Download The mechanism of Stx2 enrichment in outer membrane vesicles of

BAHL, Christopher D.
The mechanism of Stx1 incorporation in outer membrane vesicles of Escherichia coli
O157:H7 and its role in bacterial predation
Abstract
Gram negative bacteria secrete vesicles that are formed when a portion of the outer membrane
“blebs off” [1]. The lumen of these outer membrane vesicles (OMVs) contains a small portion
of the periplasm. However, it appears that the quantity of most proteins found within OMVs
does not reflect their periplasmic abundances [3]. This suggests the existence of a novel
mechanism for the selection, concentration, and packaging of proteins for secretion inside
OMVs. In the case of pathogenic bacteria, toxins are often secreted in association with OMVs,
which can aid in delivery to other cells [4]. Infection by the pathogenic Escherichia coli strain
O157:H7 is an international health concern, and the production of Shiga-like toxin (Stx) by these
bacteria is a major contributor to its pathogenicity [5]. Stx1 produced by E. coli O157:H7 has
been shown to localize within OMVs [6]. Very little is known about how OMVs are formed [7]
or how the protein cargo they carry is loaded into them. In all cases that have been investigated,
protein toxins that are packaged into OMVs are enriched [8-11]. Stx1 has yet to be directly
shown to be enriched in OMVs. We propose that Stx1 is enriched in OMVs, and that this is
achieved by associating with an as of yet unidentified outer membrane protein (OMP) while in
the periplasm. This protein sequesters it for packaging and leaves the cell with Stx1 in the
OMV. We will identify any proteins that are able to bind Stx1 within purified OMVs. The goal
is to identify the mechanism by which virulence factors that are packaged into OMVs are
selected, using Stx1 produced by E. coli O157:H7 as a model for exploration. In addition to
pathogenesis, OMVs are effective vehicles for delivery of antimicrobial factors used in bacterial
predation [12]. Since it has been shown that Stx1 can enter mammalian cells without the aid of
OMVs [13], we propose that Stx1 may not be packaged into OMVs for delivery to mammalian
tissue. Instead, Stx1 is packaged into OMVs to for delivery to bacteria that may be competing
for nutrients, and again will be used block protein synthesis [14]. We will test this hypothesis by
treating selected bacteria species with Stx1 containing OMVs. In doing so, we are seeking to
elucidate an alternate role for Stx1 that does not involve direct pathogenic effects to mammals.
Specific Aim 1: Test the hypothesis that Shiga-like toxin 1 packaging into OMVs is both a
selective process and is mediated by protein factor(s).
We will purify OMVs and periplasm from E. coli O157:H7 and characterize the quantity of Stx1
contained within them by Western blot. To identify protein components that provide packaging
specificity for Stx1, we will first identify proteins that interact with Stx1 inside OMVs by mass
spectrometry. Next, we will use gene deletions to test the role of these proteins in packaging
Stx1 into OMVs.
Specific Aim 2: Test the hypothesis that Stx1 is able to impact the growth of other bacteria.
We will assay the effect of Stx1 as a secreted toxin on 5 different bacteria by monitoring their
ability to replicate when treated with Stx1 containing OMVs.
Background and Significance
First identified as a pathogen in 1982 [15], Shiga-like toxin (Stx)-producing E. coli (STEC) is a
commonly encountered food and water borne pathogen in many underdeveloped nations [16].
The majority of human E. coli infections are caused by Stx-producing enterohaemorrhagic E.
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coli (EHEC), resulting in 5 to 7 days of bloody diarrhea, vomiting, and severe cramping before
recovery. About 15% of the time the disease progresses into life-threatening hemolytic uremic
syndrome (HUS) [17], which children and the elderly are particularly susceptible to develop
[18]. Stx production is thought to be strongly linked to STEC pathogenesis and HUS [19, 20].
Treatment of STEC infection with antibiotics increases Stx production in mice [21], and causes
more severe symptoms in humans [22] as well as increasing the risk of developing HUS [16].
The Centers for Disease Control and Prevention (CDC) estimates that roughly 70,000 STEC
infections occur every year in the United States. STEC also represents a threat to national
security as military personal deployed to third world countries are likely to encounter this
pathogen in the field from local food and water sources.
Outer membrane vesicles contain virulence factors and can be “predatory.” Many
pathogenic gram-negative bacteria secrete toxins in association with vesicles derived from the
outer membrane [4]. Among these are: Vibrio cholerae [23], Neisseria meningitidis [24],
Salmonella enterica [25], Pseudomonas aeruginosa [26, 27], and Burkholderia cepacia [8].
From E. coli alone, Stx 1 and 2 [6, 28], cytolysin A (ClyA) [9], heat-labile enterotoxin (LT) [29],
α-haemolysin [10], and lipopolysaccharide (LPS) [29, 30] have been shown to be released in
association with outer membrane vesicles (OMVs). Inclusion into OMVs has been shown to
increase toxin potency [9], enhance delivery [8, 9, 28-32], and protect from degradation [6, 8, 10,
29, 30, 33]. In addition, sequestration inside of OMVs can hide the toxins from antibodies which
can bind and inactivate them. Virulence factors are enriched in OMVs in all cases it was tested
for [8-11]. This evidence suggests that sorting and loading machinery exists that can recognize
specific proteins and sequester them for packaging into OMVs.
In addition to pathogenesis, OMVs can also be used to interact with other bacteria [34]. Among
the many interactions mediated by OMVs are: horizontal gene transfer [33, 35, 36], signaling
[37], and group antibiotic resistance [38]. However, not all of the possible interactions are
beneficial to the microbe receiving the OMVs. “Predatory OMVs” are used to inhibit growth or
even lyse other bacteria [12] and are effective against both gram-negatives and gram-positives
[1]. When in a polymicrobial environment, any advantage in the competition for limited
nutrients increases the fitness of that microbe. One way to obtain such an advantage is by killing
off the competition. When treated with an antibiotic, P. aeruginosa will package it into OMVs
for secretion and removal from the cell [39]. These OMVs are highly effective at delivering
antibiotic to other species of microbes [40] as well as mammalian cells [41]. P. aeruginosa [42]
and E. coli [3] both secrete murein hydrolases inside OMVs which can break down the
peptidoglycan layer of both gram-positive and gram-negative bacteria. In one study, OMVs
isolated from 15 different gram negative-bacteria were capable of lysing a variety of both gramnegative and gram-positive bacteria [43].
Vesicles derived from the outer membrane. The gram-negative class of bacteria is
characterized by the presence of 2 membranes surrounding the cell; an inner membrane and an
outer membrane (OM). Between these membranes is the periplasmic space [44]. Within the
periplasm is the murein sacculus [45], which is composed primarily of covalently linked
peptidoglycan. Each membrane is anchored to the murein sacculus by peptidoglycan binding
proteins. Every gram-negative microbe that has been tested secretes vesicles derived from
sections of the outer membrane that “bleb off,” capturing some of the periplasm in the process
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[46]. These vesicles maintain their membrane orientation, displaying a subset of the same
surface antigens as the bacteria it came from. OMVs have been found in every isolate of gramnegative bacteria, whether from the environment or a laboratory [47]. This has led to the
hypothesis that their production may be essential to life as a gram-negative bacterium. While
mutations have been found that reduce OMV production, no mutation has ever blocked all
production [48]. In addition, OMV production has been observed in some gram-positive bacteria
[49], mitochondria [50], and even by mammalian tissue (termed microvesicles) [51]. While
there is currently no known mechanism of OMV formation, there is evidence that they can form
spontaneously from regions of the OM that are not tethered to the cell wall [34]. Often contained
within OMVs are proteins involved in breakdown and synthesis of the murein sacculus, which
could be involved in removing OM attachment to the cell wall at the site of OMV formation.
While the direct involvement of any specific factor in the formation of OMVs has not been
observed, there are a number of systems that can impact their formation. The quorum signaling
molecule P. aeruginosa quinolone signal (PQS) can stimulate OMV formation in both P.
aeruginosa and E. coli [52], and is itself secreted in OMVs [37]. In E. coli, the σE envelope
stress response pathway can influence OMV production rates when triggered by the presence of
toxic unfolded protein in the periplasm [53]. In E. coli, P. aeruginosa, Shigella flexneri, and S.
enterica [54], destabilization or other stress to the OM has been shown to increase the production
rate of OMVs.
OMVs are dynamic structures. OMVs
produced by bacteria vary in size, density,
protein and lipid content, and production rate.
Since the production of OMVs is a way of
interacting with the extracellular milieu, this
variability allows a bacterium to respond to
changes in its environment. In Neisseria
gonorrhoeae, 2 populations of OMVs have
been characterized by density, with each of
these containing distinct profiles of DNA
binding proteins [55]. Some proteins have
been demonstrated to be enriched in OMVs
compared to their periplasmic abundance [811]. Recently, it has been shown that the
quantity of specific proteins that are packaged
into OMVs can change based on external
stimuli [56]. This has led to the hypothesis that
packaging proteins into OMVs for secretion is a
selective process [12] [29].
Unknown OMP,
sequesters Stx1
OMV
extracellular
milieu
OmpF
GFP
Tat
Stx1
MBP
periplasm
cytoplasm
Figure 1. Proposed mechanism of Stx1 packaging into
OMVs. Stx1 is produced in the cytoplasm and exported
to the periplasm by the TAT. Stx1 then interacts with
an unknown OMP, which can sequester it to the site of
OMV formation. This will allow Stx1 concentrations to
increase relative to other proteins, such as GFP and
MPB, that are incorporated due to random diffusion
throughout the periplasm. The OmpF is present in the
OM and is used as a marker for OMVs.
Stx1 produced by STEC. Although all protein
toxins secreted in OMVs are likely to be
enriched, Stx1 has a number of advantages that make it a prime candidate to use as a tool to
dissect packaging selectivity. Unlike some of the other toxins, Stx1 does not integrate into
membranes and has a stable fold. The crystal structure of Stx from Shigella dysenteriae, which
is 98% homologous to Stx1 [5], has been solved [2] and will aid in experimental design. Stx1 is
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produced and folded in the cytoplasm before being exported to the periplasm by the twin
arginine translocator (Tat) [57]. From here it is able to freely diffuse through the periplasm
before export from the cell in soluble form or packaged into OMVs. We propose the Stx1 that is
packaged into OMVs first associates with an unknown factor attached to the OM. This is likely
an OMP, and causes it to be packaged inside the OMV by sequestering it to the site of OMV
formation (Figure 1). This will allow the concentration of Stx1 in the OMV to reach higher
concentrations than proteins which are captured due to random diffusion through the periplasm,
such as maltose binding protein (MBP) or artificially introduced green fluorescent protein (GFP)
[58].
The toxin consists of 5 B subunits that form a
doughnut like ring, with 1 A subunit bound to
one face of the ring with a tail that pokes
though the hole to the other side (Figure 2).
The A subunit contains the catalytic domain,
which is an N-glycosidase. It is able to
A Subunit
90°
B Subunit
Pentamer
depurinate a specific adenine base of the
Figure 2. Structure of the Stx holotoxin determined
by x-ray crystallography displayed as a surface
eukaryotic 28S ribosomal subunit, which in turn
calculation [2]. The A subunit is shown in green and
prevents elongation during translation [59].
each B subunit in the pentameric ring is a different
Prokaryotic ribosomes are sensitive to Stx1
color. The A subunit can be seen protruding from the
activity as well [14], and the toxin is synthesized
B subunit ring on both sides.
as a propeptide to overcome this. The A subunit
is proteolytically cleaved inside a target cell or in the extracellular environment [60, 61],
producing a 27.5 kDa and a 3 kDa fragment which remain covalently linked by a disulfide bond
[62]. Reduction of this bond releases the 4.5 kDa fragment, resulting in full activation of the Nglycosidase activity of the 27.5 kDa fragment. E. coli is susceptible to this activity, as
expression of the activated A subunit is lethal [14].
The B subunit pentamer can bind the sugar component of the glycolipid globotriaosyl ceramide
(Gb3) in the membranes of mammalian intestinal epithelial cells, mediating entry through
clathrin coated pits [63]. This process occurs readily in mammalian cell culture treated with
purified Stx1 [5]. Additional studies have shown that the effect of purified Stx injected into the
digestive tract of animals is directly correlated with the expression of Gb3 by epithelial cells.
Since soluble Stx1 is able to enter mammalian cells, this brings into question the role for Stx1
packaging into OMVs. While OMV packaging may be involved in targeting Stx1 to cells that
lack Gb3, no studies have verified this to date. It is possible that OMV packaging may not play a
significant role in Stx1 delivery to mammalian tissue. Out of the many E. coli strains that
produce Stx, only a few are significant human pathogens [20]. Since Stx also functions on
prokaryotic ribosomes [14], we propose the hypothesis that Stx1 is packaged into OMVs for
delivery to other bacteria. While there is no direct evidence showing that Stx1 can be processed
to the active form by prokaryotic proteases, bacteria do possess proteases capable of cleaving the
sequence of Stx1 at the site necessary for activation [64]. Some proteases, such as OmpT [65]
and OmpP [66], are even packaged and secreted in OMVs [3]. However, even if Stx1 is not able
to be processed to the active form by bacterial proteases, the inactive form is still a functioning
toxin that is capable of depurinating ribosomes [67]. Inhibition of protein synthesis in
neighboring bacteria would increase STEC fitness in a polymicrobial environment. Many
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virulence factors produced by non-obligate pathogens have a function for survival in the
environment. Since the majority of the time these microbes are living without a mammalian
host, it is likely that Stx1 has a function not involved with mammalian pathogenesis.
Specific Aim 1: Test the hypothesis that Shiga-like toxin 1 packaging into OMVs is both a
selective process and is mediated by protein factor(s).
In this aim, we will purify OMVs and periplasm from E. coli O157:H7 and use Western blotting
to confirm that Stx1 is present at a higher concentration in the OMVs than in the periplasm. To
begin the identification of proteins that interact with Stx1 and are present in the OMVs, we will
use velocity density centrifugation to separate purified OMVs into fractions and probe for Stx1
in each by western blot. If Stx1 is found to localize to a specific fraction(s), we will use 2D gel
electrophoresis to profile the proteins present in both Stx1 containing and Stx1 lacking fractions.
Proteins that localize specifically with Stx1 will be identified by mass spectrometry. If Stx1 is
present in all fractions after centrifugation, we will immunoprecipitate purified OMVs with antiStx1 antibody and identify protein interactors by mass spectrometry. All proteins identified by
either method will be deleted using the Lambda Red system [68]. OMVs will be purified from
these mutant strains and the quantity of Stx1 within them assayed as before.
OMV purification and Stx1 measurement. If Stx1 is packaged into OMVs by being randomly
captured as it freely diffuses through the periplasm, then the concentration contained within
OMVs should equal its concentration in the periplasm. If Stx1 is present at a higher
concentration inside of OMVs than it is in the periplasm, it is said to be enriched. If Stx1 is
enriched, a selective mechanism likely exists that can sequester it during OMV formation. While
Stx1 enrichment in OMVs has not been directly shown, there is evidence to suggest it. In one
study, 22 out of 24 STEC clinical isolates clearly showed higher levels of Stx1 associated with
OMVs than was secreted when grown aerobically [6]. Additionally, the ratio of OMV associated
to soluble Stx1 was not constant between strains, indicating differential packaging between
strains. Stx1 enrichment will be verified by comparing periplasmic abundance to the OMV
associated population. We will purify both OMVs and periplasm using the methods from
Horstman and Kuehn [29] and Kesty and Kuehn [28] respectively. Stx1 is secreted as soluble
protein as well as packaged inside OMVs. The soluble Stx1 will be removed during purification
and will not skew measurement of the OMV contained population. Following purification, a
portion of the OMVs will be spread onto a lysogeny broth (LB) [69] agar plate and incubated at
37°C to ensure the solution is sterile. For all experiments in this aim, we will be using E. coli
O157:H7 grown in LB with 10 μg/mL kanamycin at 37°C unless otherwise noted. Although in
some cases exposure to antibiotic has had effects on OMV production [39], 10 μg/mL kanamycin
has been used in the past without effect [28].
We will utilize Western blotting to measure protein levels. Due to inherent variability in the
quantity of OMVs and periplasm that will be purified, we will not express values of Stx1 as a
raw value obtained directly from the Western blot. Instead, we will normalize to other proteins
and express the quantity of Stx1 as compared to their abundances. Previous studies have
quantified OMVs by measuring levels of either OmpA [9, 10], OmpF [3, 28, 29], or MBP [28].
Although under previously described circumstances they appear to remain at a constant level in
OMVs, we must account for the possibility that our intended perturbation of Stx1 packaging will
affect other proteins as well. For this reason, we will monitor the levels of all three. In addition,
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we will express GFP in the periplasm [58] and monitor its level as well. Since GFP is not
natively produced by E. coli, it is less likely interact with potential packaging machinery than an
endogenous protein. MBP and GFP will be used as periplasmic markers, and both are also
packaged into OMVs [28]. MBP is a native E. coli protein [70], and GFP can be stably
expressed to low concentrations from a plasmid [28]. These periplasmic markers will be present
in OMVs at roughly the same concentration as they are in the periplasm. By setting GFP and
MBP as the baseline and measuring Stx1 quantities in reference to their amounts, we will
compare the amount of Stx1 in the periplasm to the amount contained within OMVs. OmpF and
OmpA will be used as membrane bound markers in the OMVs. Since these proteins are each
present at consistent levels, the ratio of their abundances to each other should remain constant. A
deviation in the amount of one or more of these proteins with respect to the others will signify
that the packaging of proteins other than Stx1 into OMVs has been altered. We will then adjust
our comparison to Stx1 accordingly. Detection of such an occurrence is not possible without the
measurement of multiple control proteins.
For each Western blot, multiple dilutions of purified OMV or periplasm will be loaded onto the
gel along with multiple dilutions of the corresponding purified protein that will be probed for.
Since we will be comparing band intensities from OMVs and periplasm directly to known
quantities of purified protein, the measurements will be semi-quantitative. Stx1 will be purified
by fast protein liquid chromatography (FPLC) with the affinity resin Synsorb-Pk [71], MBP will
be purified using the pMAL Protein Fusion and Purification System from New England Biolabs
(NEB) [72], and pure GFP will be purchased from Millipore. Due to the difficulty of working
with integral membrane proteins, OmpA and OmpF will not be purified, but the levels will be
monitored. Anti-Stx1 antibody will be purchased from Santa Cruz Biotechnology, Anti-GFP
antibody purchased from Invitrogen, Anti-MBP purchased from NEB, anti-OmpA from
Heenning et al. [73], and anti-OmpF antibody from Yamashita et al. [74]. A secondary antibody
conjugated with alkaline phosphatase will allow for visualization of the protein on the blot when
treated with the alkaline phosphatase chromogen BCIP/NBT.
As a sphere increases in size, the surface area increases by a power of 2, while the volume
increases by a power of 3. Perturbation of the packaging machinery or a factor involved with
OMV synthesis may affect the size of the vesicles produced. An increase in the average vesicle
size would increase the volume of the vesicle at a much faster rate than the surface area. The
result is the level of luminal associated proteins will increase in quantity at a much faster rate
than the membrane associated proteins. This would cause the level of Stx1 to increase in relation
to the integral membrane proteins OmpA and OmpF, while its relationship to MBP and GFP will
remain the same. To distinguish between variations in vesicular size from variations in soluble
protein packaging, we will characterize the size of OMVs produced for every measurement. The
average vesicle size and size variability for each OMV preparation will be measured using
dynamic light scattering (DLS) [75].
Identification of proteins involved in Stx1 packaging. Next, we will identify the interacting
partner that is responsible for sequestering Stx1 into OMVs. To start, we will use velocity
density centrifugation to determine if E. coli O157:H7 produces multiple sub-populations of
OMVs like Neisseria gonorrhoeae [55]. Velocity density centrifugation of ETEC OMVs has
shown that vesicles from pathogenic E. coli can be separated into fractions based on density [29].
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The fractions contained differing protein contents when visualized by silver-staining after gel
electrophoresis, and we will use the same protocol from Horstman and Kuehn [29] to separate
OMVs purified from STEC. We will probe each fraction by Western blot to assay for Stx1. If
Stx1 localizes to a specific fraction or set of fractions, we will exploit this to narrow down the
number of possible Stx1 interacting proteins. In doing so, we are making the assumption that a
portion of the packaging machinery, specifically that which is responsible for providing cargo
specificity, leaves the cell with the OMV. The high quantities that can be packaged into OMVs
for certain proteins [8-11] would require constant binding during OMV formation to prevent the
cargo from escaping. We will TCA precipitate each fraction and use 2D gel electrophoresis to
profile the proteins present in each. Proteins that are present in Stx1 containing fractions, but
absent from others, will be identified by mass spectrometry. This can be done by removing the
unknown protein directly from the 2D gel. Subsequently, the genes coding for identified
proteins will be deleted using the Lambda Red system [68]. OMVs and periplasm will be
purified from these mutants, and the quantity of Stx1 contained within each will be assayed as
previously described.
In the event that Stx1 does not localize to a specific population of OMVs, we will attempt to
directly identify Stx1 interacting partners. Purified OMVs from strain O157:H7 as well as a Stx1
deletion strain will immunoprecipitated with anti-Stx1 antibody. We will use protein A magnetic
beads from NEB with their immunoprecipitation protocol [76]. Samples will be sent for mass
spectrometry in order to identify Stx1 interacting proteins. Previous proteomic analysis of E.
coli DH5α OMVs found that there are a total of 141 different proteins present [3]. We expect
that only a small population of these proteins will be immunoprecipitated with Stx1. In the event
that the OMV protein content is not identical between these two strains, we will focus first on
proteins not identified by the previous study. These strain specific proteins may be more likely
to be involved with Stx1 packaging. Next, we will focus on proteins that are or are predicted to
be membrane associated. Membrane association by the sequestering factor is a requirement of
our Stx1 packaging model. Each candidate gene will be deleted using the Lambda Red system
[68], and the resulting quantity of Stx1 contained within OMVs and periplasm will be assayed as
before.
Expected outcomes. We expect to find that the quantity of Stx1 will be greater in purified
OMVs than in purified periplasm as analyzed Western blot. We are making the assumption that
the concentration of GFP and MBP will be the same in OMVs as it is in the periplasm. This
assumption will be validated by determining the ratio of GFP to MBP. If the ratio is the same in
purified periplasm as it is in OMVs, then both proteins are likely packaged into OMVs by
random diffusion. Even if this should prove not to be the case, if the quantity of Stx1 is higher in
OMVs than in the periplasm by comparison to either of these, we will have demonstrated its
enrichment over another factor and shown that it is selectively packaged. If Stx1 is not shown to
be enriched by this method, we will proceed to the velocity density centrifugation experiments.
If Stx1 is enriched in a specific population, measurement of all OMVs at once cannot distinguish
Stx1 containing OMVs from those that lack Stx1. The result is the signal from a Stx1 containing
population will be averaged over the contents of all OMVs, and may reduce it to the point where
it is indistinguishable from proteins incorporated due to random diffusion. By blotting each
fraction individually, we will determine if there is a specific OMV population that contains Stx1,
and if it is present at higher quantity than in the periplasm. Additionally, by running 2D gel
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electrophoresis on the individual fractions, we will isolate proteins that localize specifically with
Stx1 in OMVs. Alternatively, immunoprecipitation of Stx1 will isolate proteins that directly
interact with it in OMVs. After mass spectrometry to determine the protein’s identity and
removal of the gene by deletion, we expect the mutant will have increased Stx1 quantities within
the periplasm and decreased quantities contained within OMVs when assayed by Western blot as
before. This will be the result of abrogation Stx1 packaging into OMVs.
Additional possibilities exist for Stx1 enrichment into OMVs that do not involve our proposed
mechanism of sequestration by a protein during OMV formation. One option would involve a
secretion system that can deliver proteins to the site of OMV formation. The analogy can be
drawn to filling a hot air balloon. If Stx1 is exported from the cytosol at a high enough rate near
the site of OMV formation, the slow diffusion rate though the gel like periplasm would cause the
local concentration to increase. The temporarily elevated concentration would be captured as the
OMV pinches off. This mechanism requires the site of OMV formation to be localized to the
site of Stx1 production. There is currently no evidence for specificity in the location of OMV
formation. If this process is occurring, there are likely unknown protein factors involved. If
such an OMV packaging system does exist and is not essential for life, its identification will
likely require genetic screening.
In the event that biochemical approaches of identifying Stx1 sequestering proteins are not
successful, we will take a genetic approach to identify candidate genes. We will perform a
genetic screen using random insertion transposon mutagenesis [77] with the goal of finding
genes that impact the level of Stx1 associated with OMVs. Mutant monoclonal strains will be
grown inside chambers suspended in wells containing sterile growth media. These chambers
have a 0.45 μm pore filter at the bottom. This will filter will not allow the bacteria to pass, but
will allow diffusion of small particulates, including OMVs, into the sterile growth media. The
bacteria containing chambers will be removed from the wells after overnight growth and the
trays of wells centrifuged to pellet the OMVs. The media will be removed and the OMVs
washed to remove exogenous Stx1 and GFP. GFP fluorescence will be measured in each well
before re-suspending the OMVs. This will concentrate the GFP and give a strong fluorescent
signal. The OMVs will then be re-suspended in detergent containing buffer in order to lyse the
vesicles and release Stx1. An enzyme-linked immunosorbent assay (ELISA) will then be carried
out to determine the quantity of Stx1 in OMVs produced by each strain according to Ashkenazi
and Cleary [78]. We will then normalize Stx1 measurements to the GFP fluorescence to
determine the relative abundance of Stx1 inside the OMVs. We will use this ratio to monitor
Stx1 levels inside of OMVs for the mutant population. Strains that deviate from this ratio will be
re-tested to ensure reproducibility before selection for further study. We will identify the
unknown gene that was interrupted by the transposon insertion by performing PCR using primers
against the known transposon sequence. These genes will be deleted using the Lambda Red
system [68] and the Stx1 quantities more accurately measured by the larger scale OMV
purification and analysis as performed earlier.
If we are unable to identify any proteins that interact with Stx1 that are contained within OMVs,
we must consider the possibility that Stx1 localization to OMVs is not a protein mediated
process. Other options include nucleic acids, carbohydrates, or lipids. DNA is present in many
OMVs [35], but it is not known if it is present in all OMVs. The mechanism by which DNA is
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packaged into OMVs is also unknown. DNA is not commonly found within the periplasm,
which is where it would need to be in order to sequester Stx1. The B subunit pentamer has been
shown to bind specific carbohydrate moieties [13]. LPS is a candidate due to the presence of
membrane anchored sugar groups. In the case of LT, also produced by E. coli, OMV
localization is the result of interaction with LPS [79]. Since LPS is found in the outer leaflet of
the outer membrane, LT is able to associate with the surface of OMVs as well as being packaged
inside. When Stx1 containing vesicles are treated with protease digestion enzymes, Stx1 levels
do not decrease [33]. We can conclude that Stx1 is protected from digestion because it is
protected inside OMVs, and that it does not associate with the surface of OMVs. We can
therefore discount the possibility that Stx1 binds to LPS, and conclude that it is not responsible
for sequestering Stx1 into OMVs. If it is a sugar group responsible for binding Stx1 to sequester
it, we will attempt to detect its presence by treating Stx1 containing OMVs with 8-quinaloneboronic acid [80]. This compound fluoresces at 417 nm when excited at 314 nm when bound to
a vicinal diol that is immobilized as part of a ring, which are present almost exclusively on
sugars. We will use velocity sedimentation ultracentrifugation with purified Stx1 and protein
free soluble OMV extracts to determine if there is a sugar capable of binding Stx1. LC/MS will
then be used to purify and characterize the sugar should one be found to interact with Stx1.
Another alternative is that Stx1 is interacting with a lipid. The feasibility of sequestration by
direct lipid interaction will be tested by velocity sedimentation ultracentrifugation in the presence
of digitonin using purified Stx1 and lipid extracts from OMVs. Should an interaction be
detected, HPLC fractionation of the lipid profile will allow testing in a more narrow range of
compounds. The lipids will be tested one at a time by velocity sedimentation ultracentrifugation.
Any lipids capable of binding will be identified by NMR or mass spectrometry.
Specific Aim 2: Test the hypothesis that Stx1 is able to retard the growth of other bacteria.
In a polymicrobial environment, the fitness of each individual species is directly related to its
access to nutrients. A bacteria strain is said to be “predatory” if it gains a replicative advantage
over other bacteria by lysing them or inhibiting their replication [81, 82]. Lysing other bacteria
not only prevents them from utilizing nutrients the predatory bacteria can use, but also releases
additional nutrients that are contained within the rival microbes. This becomes especially
important in a nutrient depleted environment, where lysing other bacteria may be the only
available nutrient source. Iron is often a limiting growth factor for bacteria, and Stx1 is
maximally expressed in low-iron conditions [83]. It is under these conditions that predation may
be most important in order for STEC to obtain nutrients that will allow it to grow and divide.
We will test the ability of Stx1 containing OMVs produced by E. coli O157:H7 to impact the
growth of other bacteria We will test a selection of the 17 strains used by Li et al. [43] to
examine the lytic effects of OMVs [43]. We have selected the following 5 as representative of a
variety of bacterial cell types: Bacillus subtilis ATCC 6051, Staphylococcus aureus ATCC
25923, P. aeruginosa PAO1, E. coli K-12, and Mycobacterium phlei 425. The role of Stx1 in
OMV mediated predation will be tested by removing Stx1 from OMVs as well as by using
attenuated Stx1.
The role of Stx1 in OMV based predation. Although there are many predatory factors secreted
by bacteria, we are primarily interested in OMV associated Stx1. In this set of experiments, we
will investigate the role that Stx1 and its incorporation into OMVs play in predation. Both wildtype Stx1 as well as an attenuated mutant, termed mStx1 has a double mutation in the active site
9
BAHL, Christopher D.
of the A subunit [84], will be purified as before. In addition, OMVs will be purified containing
each of these proteins as well as from a Stx1 deletion strain. The 5 test strains will be treated
with purified Stx1, mStx1, Stx1 containing OMVs, mStx1 containing OMVs, Stx1-lacking
OMVs, and Stx1-lacking OMVs with either purified Stx1 or mStx1 added in trans. These will
be added to early log phase cultures and the growth rate will be compared by measuring the
colony forming units (CFUs) from aliquots of the culture at successive time points. Counting
CFUs will allow us to determine the number of viable cells in each culture, which in turn will be
used to distinguish between normal growth, cell lysis, or retardation of replication rate.
Expected Outcomes. We know from the work by Li et al. [43] that non-pathogenic E. coli
OMVs are able to lyse all of the selected strains except S. aureus to some degree. This is likely
due to the murein hydrolases contained within the OMVs that are able to degrade the cell walls
of these bacteria. The non-pathogenic E. coli OMVs were likely unable to lyse S. aureus due to
the peptidoglycan chemotype. Since Stx1 targets the ribosomes and not the murein sacculus, its
activity should be
independent of cell wall
mStx1
Stx1
mStx1 OMVs Stx1 + mStx1 +
Treatment Stx1
structure. Thus, given the
OMVs OMVs
OMVs OMVs
+
++
+
+
+
+
Expected
previously described
outcome
bactericidal effects of Stx1
[14], we expect its presence
Table 1. Predicted results for the majority of strains in the test set when
to cause lysis or growth
treated with purified toxin or OMVs. -, little to no growth inhibition; +,
retardation of all the bacteria replicative period has increased, some cell lysis is occurring; ++, significant
in the test set. The expected lysis of cells, may by unable to measure replication period due to cell death.
results are summarized in
Table 1.
Some inhibition of growth can be expected from all treatments that contain OMVs due to the
many other antimicrobial factors contained within them. It is possible that the activity of Stx1
within the OMVs will be masked by the presence of other predatory factors. If we do not find
decreased growth rates that directly correlate with the presence of Stx1, we will repeat the
experiments using reconstituted liposomes instead of OMVs. Liposomes will replace the OMVs
as lipid based transport vehicles, and are free of additional predatory factors. We will adapt the
protocol from Rukholm et al. to generate liposomes for delivery of purified Stx1 and mStx1.
This method has been successfully used to deliver antibiotic to P. aeruginosa [85]. Liposome
experiments will be carried out as before, including treatment with empty liposomes and adding
purified toxin in trans with empty liposomes. It is possible that Stx1 is activated during
packaging into OMVs, or there is a factor packaged with Stx1 in the OMVs that promotes its
activation. Therefore, if we do not observe effects using Stx1 with reconstituted liposomes, we
will first treat purified Stx1 to mild trypsin digestion for activation [62] prior to incorporation
into the liposomes. If an effect is only seen using pre-cleaved Stx1 in reconstituted liposomes,
we will purify Stx1 from purified Stx1 containing OMVs and incorporate it into reconstituted
liposomes. This will allow us to determine if additional factors are involved in Stx1 activation.
If this population maintains the same level of activity as the trypsin pre-treated Stx1, it is likely
that Stx1 is being proteolytically cleaved by E. coli proteases in order to activate the Nglycosidase activity. If trypsin pre-treatment also increases the activity of the OMV purified
Stx1, then it is unlikely that Stx1 is activated inside of OMVs or prior to secretion.
10
BAHL, Christopher D.
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15