The lytic mechanism of Escherichia coli α-hemolysin associated to outer membrane vesicles

doi:10.4236/health.2010.25072

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Vol.2, No.5, 484-492 (2010)

doi:10.4236/health.2010.25072

Health

The lytic mechanism of Escherichia coli α-hemolysin

associated to outer membrane vesicles

——Lytic action mechanism of OMVs-associated HlyA

Vanesa Herlax1*, Maria Florencia Henning1, Ana María Bernasconi1, Felix María Goñi3, Laura Bakás1,2

1Instituto de Investigaciones Bioquímicas La Plata (INIBIOLP), CCT-La Plata, CONICET, UNLP, Facultad de Ciencias Médicas,

La Plata, Argentina; *Corresponding Author: vherlax@atlas.med.unlp.edu.ar

2Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina

3Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU), and Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain

Received 2 January 2010; revised 1 February 2010; accepted 2 February 2010.

ABSTRACT

Alpha-hemolysin (HlyA) is an extracellular toxin

secreted by Escherichia coli, targeting to plasma

membranes of eukaryotic cells. Recently it was

found that this toxin is released to external

media associated to bacterial outer membrane

vesicles (OMVs), but the hemolytic mechanism

in this way has not been studied yet. Our results

report that HlyA is the only protein present in

OMVs that is responsible for erythrocyte lysis,

and show that no fusion event is involved in the

lytic mechanism of OMVs-HlyA. Furthermore,

the specific hemolytic activity is approximately

10 fold higher than that of purified free-HlyA,

showing the same relative lysis efficiency and

specificity for erythrocytes from different

species. OMVs could be an important auxiliary

way of secretion, acting mainly as a concentra-

tion mechanism of HlyA near the target cells.

Cell lysis would occur after toxin transfer from

OMVs to target membranes, as demonstrated by

hemolysis kinetic studies, lipid mixing and

western blot assays.

Keywords: OMVs; Toxin; Lipid Mixing;

Hemolytic Activity; Erythrocytes

1. INTRODUCTION

The hemolytic toxin -hemolysin (HlyA), member of

the RTX toxin family [1], is an important virulence fac-

tor produced by several strains of Escherichia coli. It is

involved in human extraintestinal diseases, such as uri-

nary tract infections, peritonitis, meningitis and septice-

mia [2]. As in most RTX proteins, an operon composed

of 4 genes (hlyABCD) is involved in the polypeptide

synthesis, postraslational modification and secretion of

the active HlyA to extracellular media [3,4]. HlyA is

synthesized in an inactive form (ProHlyA), which is

activated in the cytoplasm to the hemolytically active

form by HlyC, a fatty acid acyltransferase [5]. Then

HlyA is secreted across both membranes by the type I

export process employing an uncleaved C-terminal reco-

gnition signal [6,7]. Although HlyA has its own machin-

ery to be export from the bacteria, Balsalobre et al. [8]

demonstrated the presence of physiologically active

HlyA in outer membrane vesicles (OMVs) of hemolytic

strains of Escherichia coli.

Outer membrane vesicles (OMVs) are constantly

being discharged from the surface of Gram negative

bacteria during bacterial growth. All Gram negative

bacteria studied up to date, including Escherichia coli,

Neisseria meningitidis, Pseudomonas aeruginosa, Hel-

icobacter pylori, Shigella flexneri and Actinobacillus

actinomycetemcomitans produce outer membrane vesi-

cles and their release is increased when bacteria are

exposed to stressful conditions such as antibiotics or

serum. One can be tempted to think that OMVs pro-

duction is the result of membrane instability, but recent

studies have demonstrated that mutations that affect

protein synthesis, localization, envelope structure and

envelope stress response pathway alter the vesiculation

levels. In this way the release of OMVs offers to the

cell an effective mechanism for removal of material as

a macromolecular complex, allowing to discard unw-

anted material or alter the composition of the envelope

under conditions where remodeling would be advantag-

eous [9,10].

Despite that in vivo, release of OMVs could not be

monitored, the presence of particles resembling these

vesicles has been detected in plasma from patients with

different infectious processes [11-18]. OMVs serve as

secretory vehicles for proteins and lipids of Gram

negative bacteria and play roles in establishing a colon-

V. Herlax et al. / Natural Science 2 (2010) 484-492

485

ization niche carrying or transmitting virulence factors

into host cells or modulating host defense and response,

acting also as long-range virulence factors that can

protect luminal cargo from extracellular host proteases

and penetrate into tissues more readily than larger bac-

teria [19]. An extensive review about the significance

of bacterial OMVs in the host-pathogen interaction was

published by Kuehn and Kesty [20]. Besides the toxin o

protein delivery, other roles were characterized for

OMVs, such as: interaction interspecies during multis-

pecies infections, DNA transfer, DNA uptake, commu-

nication interspcies [21].

OMVs are primarily composed of lipopolysaccharide

(LPS), phospholipids and various outer membrane (OM)

proteins and periplasmic components, but they do not

contain intra membrane (IM) or cytoplasmic components

[22], although OMVs isolated from E. coli O157:H7

contain DNA [23].

Considering that HlyA was found associated to

OMVs of some strain of E. coli and that erythrocytes

have been the cell type classically used in studies on the

action mechanism of this toxin, in this research we

studied the hemolytic mechanism of OMVs-associated

HlyA. The results demonstrate that the lytic action of

OMVs-associated HlyA like free-HlyA does not in-

volve fusion events, instead the toxin is transferred

from OMVs to target cells, as demonstrated by hemo-

lysis kinetic studies, lipid mixing and western blot as-

says.

2. EXPERIMENTAL PROCEDURES

2.1. Isolation of Outer Membrane Vesicles

OMVs

E. coli strains used as a source of HlyA were WAM 1824,

an overproducing hemolysin strain [24], and WAM 783,

for the unacylated inactive toxin ProHlyA [25]. Both of

them were kindly provided by R. A.Welch (University of

Wisconsin, Madison, Wisconsin).

OMVs were isolated from bacterial culture supern-

atants. Briefly, bacteria were grown at 37ºC in Luria

Bertani broth (LB). Culture samples from the decelerat-

ing growth phase were centrifuged at 10000 rpm for 20

min at 4ºC. The resulting suspension was then centri-

fuged at 47500 rpm for 2 h at 4ºC to collect the pellet

containing OMVs.

2.2. Protein Purification

Free-HlyA was purified from the supernatants resulting

from OMVs. It was concentrated and partially purified

by precipitation with 20% cold ethanol. A precipitate

containing HlyA was collected by centrifugation (1 h,

10000 rpm in a Sorvall rotor SSA 34), then it was

resuspended in 20 mM Tris, 150 mM NaCl, pH 7.0 (TC

buffer). This preparation showed, on SDS-PAGE, a main

band at 110 kDa corresponding to more than 90% of the

total protein. This band was assigned to free-HlyA. The

protein could be stored at –70°C.

2.3. OMVs Composition

2.3.1. Quantitative Assays

Quantitative assays were performed using Lowry method

for proteins [26], KDO 2-keto-3-deoxyoctonate for

LPS [27] and inorganic phosphorous for phospholipids

[28].

2.3.2. Analysis of Proteins by SDS-PAGE

Samples were electrophoresed on 10% acrylamide gels

in the presence of SDS according to Laemmli et al. [29].

Protein bands were visualized by Coomassie Blue stain-

ing [30].

2.3.3. Immunoblotting Analysis

Samples from 10% SDS-polyacrylamide gels were

transferred to nitrocellulose by the method of Towbin et

al. [31]. Blots were blocked with 3% skim milk in TBS

buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.5) at

room temperature for 2 h. They were then incubated

with a solution containing a polyclonal rabbit anti-

hemolysin antibody (1:1000) in 3% skim milk-TBS at

4°C overnight, washed with TBS buffer, and finally

reacted with peroxidase-conjugated anti-rabbit Ig anti-

body (Sigma) (1:1000) in TBS buffer with 3% skim

milk at room temperature for 2 h. After incubation and

washing as stated above, the nitrocellulose was trans-

ferred to a peroxidase substrate solution containing 6

mg 4-chloro-1-naphthol (Sigma) in 1 ml methanol, 1

ml TBS, 3 ml H2O and 8 l H2O2 for the detection of

horseradish peroxidase-conjugate antibodies on the

membrane.

2.4. Hemolytic Assays

For the hemolytic assays, free-HlyA or OMVs associ-

ated-HlyA were serially diluted in TC cold buffer con-

taining 10 mM CaCl2 on a 96-well microtiter plate. One

hundred L of the diluted suspensions were mixed with

100 μl of standardized horse or rabbit erythrocytes. The

mixture was incubated at 37°C for 30 min. The absorb-

ance of supernatants was read at 412 nM [32]. One

hemolytic unit/mL (HU/mL) is defined as 10 fold the

dilution of toxin preparation producing 50% lysis of the

erythrocyte suspension.

The standardization of erythrocytes was done just bef-

ore the assay. The erythrocytes were washed in 0.9%

NaCl and then diluted to 12.5 L in 1 mL of distilled

water to give a reading of 0.6 absorbance unit at 412 nM

[33].

V. Herlax et al. / HEALTH 2 (2010) 484-492

486

2.5. Hemolysis Kinetics

Hemolysis kinetics was determined by measuring the

decrease in turbidity (absorbance at 650 nM) of a stan-

dardized horse erythrocyte suspension exposed to free-

HlyA or OMVs associated-HlyA as a function of time at

37ºC. A series of hemolytic reactions were set up starting

with an amount of free-HlyA or OMVs associated-HlyA

that would produce 100% of hemolysis in 96-well plate

dilution assays. The real time kinetics assays for hemo-

lysis were performed in the following manner: 500 µL of

standardized erythrocytes and 500 µL of hemolysis buffer

containing 10 mM CaCl2 were placed into a cuvette.

Hemolysis started when the toxin was injected into the

cuvette, and the absorbance at 650 nM was measured.

2.6. LUVs Preparation

Large unilamellar vesicles (LUVs) composed of PC/PE/Chol

2:1:1 molar ratio were prepared by extrusion using 0.1

μM pore size membranes as described by Mayer et al.

[34].

2.7. Ghost Erythrocytes Preparation

Five mililitres of horse erythrocytes were washed with TC

buffer and osmotically lysed in 10 mM tris-HCl PH = 7.4

buffer at 4ºC for 30 min. The membranes were pelleted by

centrifugation (10 min at 10000 rpm), and washed until

the supernatant remained clear. The membranes were

finally resuspended in 3 mL of TC buffer. The phosphate

concentration of these samples was 0.53 ± 0.05 mg

phospholipid/mL (n = 3).

2.8. Labeling of Ghost Erythrocytes with

Rhodamine-Phosphatidylethalonamine

(Rh-Pe) and N-(7-Nitro-2, 1, 3-Benzoxa-

diazol-4-Yl)-Phosphatidylethanolamine

(NBD-PE)

Ghost erythrocytes were labeled with Rh-PE and NBD-PE

(Molecular Probes, Eugene, O.R.). Both fluorophores

are coupled to the free amino group of phosphatidyle-

thanolamine to provide analogues which can be incur-

porated into a lipid bilayer. 20 L of a fresh solution of

each probe (1 mg/ml in ethanol) was added to 75 L of

ghost erythrocytes. After incubating the suspension for 1 hr

at room temperature, 6 washes with TC were performed

so as to separate unbound labeled lipids. Finally the la-

beled ghost erythrocytes were resuspended in 5.4 ml of

TC.

2.9. Lipid Mixing

Membrane fusion was analyzed by lipid mixing between

OMVs and ghost erythrocytes labeled with NBD-PE and

Rh-PE. The fusion assay involves resonance energy

transfer between NBD as donor and Rh as acceptor.

When both fluorescent lipids are in ghost erythrocytes,

efficient energy transfer is observed [35]. After lipid

mixing by fusion with a population of unlabeled memb-

ranes, in this case OMVs, a decrease in efficiency of

resonance energy transfer should be observed followed

by an increase of the donor fluorescence or decrease of

the acceptor fluorescence. In our experimental design,

we followed the increase of donor (NBD-PE) fluoresc-

ence as a function of time using the excitation and emis-

sion monochromators set at 465 nM and 530 nM, res-

pectively. Slits for excitation and emission were 4 nM.

The extent of lipid mixing was determined according

to the following equation:

% lipid mixing = [(Ft – F0) / (Fmax – F0)] × 100

where F0 is the initial fluorescence value of erythrocytes

labeled with both probes, Ft is the value of fluorescence

after t minutes of incubation with OMVs and Fmax (100%

fluorescence) is the value of fluorescence after addition

of Triton × 100 to disperse maximally the probes.

3. RESULTS

3.1. Characterization of OMVs Produced by

E. coli WAM 1824

Recently, Balsalobre et al. demonstrated that physiologi-

cally active HlyA is associated with OMVs produced by

laboratory strains and also from natural and clinical E.

coli isolates. However, as the amount of OMVs and the

percentage of the soluble and OMVs associated HlyA

were likely to vary markedly depending on the strain [8],

we characterized OMVs obtained from E. coli WAM

1824 strain.

OMVs were purified from the culture filtrate of E. coli

WAM 1824. Many bilayered spherical vesicles with di-

ameters ranging from 50 to 200 nm were observed by

electron microscopy (data not shown). The lipid compo-

sition of these vesicles was analyzed by thin layer chro-

matography, showing that the two predominant lipid spe-

cies are phosphatidylethanolamine (PE) and cardiolipin

(CL), demonstrating that these vesicles arise indeed from

the bacterial outer membrane (data not shown).

The presence of HlyA in OMVs produced by E. coli

WAM 1824 is shown in Figure 1(a). This figure shows the

SDS-PAGE polypeptide profile of OMVs. A band with

an electrophoretic mobility corresponding to HlyA (MW

110 kDa) was observed whose identity was confirmed by

Western blot analysis employing polyclonal antibodies

directed against purified HlyA (Figure 1(b)). The others

bands that appear in SDS-PAGE stained by Comassie

stain, correspond to periplasmic and outer membrane

proteins also present in OMVs.

V. Herlax et al. / Natural Science 2 (2010) 484-492

487

Openly accessible at

(a) (b)

Figure 1. HlyA is present in WAM 1824 outer membrane vesi-

cles. (a) SDS-PAGE 10%, stained by Coomasie. 1: LMW, 2:

WAM 1824 OMVs (10μg prot), 3: WAM 783 OMVs (10 μg);

(b) Western-Blot, using anti-HlyA polyclonal antibodies. 1:

Rainbow molecular weight, 2: OMV’s (100 ng prot), 3:

Standard HlyA (50 ng).

3.2. Comparison of Hemolytic Activity

between Free-HLYA and OMVs

Associated-HlyA

The following experiments were done so as to compare

the hemolytic activity between the OMVs associated-

HlyA and free toxin, which was quantified by serial dilu-

tion assays against horse erythrocytes. The hemolytic ac-

tivity was 40960 and 2560 HU/ml for OMVs associated-

HlyA and free toxin, respectively. If we consider that

total protein concentration in the OMVs is 2.7 mg/mL

and 70% corresponds to HlyA (estimated from the inten-

sity of the 110 kDa band in a 10% SDS-PAGE gel, ana-

lyzed by Kodak digital Science 1D Software), so the

approximate concentration of OMVs associated- HlyA is

1.7 10-5 M. These values allow us to calculate the spe-

cific activity which is 2.37 1012 HU/mol and 2.04 1011

HU/mol for OMVs associated-HlyA and free-HlyA, re-

spectively.

In order to confirm that the hemolytic activity of

OMVs is solely due to HlyA and not to other proteins

present in the OMVs, E. coli WAM 783 (strain with a

deletion of hlyC gen that encodes HlyC, the protein

responsible for HlyA acylation) was used as a source of

OMVs. The presence of a protein corresponding to

110 kDa in 10% SDS-PAGE confirms that inactive

unacylated toxin is also associated to OMVs (Figure 1,

lane 3). OMVs obtained from this strain do not have any

hemolytic activity confirming that this activity in OMVs

is a particular property due largely or totally to the pres-

ence of HlyA.

3.3. Lipid Mixing Assays

In order to determine whether the hemolytic action mec-

hanism of OMVs associated-HlyA implies membrane

fusion, we analyzed the lipid mixing between OMVs and

ghost erythrocytes labeled with NBD-PE (donor) and

Rh-PE (acceptor). The ghost erythrocytes concentration

used in lipid mixing assays is equal to the erythrocyte

suspension used in hemolytic experiments.

6.9% FRET decrease was detected between OMVs

and labeled erythrocytes promoted by OMVs associated-

HlyA. When free-HlyA was added to labeled erythro-

cytes, 8.1% FRET decrease was obtained (Figure 2),

which cannot be assigned to lipid mixing because unla-

beled erythrocytes were absent in this experiment, so it

should be attributed to protein insertion into lipid bilayer,

increasing the distance between donor and acceptor [36].

The decrease in energy transfer in both experiments

was similar, indicating that fusion events are not inv-

olved in the action mechanism of this toxin when it is

OMVs associated; instead, a transfer process from

OMVs to the target membrane must be involved. The

presence of a high affinity specific receptor is not strictly

necessary, as the same experiments were repeated with

labeled large unilamellar vesicles (LUVs), giving similar

FRET decrease (data not shown).

3.4. HlyA Transfer from OMVs to

Erythrocytes

To study HlyA transfer from OMVs to erythrocytes, a

serial diluted hemolytic assay was carried out as desc-

ribed previously in Experimental Procedures. Samples

were centrifuged at 10,000 rpm to separate ghost erythr-

ocytes from OMVs. The pellets containing the ghosts

were resuspended in SDS sample buffer, and electropho-

resed in a 10% SDS-PAGE. Finally this gel was trans-

time (sec)

0200 400 600 800

Fluorescence at 530 nm

Figure 2. OMVs associated-HlyA does not induce membrane

fusion. Lipid mixing assays between labeled erythrocytes with

Rh-PE/NBD-PE and OMVs () or free-HlyA (▼). Solid

arrows correspond to the addition of OMVs or free-HlyA.

Dotted arrows correspond to the addition of 0.2% Triton X-100

(Fmax). Total lipid concentration was 0.1 mM. 40 HU for

free-HlyA and OMVs associated-HlyA were used.

V. Herlax et al. / HEALTH 2 (2010) 484-492

488

Openly accessible at

ferred to nitrocellulose and HlyA was detected using a

polyclonal rabbit anti-HlyA antibody. Figure 3(a) shows

that HlyA is effectively transferred to erythrocytes. The

amount of HlyA decreases with the decrease of the

OMVs concentration, according to the decrease in the

hemolytic activity observed (data not shown).

Another fact that confirms that fusion events are not

involved in the hemolytic process mediated by OMVs is

the absence of LPS in the ghost erythrocytes membranes

after their incubation with OMVs. The detection of LPS

was analyzed by a specific silver stain for LPS (BioRad

silver stain kit, catalog 161-0445) of a 16% SDS-PAGE. A

typical LPS pattern is not observed in this sample (Figure

3(b), lane 1), instead some bands of glycoproteins of

erythrocytes are observed.

(a)

(b)

Figure 3. HlyA is transfered from OMVs to erythrocytes. (a)

HlyA transference from OMVs to erythrocytes analyzed by

western blot: lane 1: standard HlyA, 2-7: samples of ghosts

erythrocytes previously incubated at 37°C during 30 min with

serially diluted OMVs, 8: ghost erythrocytes; (b) Analysis of

LPS content in ghost erythrocytes: lane 1: ghosts obtained

from the hemolysis assay with OMVs, 2: ghosts, 3: OMVs and

4: Standard LPS from E. coli 011:B4.

3.5. Hemolysis Kinetics

Typical hemolysis time courses are shown in Figure 4.

For both, OMVs associated-HlyA and free-HlyA, the

time to reach 50% of hemolysis increases as the toxin

concentration decreases. The hemolytic curves exhibit a

define lag period for both free-HlyA and OMVs associated-

HlyA. This lag period lengthens noticeably at decreasing

concentrations of OMVs associated-HlyA, but it remains

practically constant for free-HlyA as seen in Figure 5(a),

indicating that the rate limiting step in the hemolysis

process induced by OMVs associated-HlyA is the diffus-

ion of vesicles in the aqueous medium, due to their higher

molecular weight and volume compared to free-HlyA.

Initial rate of hemolysis obtained from the linear por-

tion of the kinetic curve are shown in Figure 5(b). Vir-

time

(

sec

)

02004006008001000 12001400 1600180020002200 24002600 2800

0.0

0.5

1.0

1.5

2.0

2.5

> OMV HlyA

concentration

> OMVs HIyA

concentration

DO 650 nm

(a)

time (sec)

02004006008001000 1200 1400 1600 1800 2000 22002400 2600

DO 650 nm

0.0

0.5

1.0

1.5

2.0

2.5

(b)

Figure 4. Kinetics of hemolysis induced by OMVs associ-

ated-HlyA (a) and free-HlyA (b). The real time kinetic assays

for hemolysis were performed in the following manner: 500 µl

of standardized erythrocytes and 500 µl of hemolysis buffer

containing 10 mM CaCl2 were placed into a cuvette. Hemolysis

started when the toxin was injected into the cuvette, and the

absorbance at 650 nm was measured. The arrow indicates toxin

concentration increase.

V. Herlax et al. / Natural Science 2 (2010) 484-492

489

Openly accessible at

g protein

0510 15 20 25 30 35 40

lag period (sec)

200

400

600

800

1000

1200

1400

1600

HlyA-OMV

free HlyA

(a)

g protein

0510 15 20 25 30 35 40

hemolysis rate (DO/sec)

0,000

0,001

0,002

0,003

0,004

0,005

0,006

HlyA-OMV

free HlyA

(b)

Figure 5. Initial hemolytic rate (a) and lag time (b) as a func-

tion of OMVs associated-HlyA (●) and free-HlyA (■) concen-

tration. Initial rates (linear portion) and lag time were obtained

from Figure 5 at different toxin concentrations.

tually constant values were obtained with OMVs associ-

ated-HlyA for all the concentrations studied, while a

decrease in initial rates as concentrations decrease was

observed for free-HlyA.

3.6. Erythrocytes Specificities of Free-HLYA

and OMVs Associated-HlyA

To characterize the hemolytic efficiency of OMVs assoc-

iated-HlyA on different species of mammalian cells, we

chose rabbit and horse erythrocytes, which were the

most commonly used and characterized in previous stu-

dies related with the hemolytic action mechanism of this

toxin. In Figures 6(a) and (b) it can be seen that in both

cases, rabbit erythrocytes are more sensitive than those

of horse, as demonstrated in earlier results on the char-

acterization of the lytic action of HlyA [37]. The D50

values (amount of toxin that produces 50% of hemolysis)

are 0.46 and 2.81 nM for OMVs associated-HlyA for

rabbit and horse erythrocytes respectively.

The dose-response curves indicate a higher lytic effic-

iency of OMVs associated-HlyA (Figure 6(a)) as com-

pared to free-HlyA (Figure 6(b)), shown by the D50 on

horse erythrocytes of 2.81 and 22.5 nM, respectively.

Moreover, analysis of curves of hemolytic activity

against concentration using SigmaPlot (Jandel Scientific,

San Rafael, CA) fitted a sigmoidal curve in both cases.

In the case of horse cells, data fit a sigmoid 4 parameter

curve, while the rabbit ones fit a logistic 3 parameter

curve, demonstrating a different cooperativity in the

hemolysis process. The same behavior is observed for

OMVs associated-HlyA as for free-HlyA.

4. DISCUSSIONS

Recently, it was demonstrated the presence of active

HlyA associated with OMVs produced by natural and

clinical E. coli isolates. Despite a great deal of published

log g Hly in OMV

0.0001 0.0010.010.11101001000

100

120

% hemolysis

log μg HIy in OWVs

log μg HIy in OMVs

(a)

log g free-Hly

0.0001 0.0010.010.11101001000

% hemolysis

100

120

(b)

Figure 6. Hemolysis induced by OMVs associated-HlyA and

free-HlyA on erythrocytes of different species. Hemolytic act-

ivity of OMVs associated-HlyA (a) and free-HlyA (b), tested

with rabbit erythrocytes (●) and horse erythrocytes (○).

V. Herlax et al. / HEALTH 2 (2010) 484-492

490

data regarding the activity of soluble HlyA secreted by E.

coli, nothing was known regarding the action mechan-

ism of OMVs associated HlyA. First, we demonstrated

in this research, the presence of HlyA associated to

OMVs produced by a laboratory E. coli strains (WAM

1824). We demonstrated the presence of HlyA associated

to these vesicles as seen in SDS-PAGE polypeptide prof-

ile and Western blot analysis employing polyclonal ant-

ibodies directed against purified HlyA (Figure 1). The

toxin associated to OMVs has a specific activity one

order of magnitude higher than the free-HlyA. It is imp-

ortant to note that this protein is the only one respons-

able of the hemolytic activity as OMVs produced by

strain WAM 783 (strain encoding the inactive form of

HlyA, ProHlyA) are not hemolytic.

In order to determine whether the hemolytic action

mechanism of OMVs associated-HlyA implies mem-

brane fusion, we analyzed the lipid mixing between

OMVs and labeled ghost erythrocytes. The small FRET

decrease obtained could be attributed to protein insertion

into lipid bilayer, as similar values were obtained in the

presence of free-HlyA (Figure 2). These values are sim-

ilar to those obtained in the study of HlyA insertion area

using also FRET experiments with labeled membranes

[36]. As for free-HlyA [38], fusion events are not inv-

olved in the action mechanism of the toxin associated

with OMVs; instead, a transfer process from OMVs to

the target membrane must be involved. The transference

of HlyA as a mechanism was confirmed by the presence

of HlyA and by the absence of LPS in the pellet obtained

after incubation of ghost erythrocytes with OMVs (Fig-

ure 3). These results effectively demonstrate that the

hemolysis is caused by the transference of HlyA and not

OMVs-cell membranes fusion.

Although fusion events have been described for leuk-

otoxin from A. actinomycetemcomitans, another RTX

toxin, this could be specific for this RTX toxin [39], as

the secretion of leukotoxin differs from that of other

RTX proteins in that it remains associated with intact

cells even in the presence of functional ltxBD genes [40].

The common mechanism of the lytic action of HlyA

implies the diffusion of a soluble secreted toxin from

bacteria, through aqueous medium, to reach the target

cells. Now, our results demonstrate that a large number

of toxin molecules are concentrated and transported by

OMVs and then transfer to target cells.

The different behavior in the kinetics of hemolysis

observed in Figure 4 for OMVs associated-HlyA and

free-HlyA can be explained considering that, in the first

case, the limiting step is the slow diffusion of OMVs in

the aqueous medium that increases the lag time. Once

OMVs reach the erythrocyte, a large number of toxin

molecules which are concentrated on OMVs, are all tog-

ether transferred to target cells either as a monomer or as

a preassembled oligomer. On the contrary, the limiting

step for free-HlyA is the number of toxin molecules that

reach the target cells, due to the fact that they decrease

as a function of toxin concentration. This can explain the

high specific hemolytic activity for OMVs-associated

HlyA in comparison with the hemolytic activity of free-

HlyA. The fact that HlyA can be transfer in an oligo-

meric structure is possible because we have recently

demonstrated that an oligomer is necessary for the occu-

rrence of hemolysis [41]. What’s more, the adoption of a

cytolytically active oligomeric conformation in OMVs

was described for enterobacterial ClyA cytotoxin [18].

The transference is direct from the OMVs to the eryt-

hrocytes without the delivery of the toxin to the medium.

This is demonstrated by the differences found in the

hemolysis kinetic studies (Figure 4). This conclusion is

also supported by results published by Balsalobre et al.,

who demonstrated that HlyA is tightly associated to

vesicles appearing in a soluble form only after the mem-

brane structure of the OMVs is disrupted by detergents

[8]. This transfer process does not need the presence of a

specific high affinity receptor because the same effect

was observed when LUVs labeled with Rh-PE and

NBD-PE were used (data not shown).

Rabbit and horse erythrocytes, were the most comm-

only erythrocytes used and characterized in previous

studies related with the hemolytic action mechanism of

this toxin. In Figures 6(a) and (b) it can be seen that in

both cases, rabbit erythrocytes are more sensitive than

those of horse, as demonstrated in earlier results on the

characterization of the lytic action of HlyA [37]. For

rabbit erythrocytes a smoother hemolytic curve was ob-

tained in comparison to that of horse erythrocytes. This

fact may be due to the presence of high proportion of

high affinity receptors in the horse erythrocytes in com-

parison to those of rabbit. The higher lytic efficiency of

the toxin with rabbit cells could be due to the presence

of a large number of low affinity binding sites i.e. mem-

brane phospholipids, which facilitate the concentration

of the toxin in the membrane (Herlax et al., unpublished

results).

Currently, the presence of a receptor is a contradictory

point in the elucidation of the action mechanism of this

toxin, probably due that experiments were done with

erythrocytes from different mammalian species. Glyco-

phorin was described as a receptor in horse and human

erythrocytes [42], while another group proposed that the

hemolytic process does not depend on the receptor pres-

ence [43]. Anyway, the same behavior for HlyA against

to erythrocytes of different species is observed for

OMVs associated-HlyA as for free-HlyA.

Finally, it is important to remember that our previous

results support the hypothesis that HlyA is secreted to

the external medium as a LPS-HlyA complex, in which

the main action of LPS is to maintain the protein stabil-

ity in solution, while it only indirectly affects HlyA lytic

V. Herlax et al. / Natural Science 2 (2010) 484-492

491

Openly accessible at

activity [44]. The presence of LPS in the toxin sample

used in those studies, which was obtained by precipita-

tion techniques from a culture supernatant without the

ultracentrifugation step, could be due to some OMVs

contamination. This is also supported by the presence of

phospholipid in toxin samples purified by methods such

as ammonium sulphate precipitation and exclusion chro-

matography [45]. Due to the fact that OMVs associated-

HlyA represents a very high percentage of toxins in the

culture supernatants, results on biological effects of

HlyA published up to date, mainly those in which sublytic

concentrations were used, should be revised as small

amounts of LPS present in the OMVs can stimulate the

release of inflammatory mediators [46].

In conclusion, our results demonstrate that OMVs

constitute an alternative secretion mechanism for HlyA

that result in the toxin reaching higher concentrations

without altering its lytic action mechanism.

The observation that certain virulence factors are enri-

chhed in vesicles suggests that OMVs may have a key

role in bacterial pathogenesis by mediating transmission

of active virulence factors and other bacterial envelop-

ment component to host cells. Numerous OMVs associ-

ated virulence factors have been shown to induce cyto-

toxicity confer vesicle binding to and invasion to host

cells and modulate the immune response. On the other

hand, similar to bacterial-host cell interactions, OMVs-

cell interactions can be altered by manipulating the exp-

ression of outer membrane proteins in bacteria. The ma-

nipulation of OMVs adherence and the possibility to

create chimeras between the N-terminal half of HlyA,

which contains the pore forming domain, and binding

domains to specific proteins in the surface of a cell of

interest (e.g. cancerous cells) should be useful for num-

erous applications, redirecting this engineering OMVs to

specific cell types in order to achieve a desired therapeutic

response.

5. ACKNOWLEDGEMENTS

This work was partially supported by grants from UNLP, CICPBA and

ANPCyT-Argentina. LSB is a member of the “Carrera del Investigador”,

CICPBA, Argentina; VH is a member of the “Carrera del Investigador”,

Consejo Nacional de Investigaciones Científicas y Tecnológicas

(CONICET), Argentina and MFH is a Fellow from CONICET, Argentina.

AMB is a member the “Carrera del Técnico y Profesional de Apoyo”,

CONICET.

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