final paper for research with color
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Identification of a Rationally Designed LPS Molecule that Outcompetes
Naturally Occurring Pro-inflammatory Forms of LPS
Kaitlyn V. Smith1, Erin Harberts1,2, Kelsey Gregg2, Francesca Gardner2, and Robert K. Ernst2
1Biology Department, Loyola University2Department of Microbial Pathogenesis, University of Maryland School of Dentistry
AbstractLipopolysaccharide (LPS), a bacterial membrane component, signals through Toll-like
receptor 4 (TLR4) to cause the release of pro-inflammatory cytokines that result in septic shock.
LPS is present on all Gram-negative cell walls and its structure is variable, resulting in altered
binding to TLR4 receptors. To modify the LPS expressed, a technique called Bacterial Enzyme
Combinatorial Chemistry (BECC) can be used. Using BECC, LPS modifying enzymes from
mixed bacterial species are transferred into a recipient non-inflammatory Yersinia pestis strain
that ultimately alters the final LPS structure. This study investigates the induction of
inflammatory cytokines by specific BECC LPS molecules with an altered affinity for TLR4.
THP-1 Dual cells, human monocytic derived macrophage, were used to screen candidate
molecules for activation of a pro-inflammatory transcription factor, NFκB. These cells have the
gene for an alkaline phosphatase molecule, SEAP, under the NFκB promoter. Levels of SEAP in
cell culture supernatant can be easily quantified and used to calculate the EC50, a value defining
the concentration at which the agonist reaches 50% of maximum signaling. Cells were then co-
cultured with BECC LPS and pro-inflammatory LPS to investigate the possibility that septic
shock causing inflammatory signaling could be outcompeted. Using this screening method, LPS
from Y. pestis with the enzymes LpxF, which removes a phosphate, and PagP, which adds a fatty
acid, added was found to specifically compete with pro-inflammatory forms of LPS and increase
the EC50 400 fold. Levels of pro-inflammatory cytokines, IL-8, TNF-α and RANTES, and anti-
inflammatory IL-10 were measured by ELISA and shown to correlate with NFκB activation.
Ultimately this molecule may have vast impact on the treatment of septic shock and may lower
the mortality and burden of blood borne bacterial infections.
1 IntroductionOn average one million Americans are diagnosed with the condition of sepsis (Hall,
2011). However more concerning is that between 28-50% of these cases end up succumbing to
this condition (Wood, 2004). The NIH defines sepsis as full body inflammation accompanied by
fever, confusion, rapid heartbeat and breathing. Septic shock is not directly cause by a pathogen,
but instead arises from other medical conditions like infections or invasive surgery.
Research has shown that the main causative agent of sepsis is a molecule called
lipopolysaccharide (LPS) and is present on all Gram-negative cell walls and consists of three
main regions the o-antigen, core sugars, and lipid A. Lipid A anchors LPS into the cell
membrane and is the cause of the inflammation in septic shock. A study preformed in 2013
showed that the structure is not static, but varies vastly from species to species (Needham). It is
these variations that cause some LPS molecules to bind with a higher affinity to TLR4 receptors
and trigger pathways that release varying levels of proinflammatory cytokines. Releasing lower
levels of cytokines results in a lower inflammatory response, which is beneficial to humans.
Recent studies have shown that the interaction of lipopolysaccharide (LPS) and Toll- like
receptor 4 (TLR4) triggers the release of proinflammatory cytokines that directly affect the
body’s inflammatory response (Yong-Chen Lu, 2008). The signal transduction of TLR4
receptors is split into two separate pathways, the MyD88-Dependent and MyD88- Independent,
each resulting in different products (Yong-Chen Lu, 2008). Young-Chen Lu studies elaborates
on the dependent pathway describing the kinase pathway that eventually leads to the activation
of NF- κB transcription factor which up-regulates the transcription of proinflammatory cytokines
(2008).
Research on lipid A remolding preformed by Brittany Needham discusses the many ways
and possible reasons lipid A comes in such varying forms (2013). As stated above the structure
of LPS is not static and this is because of the lipid A region, which varies in structure due to the
wide range of lipid A modifying enzymes found across species (Needham, 2013). Though
another important thing to note is that these enzymes all work at specific temperatures and is
possibly an immune evasion tactic. Most LPS is produced in its host between 21-27O C and is
more inflammatory then the form that is produced at 37o C. This less inflammatory form at
human temperature probably helps the infection invade the host fully before the innate immune
system can be activated (Needham, 2013).
In most studies focused on cytokines released due to LPS a monocytic cell are cultured
for use and testing. THP-1 cells are commonly used because they come from varying sources
ranging in sex and ethnicities (Folkard, 2014). THP-1 cells express TLR4 receptors and allow
for LPS to signal through the NF- κB transcription factor. Activating NF- κB is useful in these
studies because its activation can be used as a marker to confirm that cytokines are being
produced and an estimate of at what concentration. A study conducted by Folkard looked at how
a diet high in vegetables is associated with a lower inflammatory response. They concluded it
was because of molecules in vegetables change the TLR4 receptors shape causing LPS to not be
able to bind as tightly, which alters the amount of cytokines that can be produced (2014). This
study does not focus on the fact that different lipid A structures also lead to the release of varying
amount of cytokines, but only on how modifying a molecules ability bind can help combat high
inflammatory responses.
While it seems that changing the receptors shape is an effective way to combat a high
inflammatory response it can be quite difficult to do. In recent studies, changing the structure of
the lipid A to make it a molecule that has a lower-efficacy has become the focus (Zhang, 2006).
The enzymes mentioned early that modify LPS structure do so by removing or adding acyl side
chains off of the two main carbon rings of the structure (Yong-Chen Lu, 2008). The studies
conducted by Zhang showed some proof that modifying lipid A can create molecules that have a
lower efficacy when signaling through a TLR4 receptor and can also change the type cytokines
produced (2006).
The two previous mentioned studied both have results that opened the doors for research
on combating naturally occurring LPS. Both strategies work well separately, but what if they
were combined. Folklard proposed a change in the TLR4 receptor, but could modifying the
structure of the lipid A to bind tighter receptor be more successful. Creating lipid A molecules,
using a process called bacterial enzymatic combinatorial chemistry(BECC) that have high
affinity for TLR4 receptors and low efficacy for releasing proinflammtory cytokines could be
the key. A molecule that could bind tigher to TLR4 receptors could out compete naturally
occurring LPS, while also releasing less cytokines would be medical applicable for those
suffering from sepsis. This study looked at the possibility to use BECC to create an LPS
molecule with high affinity for TLR4 and low efficacy of proinflammatory cytokine release,
which would be capable of outcompeting septic shock-causing LPS.
2 Methods
2.1 Cell Culture
The cells cultured for this study were THP-1 cells from Invivogen. They were
selected for the presence of a SEAP reporter under NF- κB, whose activity would help to
determine whether or not cytokines were being released. The cells also have a Luc reporter
under IRF. They were cultured in a tissue lab and maintained in a suspension solution of
complete RPMI including fetal bovine serum, as proteins, along with penicillin and
streptomycin. Every week the cells were split and every few passes of the cells they would be
given a target antibiotic to maintain their health. The cells were counted each time before they
were plated to check their viability and ensure there was enough to fill each well with at least
100 cells. Vitamin D was added to the plate solution to jumpstart the cells and get them to attach
in a single cell layer across the bottom of each well. Before molecules were added to the wells to
be tested the plates were observed under a microscope to ensure the cells were fully adherent and
evenly distributed in each well. This helped shrink the margin of error by making each testing
surface, the well, the same across the plates.
2.2 Creating BECC Molecules
Molecules for this study were created using a method created in the University of
Maryland Baltimore’s dental school called bacterial enzymatic combinatorial chemistry
(BECC). As stated earlier enzymes play a major role in the modification of LPS molecules
and are active in very specific temperatures. BECC is a method, which takes advantage of all
the varying species enzymes and their temperature specificity. Using Yersinia pestis as
blank canvass and enzymes were transfected in the form of plasmids into the cells and then
cultured at specific temperatures. The plasmids caused Y. pestis to express new and
modified forms of LPS with varying lipid A structures for each new combination.
The new molecules were given a number and the enzymes and temperature used to
create them was recorded. In order to use the lipid A for testing it was removed from the
membranes of the cells using a standard hot phenol extraction. The LPS was then dried
using a lyophilizer and stored. The structures for each molecule were drawn in order to
determine similar structures to be tested along side each other. All of the molecules were
put over THP-1 cells in a 5 serial log dilution starting at 1000 ng all the way down to .01 ng
two wells being treated with each dilution. The plates were developed using QUANTI blue
media, which reacts in response to the SEAP reporters under NF- κB transcription factors the
more active they are correlates to the amount of cytokines released.
2.3 Testing BECC Created Lipid A Against Naturally Occurring Forms For Competitive Inhibition
A
B
Figure 1. A) This is the diagram of the standard format of a 96 well plate of THP-1 cells when culturing them for the initial competitive inhibition analysis of each molecule. B) This is the diagram of the standard format of a 96 well plate of THP-1 cells when culturing them for further testing of a molecule, which showed competitive inhibition properties. This set-up will allow for analysis to determine if there is dose dependency of the competitive molecules.
Using the graphs created in section 2.2 all the molecules that showed potential were
selected for testing of their competitive inhibition. Serial dilutions of each molecule were
pipetted by hand under a tissue culture hood to ensure sterility. They were created in serial
dilutions across a five -log scale ranging from 1000 ng to .01 ng. A 96 well plates of cultured
THP-1 cells were used to test all the molecules production of cytokines. The first two columns
were full controls and each should be filled with 200μL of media. Starting in column 3 row A
was always run as a serial dilution of 100μL Wild Type Salmonella and 100μL of media. Rows
B and C were serial dilutions of two of the selected molecules to be tested and were 100μL of the
appropriate dilution and 100μL of media. Row D would then include 100μL of molecule one at
a locked concentration of 10 ng and the same serial dilution of WT Salmonella. Row E consists
of 100μL of a 10 ng of WT Salmonella locked and 100μL of the serial dilution of molecule one.
Row F and G are identical to Rows D and E except molecule one is replaced with molecule 2.
Row H is a full control each well filled with 200μL of media. Please see Figure lA for a picture
summary of this standard set up. They plates were incubated and developed using QUANTI-Blue
and data collected to be formatted into graphs.
Molecules that showed competitive inhibition against WT Salmonella were then tested
for dose dependency. The dilutions of each molecule were created the same way and were tested
over 96 well plates of THP-1 cells. Row A was cultured with 100μL of the serial dilution of
WT Salmonella and 100μL of media. Row B was cultured with 100μL of the molecule being
tested and 100μL of media. Row C-G each are treated with 100μL of serial dilution WT
Salmonella. Then each row was treated with 100μL of one of the locked concentrations of
molecule one. They concentrations for each row are as follows: C- .01 ng, D-1 ng, E-10 ng, F-
100 ng, and G- 1000 ng. Row H was cultured with 100μL of 100 ng of WT Salmonella 100μL
of serial dilution of molecule one. The setup for these plates is summarized in Figure 1B Plates
were analyzed in the same way mentioned above.
When a successful competitive inhibitor was identified its structure was analyzed and
used to determine molecules with similar structures to be tested using the same methods
mentioned above.
3 Results
Molecules were created using BECC, the process mentioned in the methods sections.
This graph is one example of many used to select molecules for further testing (Figure 2). Figure
2 was analyzed and 470@26 provided satisfactory evidence that it was both a molecule that had
high affinity and low efficacy. It was selected for further testing of whether or not it could be a
competitive inhibitor of naturally occurring LPS. Three other molecules were selected for first
round testing: 438 @ 37, TBE-47 @ 26, and TBE-47 @ 37.
Molecules TBE-47 @ 26 and TBE-47 @ 37 were cultured on the same plate in the
standard format (Figure 1A). After being developed and analyzed there was no visible and
statistical evidence that either molecule was a competitive inhibitor of the WT Salmonella. It
was reasoned that this meant it was probably not able to outcompete many natural forms of LPS
and it was decided there would be no further testing on these structures.
Then molecules 470 @26 and 438 @ 37 were cultured on the same plate in the
standard format (Figure 1A). The plate of both molecules was developed. After
development 438 @ 37 showed no visible or statistical evidence that it was a competitive
inhibitor of natural occurring LPS and was excluded for further test. 470 @ 26 upon
analysis showed that when a serial of dilution of WT Salmonella was treated with 10 ng of
470@26 had an EC50 that shifted almost 200 fold (Figure 3A ). There was also visible
Figure 2. Serial Dilutions of LPS BECC Molecules Compared to Wild Type Salmonella. This graph was created in PRISM from data read from a plate of THP-1 cells treated with the LPS molecules with -1 representing the .01 ng concentration and 3 the 1000 ng. The point of inflection of each line is representative of the 50% effective concentration (EC50) and the height of the line against the Y-axis shows the molecules efficacy.
evidence of this when the plate was developed (Figure 3B). This was strong evidence that
this BECC molecule is competitive inhibitor of WT Salmonella and possibly may other forms
of naturally occurring LPS. With this strong evidence it was determined that 470 @ 26
would be tested for dose dependency .
A
B
A
B
Molecule 470 @ 26 was
determined to be a competitive
inhibitor of WT Salmonella. A
plate was then cultured in the
standard dose dependent format
(Figure 1B). Once developed and
analyzed using PRISM there was both strong visible and statistical evidence that 470 @ 26 was
dose dependent in its inhibition. The wells treated with the same concentration of WT
Figure 4 A. This is the developed plate that was set up in the dose dependency format (Figure 1B) using molecule 470 @26 as the ‘BECC Molecule’. It provides strong visible evidence that 470 is a competitive inhibitor and works in a dose dependent manner. B. This graph is reflective of the wells shown in image A. Created in PRISM and upon analysis it can be seen that 470 successfully outcompetes WT Salmonella in concentrations of 10, 100, 1000 ng/mL. The scale of the X-Axis is a log scale -1 correlates to a concentration of .01 ng and 3 1000 ng/mL. The Y-Axis correlates to the efficacy of each molecule or combination.
Salmonella as 470 @ 26 show a decrease in color when compared to the well of just WT
Salmonella at the same concentration this provides us with strong evidence that 470 @26 is
outcompeting WT for the receptors along with releasing a less inflammatory response. This is
further supported when looking at the data in graph format (Figure 4B).
Looking at the graph it is evident that at concentrations of 100 and 1000 ng/mL are strongly dose
dependent and competitively inhibit WT Salmonella, while producing less cytokines causing a
lower inflammatory response (Figure 4B). This evidence made 470 @ 26 on a list for further
testing. The structure of 470 @ 26 was also analyzed and similar structures were identified to be
the next tested.
Structurally similar plate
References:
Margaret Jean Hall, Ph.D.; Sonja N. Williams, M.P.H.; Carol J. DeFrances, Ph.D.; and Aleksandr Golosinskiy, M.S.National Center for Health Statistics Data Brief No. 62 June 2011. Inpatient care for septicemia or sepsis: a challenge for patients and hospitals
Wood KA, Angus DC. Pharmacoeconomic implications of new therapies in sepsis. PharmacoEconomics. 2004;22(14):895-906.http://www.nigms.nih.gov/education/pages/factsheet_sePSIs.aspx