Download Gene expression analysis to evaluate the effect of p38 specific

Gene expression analysis to evaluate the effect of p38 specific inhibitor SB203580 on
SEB-induced apoptosis related pathways
By: Lisa Hesse, Advisor—Dr. Chanaka Mendis
Abstract
Staphylococcal enterotoxin B (SEB) is a bacterial toxin that has been thoroughly
investigated. However, little is known about the cascades of signaling events that explain
its patho-mechanism. This research involves the pathogenic nature of SEB, which can
cause death in human peripheral blood mononuclear cells (PBMC) by inducing multiple
signal transduction pathways. Inhibiting crucial pathway inter-connector (p38) may alter
unwanted, SEB-induced, cellular activities. This study focuses on obstructing signaling
pathways using inhibitor SB203580 and analyzing alterations to known expression
patterns of genes associated with apoptosis [the Caspase genes and Heparanse precursor
(HEP)]. As our experimental design specifically targets p38, we believe our target is
unique and has the ability to sustain longer lasting inhibitory effects. We are also
confident that inhibiting such a component will have a maximum effect on a cell module,
terminating the “leaking effect,” which may benefit other experimental modules focused
on disease prevention and rapid diagnostics.
Introduction
Staphylococcal enterotoxin B (SEB) is a toxin secreted by the bacterium Staphylococcus
aureus and causes a person to have symptoms of food poisoning. These flu-like symptoms, if
overlooked for a prolonged period of time, can cause a lot of harm to a person, and possibly lead
to death. S. aureus is found in improperly refrigerated meats and dairy products and will survive
unless food is cooked thoroughly. Once in the body, the bacterium releases the SEB toxin,
triggering a multitude of cell signaling pathways leading up to the flu-like symptoms associated
with food poisoning. The genes activated in these cell pathways have been sequenced and are
well known; however, a way to inhibit the activation of such genes has yet to be perfected. It is
known that the structure of SEB contains two units of the amino acid cysteine in the middle of
the whole protein forming a disulphide bridge when the toxin is mature with an intervening
variable loop (Jett 1092). SEB is different form the other staphylococcal enterotoxins (SEs) such
as SEA, C1, C2, C3, D, E, G, H, I, and J due to its affinity for different cell types as well as for
the binding sites of the class II major histocompatibility complex (MHC II) and T-cell receptors
(Jett 1093). It has been found that multiple conserved residues on the smaller domain structure
of the toxin as well as in the N- and C-terminal regions allow SEB to bind to the β subunit of
MHC II (Jett 1093). The binding of SEB to MHC II activates polyclonal T lymphocytes, the cells
of the immune system that are responsible for killing viral infected cells (Blazes).
In an attempt to determine the exact pathways SEB induces within a cell, the various
gene expressions of genes known to be associated with apoptosis will be analyzed and quantified
when compared to the expression of a housekeeping gene. This project focuses in on the p38
inter connector, which is known to have an influence on the apoptosis pathway induced by the
SEB toxin. By introducing an inhibitor to this interconnector, SB203580, the effect of blocking
the pathway may allow for a complete specific gene pathway to be determined. Once the
pathway is mapped, the information has the potential to provide medicinal breakthroughs in the
fields of cell death and aging.
Methods
QUANTIFICATION OF RNA AND PROTEIN SAMPLES
RNA and protein samples were isolated from three types of human PBMCs (Peripheral Blood
Mononuclear Cells): 1) control (not treated with anything at 2 and 6 hours), 2) toxin exposed
cells with inhibitor for 2 and 6 hrs, and 3) toxin exposed cells without the inhibitor at 2 and 6
hours (WRAIR, Silver Spring, MD). These samples were then quantitated using a UV-VIS
spectrometer (PerkinElmer Lambda 900 UV/VIS/NIR Spectrometer).
REVERSE TRANSCRIPTION (RT-PCR)
RT was done using an iScript™ cDNA Synthesis Kit (BIO-RAD) in a Thermocycler (Eppendorf
Mastercyler Personal).
POLYMERASE CHAIN REACTION (PCR)
PCR was carried out using primers of genes of interest designed through various kinds of primerdesign software in a Thermocycler (Eppendorf Mastercycler Personal) using a PCR master mix
kit (Roche Diagnostics, Indianapolis, IN)
VISUALIZATION & QUANTIFICATION
All PCR samples were analyzed on 1% agarose gels using syber green for fluorescence and were
visualized through an in house imaging apparatus and quantitated by Image J analysis.
ELISA
All samples were either run using the Caspase-3, Caspase-8, or Caspase-10 Colorimetric Assay
Kits from BioVision (Mountain View, CA). The type of instrument used was a Microplate
Autoreader EL311 Bio-Tek Instrument.
Results and Discussion
Previous research has discovered that SOD genes regulate caspase-1 activation through
reversible oxidation and glutathionylation of cysteine residues (Meissner). It has also been
discovered that USP has been shown to play a large part in caspase regulation and cell survival
through multiple ubiquitin pathway proteins and their effect on targeted degradation via the
ubiquitin-proteasome pathway, specifically with its effect on the gene IAP (Steller). With this
knowledge, a set of genes was selected in order to attempt to piece a signaling pathway together.
From the results obtained thus far, Caspase 1, 6, 7, 9, 10, HEP, SOD, USP, STK17A, and
REQ were all up-regulated at both the 2 and 6 hour exposures by SEB (Table 1). The results also
showed that the p38 inhibitor SB203580 was able to alter the expression of all SEB induced
apoptosis related genes (Table 1). From these results, it is clear that SEB affects the intrinsic
apoptosis pathway. The intrinsic apoptosis pathway consists of the induction of Caspase 9
followed by the induction of Caspase 3, 6, and 7. On the other hand, the extrinsic apoptosis
pathway consists of the induction of Caspase 8 followed by the induction of Caspase 10, 3, 6,
and 7. Due to the values of 16.9 ± 2.0 (SEB 2 hr) and 10.4 ± 0.4 (SEB 6 hr) it can be said that
SEB affects the apoptosis pathway due to the up-regulation of Caspase 9.
In conjunction with the testing of RNA samples with the genes of interest, protein testing
has begun with Caspase 3 and Caspase 8 (Table 2). As done with the RNA data, calculations are
performed to normalize the absorbance readings and from this data no conclusive results as to
the apoptosis pathway affected can be obtained due to the fact that the protein of Caspase 9 has
not been tested yet.
Gene
Time Counts 2 hr
Time Counts 6 hr
24 hr p38 (inhibitor)
Caspase 1
8.8 ± 0.1
6.2 ± 0.1
1.5 ± 0.4
Caspase 6
4.0 ± 0.4
6.3 ± 0.3
0.9 ± 0.5
Caspase 7
5.8 ± 0.4
5.5 ± 0.2
1.4 ± 0.6
Caspase 9
16.9 ± 2.0
10.4 ± 0.4
4.5 ± 3.3
Caspase 10
6.8 ± 0.4
8.5 ± 0.3
1.5 ± 0.4
HEP
11.2 ± 0.0
11.5 ± 0.3
2.1 ± 0.1
SOD
7.0 ± 0.0
7.6 ± 0.3
1.5 ± 0.1
USP
8.1 ± 0.2
6.8 ± 0.0
1.6 ± 0.3
STK17A
13.0 ± 0.1
13.0 ± 0.1
3.2 ± 0.2
REQ
4.8 ± 0.0
8.1 ± 0.0
1.8 ± 0.2
Table 1: These calculated regulation values were obtained using the absorbance value of the gene of interest and the housekeeping gene.
Gene
Caspase 3
Caspase 3
Caspase 3
Caspase 3
Caspase 3
Caspase 3
Caspase 3
Caspase 8
Caspase 8
Caspase 8
Caspase 8
Caspase 8
Caspase 8
Caspase 8
Sample
SEB-SP 2H A
5-LPS SP A
SEB 12 H B
5-LPS PD B
LPS 2H B
SEB-SB 2H A
LPS 2-C A
SEB-SP 2H A
5-LPS SP A
SEB 12 H B
5-LPS PD B
LPS 2H B
SEB-SB 2H A
LPS 2-C A
Calculated Values: #/C
Absorbance at 405 nm
1.679
0.047
1.393
0.039
1.821
0.051
0.893
0.025
1.893
0.053
2.250
0.063
1.000
0.028
2.706
0.046
1.529
0.026
1.941
0.033
1.176
0.020
1.765
0.030
1.588
0.027
1.000
0.017
Table 2: Elisa absorbance readings which were prepared using Caspase 3 and 8 kits.
Conclusion and Future Plans
While there are still many genes to be tested in potential apoptosis pathways of human
peripheral blood mononuclear cells, this project yielded promising results. Since a lot of the
genes tested have been effectively down-regulated by the p38 pathway inhibitor SB203580, the
potential for mapping out the apoptotic pathway of the SEB toxin is growing with each
additional gene studied. Currently, reproducible results are in the process of being obtained, so
that observations can be confirmed, along with other genes that are associated with apoptosis:
GRIM 19, NGFRAP1, MAPKAPK5, PDCD4, MIHC, Caspase 3, and Caspase 8. As the data
pool grows, the apoptotic pathway can be traced and confirmed, thus being a vital study to the
hopes of inhibiting cell death in affected cells.
References
Blazes, David L., Lawler, James V., Lazarus, & Angeline A. “When Biotoxins are Tools of
Terror”. Postgraduate Medicine, Vol. 112:2. Section Symposium: Third of Three Articles
on Bioterrorism
Jett, Marti, Ionin, B., Das, R., & Neill, R. “The Staphylococcal Enterotoxins”. Walter Reed
Army Institute of Research, Silver Spring, MD, USA. 2001
Meissner, Felix, K. Molawi, A Zychlinsky. “Superoxide dismutase 1 regulates caspase-1 and
endotoxic shock” Nature Immunology, Vol. 9:8 pp: 866-872. August 2008.
Mendis, Chanaka, Campbell, K., Das, R., Yang D., & Jett, M. “Effect of 5-Lipoxygenase
inhibitor MK591 on early molecular and signaling events induced by staphylococcal
enterotoxin B (SEB) in human peripheral blood mononuclear cells (PBMCs)” FEBS
Journal, May 2008
Steller, H. “Regulation of apoptosis in Drosphilia” Cell Death and Differentiation, (15) 11321138. 2008