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