two timepoints will be tested to measure mucin
expression in early (typically 3-6 hours) and late (typically 12-24 hours) infection with T. gondii.
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The trophozoites will be purified using PBS and a parasite filter, and then counted in a
hemocytometer. A three-to-one ratio of parasites to cells will be transferred to four of the six
petri dishes by means of dilution if necessary. The other two of the six petri dishes will not be
infected (control group).
All of the following procedures will be performed according to the RNA Extraction and
Quantification assay from the Monroy lab. RNA will be extracted and quantified from all petri
dishes to measure the genetic expression of the various mucin proteins in the cells. RNA will be
extracted from two of the four petri dishes infected with T. gondii between a 3-6 hour time-point.
RNA will then be extracted from the other two of the four petri dishes infected with T. gondii
between a 12-24 hour timepoint. RNA from the two uninfected petri dishes will be extracted
during the same timeframes as the other petri dishes. Thus, we will have two samples of RNA
from non-infected cells, two from 3-6 hour infected cells, and two from 12-24 hour infected cells
Before Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) will be performed
on the samples obtained from the petri dishes, PCR and gel electrophoresis will be performed on
previously extracted MODE-K cDNA using primers coding for MUC1, MUC2, MUC4,
MUC5B, and MUC13 genes to confirm primer function. Once primer function is confirmed, RTPCR will be performed to quantify MUC1, MUC2, MUC4, MUC5B, MUC13 genes of the
samples obtained from the petri dishes. After RT-PCR, agarose gel electrophoresis will be used
to compare the expression of each of the MUC genes.
T-testing will be used to measure the differences in gene expression between MUC1,
MUC2, MUC4, MUC5B, and MUC13 through gel electrophoresis results for each sample of
RNA obtained from the petri dishes. Additionally, analysis of variance will be used to compare
the values obtained from the two 12-24 hour, two 3-6 hour, and two non-infected RNA samples
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through gel electrophoresis results. If expression of mucin genes during T. gondii infection is
observed, then an in vivo study will be designed to measure mucin expression during infection in
a mouse model. In this study, we will test for correlation between parasite load and mucin
expression. In addition, we will observe TLR and cytokine expression in relation to mucin
expression during infection.
Anticipated Results
Differences in mucin gene expression between early, late, and no infection are expected
to be observed. However, the expression of all the mucin genes between the non-infected, 3-6
hour, and 12-24 hour infected cells is difficult to predict. Nonetheless, it is anticipated that all
mucin genes will be either up or down-regulated during infection when compared to cells with
no infection. As stated earlier, down-regulation may occur due to Toxoplasma gondii’s ability to
manipulate host immune responses (Peng et al., 2011). Additionally, it is expected that mucin
expression will be significantly different in the 12-24 hour infected cells when compared to the
3-6 hour infected cells due to a prolonged infection. Another possibility would be up-regulation
of MUC2 and MUC5B gene expression due to their gel-forming characterization, and downregulation of MUC1, MUC4, and MUC13 considering their cell-surface characterization. Gelforming mucins may be up-regulated during infection due to mucin secretion being the primary
immune response which leads to parasite expulsion (Hasnain et al., 2010). Cell-surface mucins
may be down-regulated due to immune response manipulation from T. gondii (Peng et al., 2011).
Whether up-regulation or down-regulation occurs, both results will be valuable in understanding
T. gondii’s strategy to host invasion and survival.
In obtaining these results, it is hoped that a better understanding of T. gondii and mucin
expression is achieved. Through this understanding, therapeutic methods can begin to be
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formulated by moving to an in vivo mouse model. Additionally, more information concerning
parasite-mucin interactions can be made available for other researchers.
Timeline
Future weeks
Procedures to perform
11/7-11/11
Count MODE-K cells and
transfer to petri dishes with
media. Infect cells in petri
dishes with T. gondii. Extract
RNA from all samples and
store for later use
11/14-11/18
Test primers to see if they
function through PCR and
gel electrophoresis
11/21-11/25
Perform RT-PCR on all the
samples to obtain cDNA
11/28-12/2
Perform gel electrophoresis
on samples
12/5-12/9
Analyze results from gel
electrophoresis
1/16-2/10
Design the in vivo study (if
adequate results are
obtained from the
preliminary study)
Perform the in vivo study
2/13-3/3
3/5-3/16
Analyze results from in vivo
study
3/19-4/6
Complete and report findings
from in vivo study
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