INFECTION AND IMMUNITY, Oct. 2007, p. 4719–4727
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 75, No. 10
Mannheimia haemolytica Leukotoxin Binds to Lipid Rafts in Bovine
Lymphoblastoid Cells and Is Internalized in a Dynamin-2- and
Dhammika N. Atapattu and Charles J. Czuprynski*
Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin
Received 13 April 2007/Returned for modification 15 June 2007/Accepted 24 July 2007
Mannheimia haemolytica is the principal bacterial pathogen of the bovine respiratory disease complex.
Its most important virulence factor is a leukotoxin (LKT), which is a member of the RTX family of
exotoxins produced by many gram-negative bacteria. Previous studies demonstrated that LKT binds to the
?2-integrin LFA-1 (CD11a/CD18) on bovine leukocytes, resulting in cell death. In this study, we demon-
strated that depletion of lipid rafts significantly decreases LKT-induced bovine lymphoblastoid cell (BL-3)
death. After binding to BL-3 cells, some of the LKT relocated to lipid rafts in an LFA-1-independent
manner. We hypothesized that after binding to LFA-1 on BL-3 cells, LKT moves to lipid rafts and
clathrin-coated pits via a dynamic process that results in LKT internalization and cytotoxicity. Knocking
down dynamin-2 by small interfering RNA reduced both LKT internalization and cytotoxicity. Similarly,
expression of dominant negative Eps15 protein expression, which is required for clathrin coat formation,
reduced LKT internalization and LKT-mediated cytotoxicity to BL-3 cells. Finally, we demonstrated that
inhibiting actin polymerization reduced both LKT internalization and LKT-mediated cytotoxicity. These
results suggest that both lipid rafts and clathrin-mediated mechanisms are important for LKT internal-
ization and cytotoxicity in BL-3 cells and illustrate the complex nature of LKT internalization by the
Mannheimia haemolytica is the principal bacterial agent re-
sponsible for the bovine respiratory disease complex (50). M.
haemolytica produces a leukotoxin (LKT) that is considered its
most important virulence factor. The LKT belongs to the RTX
(repeats in toxin) family of toxins that contain glycine-rich
repeats in the primary structure and are produced by a variety
of gram-negative bacteria (47). LKT binds to the ?2-integrin
LFA-1 (CD11a/CD18) on bovine leukocytes, resulting in
apoptosis or cytolysis of bovine cells (2, 25, 32). This response
is contrary to the usual outcome of cell signaling via CD11a/
CD18, which generally leads to cell activation and survival
rather than cell death (11, 24).
It has been shown that upon stimulation, LFA-1 receptors in
human neutrophils associate in detergent-resistant membrane
microdomains called lipid rafts (14). Lipid rafts are choles-
terol- and sphingolipid-enriched membrane structures that are
approximately 50 to 100 nm in diameter (39). Coalescence of
lipid rafts can result in membrane domains up to 400 nm in
diameter that involve up to 30 to 50% of the cell surface,
depending on the cell type (22, 42). Lipid rafts have been
implicated in several important cellular functions, such as in-
ternalization of receptors and their ligands, signal transduc-
tion, and cholesterol transport. The relationship between
membrane receptors and lipid rafts is highly variable. For ex-
ample, epidermal growth factor and ?2adrenergic receptors
move away from lipid rafts following receptor activation. In
contrast, insulin and angiotensin II receptors move into lipid
rafts following ligand binding, and nerve growth factor and
platelet growth factor receptors do not associate with lipid rafts
Numerous bacterial toxins, bacteria, viruses, and parasites
use lipid rafts and raft-associated caveolae to bind to cells and
be internalized. Examples include Vibrio cholerae toxin, Staph-
ylococcus aureus ?-hemolysin, aerolysin, diphtheria toxin, Sal-
monella enterica serovar Typhimurium, Shigella flexneri, Bru-
cella species, echovirus 1, and simian virus 40 (34, 41).
Alternatively, some bacterial toxins, such as Pseudomonas exo-
toxin A, V. cholerae toxin, and anthrax toxin, target the clath-
rin-mediated endocytic pathway for their internalization (21,
36). One protein common to both the clathrin- and caveola-
mediated endocytic pathways is dynamin, a large GTPase that
is responsible for budding of clathrin-coated pits and for clip-
ping the neck of caveolae from the cell membrane (37, 38). The
three mammalian forms of dynamin (dynamin-1, dynamin-2,
and dynamin-3) are generally localized in the plasma mem-
brane, trans-Golgi network, and endosomes, respectively (6,
In this study, we hypothesize that M. haemolytica LKT binds
to lipid rafts and clathrin-coated pits, followed by internaliza-
tion into bovine lymphoblastoid (BL-3) cells. We show here
that after binding to LFA-1, LKT relocates to lipid rafts in an
LFA-1 independent manner. This results in asymmetrical ag-
gregation of lipid rafts on cells. Raft aggregation and internal-
ization of LKT lead to dynamin-, clathrin-, and actin-depen-
dent cytotoxicity of BL-3 cells. These data demonstrate the
complex participation of the cytoskeleton in LKT-mediated
death of BL-3 cells.
* Corresponding author. Mailing address: Department of Pathobio-
logical Sciences, University of Wisconsin, 2015, Linden Drive, West,
Madison, WI 53706. Phone and fax: (608) 262-8102. E-mail: czuprync
?Published ahead of print on 6 August 2007.
MATERIALS AND METHODS
LKT production and purification. Crude LKT was prepared and purified from
logarithmic-growth-phase culture filtrates as described previously (3). One unit
of LKT activity was defined as the LKT dilution causing 50% killing of 106BL-3
cells when they were incubated at 37°C for 1 h, as determined by trypan blue
exclusion. LKT was stored at ?80°C until it was used. Culture filtrate from an
lktC mutant of M. haemolytica (SH 1562) that produces an LKT protein with no
biological activity was prepared in a manner similar to that used to prepare
culture filtrate from wild-type M. haemolytica strain A1 (generously provided by
S. K. Highlander, Houston, TX).
Cell line and cell culture. BL-3 cells were used as a target for all cytotoxicity
experiments in this study (kindly provided by Ronald Schultz, Madison, WI).
These nonadherent cells were grown at 37°C in the presence of 5% CO2in RPMI
medium supplemented with 10% fetal bovine serum (Gibco BRL, Burling-
LKT cytotoxicity assays. BL-3 cells (106cells per ml) were washed, resus-
pended in antibiotic-free RPMI medium, and incubated at 37°C with 0.2 or 0.5
U of LKT for the indicated time periods. Cell metabolism was quantified using
the Cell Titer 96 AQ one-assay system (Promega Corporation, Madison, WI).
The optical density of the reaction mixture was measured at 450 nm using an
automated enzyme-linked immunosorbent assay reader (Mini plate EL 312; BIO
TEK Instruments Inc., Winooski, VT).
Antibodies and fluorescent probes. A neutralizing monoclonal antibody
(MAb) against LKT (MM601) was a generous gift from S. K. Srikumaran (Wash-
ington State University, Pullman). Anti-LFA-1 MAb (VMRD, Pullman, WA),
anti-flotillin antibody (BD Bioscience, San Jose, CA), anti-dynamin-2 MAb (US
Biologicals, Swampscott, MA), rhodamine-conjugated cholera toxin subunit B
(CTB-Rho) (Molecular Probes, Carlsbad, CA), and anti-clathrin heavy chain
MAb clone X22 or anti-clathrin light chain MAb clone CON.1 (Calbiochem, San
Diego, CA, and Lab Vision, Fremont, CA) were used in this study. Rhodamine-
labeled transferrin (kindly provided by Linda Schuler, Madison, WI) was used to
determine clathrin-mediated internalization in BL-3 cells.
Lipid raft depletion and sequestration. To deplete lipid rafts, BL-3 cells were
incubated with serum-free RPMI medium containing 5 mM methyl-?-cyclodex-
trin (MCD) at 37°C for 30 min. To reconstitute cholesterol in MCD-treated BL-3
cells, the cells were incubated with 10 mM MCD-cholesterol (Sigma Chemicals,
St. Louis, MO) in RPMI medium for 30 min at 25°C (18). Raft sequestration was
achieved by treating BL-3 cells with filipin (4.5 ?g/ml) for 30 min at 37°C.
Dynamin-2 knockdown. Dynamin-2 in BL-3 cells was knocked down with a
small interfering RNA (siRNA) designed for the sequence 5?-GGGATGTCCT
GGAGAACAA-3?, using the T7 RiboMAX Express RNA interference system
(Promega Corporation, Madison, WI). BL-3 cells were transfected with siRNA
using the X-treme transfection reagent (Roche, Nutley, NJ) for 6 h, followed by
further incubation for 72 h at 37°C. Transfected BL-3 cells were used for cyto-
toxicity and immunofluorescence studies. Cells transfected with a scrambled
siRNA (5?-GCCCTGTTCTATAAATATC-3?) served as a negative control. The
reduction in dynamin-2 mRNA knockdown was assessed by an endpoint reverse
transcription-PCR assay (with primers F [GGG AAC CTC TGA CCT CTC
CAA] and R [CTG GTG CTG ATG TCC CCA AT]) and with immunoblotting.
Clathrin coat inhibition. Clathrin coat formation was inhibited using domi-
nant negative Eps15 plasmid constructs (D111); control cells were incubated with
a control plasmid (?D111). Both plasmids were generous gifts from Linda
Schuler (Madison, WI). Plasmids were transfected into BL-3 cells by incubation
for 6 h at 37°C with the FuGENE HD transfection reagent according to the
protocol supplied with the reagent (Roche, Nutley, NJ).
Chemical inhibition of clathrin internalization was achieved by K?depletion.
Briefly, cells were incubated three times for 10 min at 37°C in isotonic K?-free
depletion buffer (140 mM NaCl, 20 mM HEPES-NaOH [pH 7.4], 1 mM CaCl2,
1 mM MgCl2, 1 mg/ml glucose, 0.5% bovine serum albumin). Potassium-de-
pleted BL-3 cells were incubated with LKT or rhodamine-labeled transferrin (a
generous gift from Linda Schuler, Madison, WI). In some experiments actin
depolymerization was achieved by incubating BL-3 cells in RPMI medium with
cytochalasin D (2 ?g/ml; Sigma) for 1 h at 37°C.
Transferrin internalization studies. To demonstrate inhibition of clathrin
internalization, BL-3 cells were depleted of K?by incubation at 4°C for 1 h in
K?-free buffer. Control cells were incubated in normal RPMI medium or in
K?-replete buffer. The cells were then incubated with rhodamine-conjugated
transferrin (30 ?g/ml) or LKT (0.5 U) for 60 min at 37°C and visualized by
Staining for cytoskeletal actin with phalloidin-FITC. BL-3 cells were stained
with phalloidin-fluorescein isothiocyanate (FITC) according to the protocol pro-
vided by Molecular Probes (protocol MP00352). Briefly, BL-3 cells were fixed
with 2% paraformaldehyde for 10 min, washed three times with phosphate-
buffered saline (PBS), and permeabilized with cold acetone for 10 min. Cells
were washed three times with PBS and stained with 20 ?M phalliodin-FITC for
20 min at 25°C. Following this the cells were washed three times with PBS and
visualized by fluorescent microscopy at an emission wavelength of 480 nm.
Detergent extraction and flotation of lipid rafts. Detergent extraction and
flotation of BL-3 cell membranes were performed using a slight modification of
a previously described method (30). Briefly, 106BL-3 cells were incubated with
0.5 U of LKT for 30 min at 37°C. The cells were washed three times with PBS and
lysed using a mechanical homogenizer, and the lysate was incubated on ice for 30
min in TNE buffer (25 mM Tris-HCl [pH 7.4], 150 mM NaCl, 5 mM EDTA, 1
mM dithiothreitol, protease inhibitor mixture, 1% Triton X-100). Samples were
mixed with 40% Optiprep (Nycomed, Altana Inc., New Jersey) and overlaid with
30, 20, 10, and 0% Optiprep to obtain a discontinuous gradient. Samples were
then centrifuged at 55,000 rpm in a TLS 55 rotor (Beckman, Fullerton, CA) for
4 h at 4°C. Five fractions were collected from the top to bottom of the tube
(designated fractions 1 to 5 from the top to the bottom of the tube). Proteins
were precipitated with a chloroform-methanol extraction method and analyzed
by Western immunoblotting (48).
Raft patching, clathrin staining, and immunofluorescence confocal micros-
copy. BL-3 cells were incubated with 0.5 U of LKT for 30 min, washed three
times with PBS, and fixed with 2% paraformaldehyde for 10 min at 25°C. Cells
were washed three times with PBS and incubated with CTB-Rho for 1 h at 25°C,
which was followed by incubation with anti-LKT-FITC for 1 h. Cells were washed
three times with PBS and examined using laser confocal microscopy. Clathrin
staining was done in a similar manner using anti-clathrin heavy chain MAb.
Images were analyzed with Image J software (NIH; http://rsb.info.nih.gov/ij).
Determination of LKT internalization by flow cytometry. BL-3 cells were
incubated with 0.5 U of LKT for the indicated times. Cells were washed three
times with PBS and fixed with 2% paraformaldehyde for 10 min at 25°C. Some
samples were permeabilized with 1% Triton X-100 for 15 min. Cells were then
stained with anti-LKT MAb (MM601), followed by FITC-labeled anti-mouse
immunoglobulin G (IgG). Flow cytometric analysis was performed with both
groups of cells (FACScan; Becton Dickinson, Franklin Lakes, NJ), and the cell
population of interest was gated on the basis of forward- and side-scatter light
characteristics. The fluorescent intensity of this population was determined and
used for the analysis. Heat-inactivated LKT (inactivated by heating at 100°C for
10 min) and M. haemolytica producing a biologically inactive lktC mutant toxin
were used as controls (data not shown).
Statistical analysis. Group means were compared by analysis of variance,
followed by the Tukey-Kramer pairwise comparison test, using the Instat statis-
tical package (GraphPad, San Diego, CA). The level of statistical significance
was set at P ? 0.05.
Lipid raft depletion inhibits LKT-mediated cytotoxicity. We
first investigated the role of lipid rafts in LKT-mediated cyto-
toxicity by depleting and sequestrating cholesterol in BL-3 cells
using MCD and filipin, respectively. We observed 57 and 50%
reductions in cytotoxicity following MCD and filipin treat-
ment, respectively. Cholesterol reconstitution of MCD-treated
BL-3 cells restored LKT-mediated cytotoxicity to control levels
LKT colocalizes with lipid rafts and clathrin in BL-3 cells.
Next we used discontinuous Optiprep gradient ultracentrifu-
gation to fractionate lipid raft and nonraft membrane fractions
from LKT-treated BL-3 cells. LKT was detected by immuno-
blot analysis of the lipid raft samples recovered from the less
dense fractions of the Optiprep gradient. As determined by
densitometry, approximately 47% of the LKT was associated
with the lipid raft fraction. A small amount of LKT (4%)
colocalized with LFA-1, and the remainder associated with
neither LFA-1 nor raft fractions (Fig. 2). In contrast, 98% of
the LKT was found in the bottom fraction of the gradient when
we used membrane lysates from cholesterol-depleted BL-3
cells (MCD treated) that lacked lipid rafts (Fig. 2A). The
relatively small portion of LKT associated with LFA-1 suggests
4720 ATAPATTU AND CZUPRYNSKIINFECT. IMMUN.
that binding of LKT to LFA-1 is a transient process. LKT not
associated with either lipid rafts or LFA-1 was found in the
higher-density membrane fraction, associated with clathrin
(Fig. 2C) (23).
Unclustered lipid rafts are too small to be visualized by light
microscopy (51). Once stimulated, they form aggregates up to
400 nm in diameter that enable them to be visualized by flu-
orescent tagging of raft-associated glycosylphosphatidyliniso-
tol-anchored proteins. Using CTB-Rho (which binds to a GM1
ganglioside receptor) as a marker for lipid rafts, we observed
colocalization of LKT with lipid rafts on BL-3 cells by confocal
microscopy. Confocal micrographs revealed a focal distribu-
tion of LKT on lipid rafts, with raft aggregation and localiza-
tion visible at one pole of the cell. However, not all cells
stained positive for either LKT or CTB-Rho, which may indi-
cate that there is heterogeneity of ganglioside receptor expres-
sion on the surface of BL-3 cells (Fig. 3). Staining for clathrin-
demonstrated colocalization of clathrin with LKT in BL-3 cells.
LKT is internalized into BL-3 cells. Binding of proteins to
lipid rafts may result in trafficking of proteins into the cell.
Therefore, we next investigated whether LKT remains on the
cell surface of BL-3 cells. Surface staining for LKT on BL-3
cells began to decrease by about 30 min, while the total signal
for LKT (determined by staining permeabilized BL-3 cells)
remained unchanged (Fig. 4). These findings suggest that once
bound to the cell, LKT is internalized and remains within the
cell for at least 1 h.
LKT-mediated cytotoxicity and internalization is dynamin,
clathrin, and actin dependent. Next we investigated possible
pathways by which LKT might be internalized into BL-3 cells.
Using siRNA to knock down dynamin-2 expression, we re-
duced LKT-mediated cytotoxicity by approximately one-half
the value for control LKT-treated BL-3 cells. Using FITC-
conjugated anti-LKT MAb and fluorescent microscopy, we
FIG. 1. Depletion and sequestration of lipid rafts in BL-3 cells
inhibit LKT-mediated cytotoxicity. BL-3 cells were pretreated for 30
min at 37°C with either 5 mM MCD (to deplete cholesterol) or 4.5
?g/ml filipin (to sequester lipid rafts). Cells were then incubated with
LKT (0.5 U) for 1 h. Cell viability was measured in a cytotoxicity assay
as described in Materials and Methods. Some MCD-treated BL-3 cells
were incubated with MCD-10 mM cholesterol (to reconstitute lipid
rafts) before exposure to LKT. BL-3 cells incubated with MCD, filipin,
or MCD-cholesterol alone were included as controls. Data are ex-
pressed as means and standard errors of the means of three separate
experiments. An asterisk indicates that the P value is ?0.05 for a
comparison with BL-3 cells exposed to LKT alone.
FIG. 2. LKT associates with detergent-resistant membrane fractions (lipid rafts) in BL-3 cells. A total of 106BL-3 cells were incubated with
LKT (0.5 U) for 30 min at 37°C and then lysed with TNE buffer. The lysates were centrifuged at 10,000 ? g for 15 min. Detergent-resistant
membrane fractions were isolated on Optiprep discontinuous gradients, and five fractions were collected from the top to the bottom (designated
fractions 1 to 5). In panel A, protein was extracted from each fraction, and Western immunoblotting was performed for LKT, LFA-1, and flotillin.
BL-3 cells depleted of cholesterol by MCD treatment and lipid raft fractions from non-LKT-treated BL-3 cells were included as controls and
probed with anti-LFA-1 and anti-flotillin antibodies. In panel C, protein was extracted from each fraction, and Western immunoblotting was
performed for LKT and clathrin light chain. This allowed us to characterize LFA-1 and LKT in raft and nonraft fractions. In panel B, the intensities
of LKT bands in raft and nonraft fractions in panel A were quantified using LabWorks analysis software and are expressed as means and standard
errors of the means of three separate experiments.
VOL. 75, 2007M. HAEMOLYTICA LKT TRAFFICKING BY LIPID RAFTS4721
also demonstrated a significant reduction in LKT internaliza-
tion by siRNA-treated BL-3 cells (Fig. 5).
Dynamin-2 is a membrane scission protein that participates
in both lipid raft caveola-dependent and clathrin-dependent
vesicle trafficking in cells. We assessed the role of dynamin-2 in
LKT-mediated cytotoxicity by several methods. We first ad-
dressed the role of the Eps15 protein, which along with the
AP-2 adaptor complex forms an integral part of the clathrin-
coated pit. To do this, we used a dominant negative Eps15 DIII
protein that lacks the Eps15 homology domains and coiled coil
regions that are required for the assembly of clathrin pits (4, 5).
LKT-mediated cytotoxicity was significantly reduced when we
inhibited clathrin coat formation and internalization by trans-
fecting BL-3 cells with a plasmid (Eps15 DIII) that expresses a
dominant negative Eps15 protein (Fig. 6). Immunofluores-
cence microscopy confirmed that LKT internalization was also
significantly decreased in Eps15 DIII plasmid-transfected cells
compared to cells transfected with a control ?DIII Eps15 plas-
mid. Inhibition of internalization of transferrin, which is trans-
ported exclusively by a clathrin-mediated pathway, confirmed
that clathrin coat formation was inhibited in dominant negative
Eps15 protein-expressing BL-3 cells. We also used K?deple-
tion to investigate clathrin-mediated internalization of LKT.
K?depletion in BL-3 cells resulted in a 42% reduction in
cytotoxicity (Fig. 7) and a significant reduction in LKT inter-
nalization, as determined by immunofluorescence microscopy
(data not shown).
Finally, we investigated the role of cytoskeletal actin poly-
merization in LKT internalization. Treating BL-3 cells with the
selective inhibitor cytochalasin D reduced LKT-mediated cy-
totoxicity from 61% (control cells) to 18% (cytochalasin D-
treated cells) and similarly reduced internalization of LKT into
BL-3 cells (Fig. 8).
The observations made in this study, together with other
recent reports, demonstrate that the molecular interactions
between RTX toxins and their target cells are complex. Orig-
inally, the primary mechanism of RTX toxin-induced cytotox-
icity was thought to be pore formation in the cytoplasmic
membrane. Later studies demonstrated that the M. haemo-
lytica LKT binds to LFA-1 (CD11a/CD18) before inducing cell
death by apoptosis (3, 10, 20, 25). Binding of LKT to LFA-1
results in phosphorylation of the cytoplasmic tail of CD18 (26).
Signaling through ?2-integrins usually transmits a survival sig-
nal. These disparate findings suggest that LKT may stimulate a
cascade of events that may induce cell death by an uncharac-
FIG. 3. LKT colocalizes with lipid rafts and clathrin pits on BL-3 cells. A total of 106BL-3 cells were incubated with LKT (0.5 U) for 30 min
at 37°C. Cells were then fixed with 2% paraformaldehyde for 10 min, blocked with 3% bovine serum albumin for 20 min, and incubated for 1 h
at 25°C with FITC-conjugated anti-LKT MAb, CTB-Rho (which binds to GM1 associated with lipid rafts), or Texas Red (TR)-conjugated
anti-clathrin heavy chain antibody. Cells were visualized with a confocal microscope. Panel A shows a single cell in which LKT (green) colocalized
with lipid rafts (red). Panel B shows colocalization of LKT with clathrin (red). In panel C, the mean lipid raft fluorescence intensities of images
from 50 randomly selected cells were quantified for LKT-induced raft aggregation, using Image J software (NIH; http://rsb.info.nih.gov/ij).
Bars ? 10 ?m.
FIG. 4. Surface fluorescence for LKT on BL-3 cells decreased with
time, while total fluorescence remained unchanged. A total of 106
BL-3 cells were incubated with LKT (0.5 U) for the indicated times (5
to 60 min) at 37°C and then fixed with 2% paraformaldehyde. Fixed
BL-3 cells were stained with FITC-conjugated anti-LKT antibody for
60 min at 25°C, and the surface fluorescence was analyzed by flow
cytometry (Œ). To quantify total fluorescence of cells (f), LKT-treated
BL-3 cells were permeabilized with cold acetone (?20°C) for 10 min
before labeling with FITC-conjugated anti-LKT antibody. The cells
were then analyzed by flow cytometry. Fluorescence is expressed as the
means ? standard errors of the means of three separate experiments.
An asterisk indicates that the P value is ?0.05.
4722 ATAPATTU AND CZUPRYNSKIINFECT. IMMUN.
terized pathway, perhaps in a receptor-independent manner
(16, 31). More recently, it has been shown that a related RTX
toxin, the hemolysin of Escherichia coli (HlyA), can bind to
target cells independent of its receptor. The physical proper-
ties of the cell membrane have been suggested to play a major
role in HlyA-induced cell death (46).
In this study, we demonstrate that early association of LKT
with lipid rafts and clathrin-coated pits results in LKT inter-
nalization in a dynamin-2- and actin-dependent manner. These
events are essential components of the pathway by which LKT
induces BL-3 cell death. Recent studies of the Actinobacillus
actinomycetemcomitans RTX toxin (LTX) showed that it too
binds to lipid microdomains in a similar manner (15). In our
study, we showed that diminution of lipid rafts by cholesterol
depletion significantly reduced LKT-mediated cytotoxicity.
Membrane fractionation demonstrated that a substantial pro-
portion of the M. haemolytica LKT (?47%) was associated
with lipid rafts. However, an estimated 49% of LKT was asso-
ciated with neither LFA-1 nor the lipid raft-containing mem-
brane fractions. Based on previous reports that clathrin local-
izes mostly in the denser and lower fractions of an Optiprep
density gradient, we suggest that the LKT contained in the cell
membrane fraction that lacks rafts or LFA-1 might be associ-
ated with clathrin-coated pits (23). LFA-1 was not associated
with lipid rafts in either LKT-treated or control BL-3 cells.
Consistent with previous reports, we also demonstrated that
lipid raft depletion of BL-3 cells (by MCD treatment) did not
alter LFA-1 expression (data not shown) (35). These findings,
therefore, suggest that LFA-1 might provide only a temporary
docking site for the initial binding of LKT. Once LKT disso-
ciated from LFA-1, the former moved to lipid rafts, while
LFA-1 remained in the raft-free cell membrane fraction (Fig.
2). This explanation is consistent with previous observations by
Leitinger and Hogg, who found that LFA-1 is excluded from
lipid rafts until the cells are stimulated (33). Movement of
LFA-1 and associated protein complexes (integrin-associated
proteins) to lipid rafts depends on both ligand binding and
release of cytoskeletal constraints. There are other examples of
bacterial toxins translocating from one protein to another on
target cell surfaces. For example, tetanus toxin binds to a
receptor complex which consists of polysialogangliosides
(GD1b and GT1b) and glycosylphosphatidylinositol-anchored
proteins on the neuronal cell surface. Once tetanus toxin binds
to GD1b, it dissociates from this receptor and is then internal-
ized via a clathrin-dependent pathway (8). More detailed stud-
ies are, however, needed to definitively demonstrate lateral
translocation of LKT from LFA-1 to lipid rafts.
Using confocal microscopy, we demonstrated that lipid raft
aggregation occurred asymmetrically on the BL-3 cell, at sites
where LKT also appeared to accumulate. Interestingly, not all
FIG. 5. siRNA knockdown of dynamin-2 reduces LKT-mediated cytotoxicity and internalization in BL-3 cells. Dynamin-2 was knocked down
by transfecting BL-3 cells with siRNA (0.2, 0.5, or 2.0 ?g) for 6 h, followed by further incubation in RPMI medium with 10% fetal bovine serum
for 72 h. Control cells were transfected with a scrambled siRNA sequence for the same time period. To assess the degree of dynamin-2 RNA
knockdown, RNA was extracted, and a one-step reverse transcription-PCR assay with dynamin-2-specific primers was performed. In panel A the
products were visualized by electrophoresis in a 1% agarose gel. In panel B, dynamin-2 siRNA- or scrambled siRNA-transfected BL-3 cells (106)
were incubated with LKT (0.5 U) for 1 h, and cytotoxicity was measured as described previously. The data are the means and standard errors of
the means of five separate siRNA transfection experiments. An asterisk indicates that the P value is ?0.05 for a comparison with the scrambled
siRNA control. Panel C shows an immunoblot analysis of dynamin-2 knockdown from BL-3 cells transfected with 2 ?g of dynamin-2 siRNA. In
panel D, BL-3 cells transfected with dynamin-2 siRNA (2.0 ?g) or with scrambled siRNA were fixed, permeabilized with cold acetone, blocked
with 3% bovine serum albumin, and incubated with anti-LKT MAb. A goat anti-mouse IgG-Texas Red-conjugated secondary antibody was added,
and the LKT signal was visualized by fluorescent microscopy at excitation and emission wavelengths of 595 and 614 nm, respectively. The arrows
indicate internalized LKT. Bars ? 10 ?m. Conl, control.
VOL. 75, 2007 M. HAEMOLYTICA LKT TRAFFICKING BY LIPID RAFTS 4723
BL-3 cells stained positive for both LKT and lipid rafts (the
latter was assessed by CTB-Rho staining). This finding proba-
bly reflects the unsynchronized growth of BL-3 cells, which are
rapidly multiplying blast cells. Cells at different stages of the
cell cycle may express different amounts of membrane proteins
and lipid rafts (19). Some recent studies show that the lipid raft
marker GM1, which binds CTB, also resides in clathrin-coated
pits on the cell surface (28). Internalization of CTB-Rho,
therefore, could occur via two possibly independent pathways.
This would result in heterogeneous staining for CTB-Rho
among the BL-3 cells. Aggregation of lipid rafts and clathrin
coats with LKT could be an important early event that facili-
tates pore formation, LKT internalization, or signaling that
leads to cell death.
FIG. 6. Expression of a dominant negative Eps15 protein in BL-3 cells inhibits clathrin-dependent internalization of LKT and LKT-mediated
cytotoxicity. Panel A is a schematic representation of the dominant negative mutant Eps15 protein (DIII) and the control Eps15 protein (DIII?2)
expressed in BL-3 cells. In the Eps15 (DIII) construct, the Eps15 homology domains (EH) and N-terminal coiled coil domains that are required
for clathrin coat assembly have been deleted. In panel B, BL-3 cells were transfected with 6 ?g of Eps15 DIII or the control Eps15 DIII ?2 plasmid
for 6 h at 37°C and then incubated with LKT (0.2 U) for 1 h at 37°C. Cytotoxicity was measured as described previously. The data are the means
and standard errors of the means of three separate experiments. The asterisk indicates that the P value is ?0.05. In panel C, BL-3 cells transfected
with the Eps15 DIII or Eps15 DIII ?2 plasmid were stained for cytoplasmic LKT and for transferrin (which is internalized by the clathrin-mediated
pathway). BL-3 cells incubated with LKT were fixed, permeabilized, blocked with 3% bovine serum albumin, and incubated with FITC-conjugated
anti-LKT MAb and rhodamine-conjugated transferrin. Images were visualized by confocal microscopy. The control plasmid-transfected BL-3 cells
demonstrated internalization of transferrin (red) and LKT (green), while the Eps15 DIII plasmid-transfected BL-3 cells showed reduced
internalization of LKT and virtually no signal from transferrin. Cells were counterstained with 4?,6?-diamidino-2-phenylindole (DAPI) nuclear stain
(blue). The images in panel C show representative cells from three separate experiments that were performed. Bars ? 10 ?m.
4724 ATAPATTU AND CZUPRYNSKIINFECT. IMMUN.
Binding of protein toxins to lipid rafts could trigger toxin
internalization and trafficking of ligand-receptor complexes
within cells (21, 49). Internalization of LFA-1 could occur
either independent of or in association with lipid rafts (14).
Our observation that surface staining for LKT on BL-3 cells
decreases with time suggests that once bound to BL-3 cells,
LKT molecules are internalized and do not recirculate to the
cell surface within 60 min. Because only about 4% of LKT was
associated with LFA-1 at any single time point, we infer that
LKT internalization is largely LFA-1 independent and perhaps
dependent on both lipid raft and nonraft membrane fractions.
However, recent studies showed that LFA-1 is internalized and
recirculated within peripheral blood neutrophils (14). Thus, we
cannot definitively exclude the possibility that a small fraction
of LKT trafficked into the BL-3 cells along with LFA-1.
Our investigation provided evidence for the importance of
dynamin-2 in lipid raft- and clathrin-mediated LKT internal-
ization. Dynamin-2 is a large GTPase that is responsible for
clipping off both caveolae and clathrin vesicles from the cell
membrane (45). Our data suggest that dynamin-2 plays a role
in LKT internalization, possibly by modulating the cytoskele-
ton-microtubule network. This in turn could lead to formation
of transport vesicles that contain LKT. The role of clathrin in
trafficking of other bacterial toxins into the cell is well docu-
mented (1, 21). Furthermore, there is now evidence that de-
pletion or sequestration of cholesterol also depletes cells of
clathrin-coated pits (44). The close relationship between lipid
rafts and clathrin coats makes it difficult to distinguish between
these two pathways of protein trafficking (8, 29). Factors that
determine whether LKT is transported via one pathway or the
other are not yet defined, nor is it certain that these pathways
are mutually exclusive. Perhaps the dynamics of LKT move-
ment on the cell membrane determines the pathway by which
LKT is internalized into BL-3 cells.
It was interesting to note that knocking down dynamin-2 had
approximately the same impact on LKT-mediated cytotoxicity
of BL-3 cells (50% reduction in cytotoxicity) as inhibiting
clathrin-coated vesicle formation had (42 to 66% reduction in
cytotoxicity). Attempts to inhibit both pathways (combined
dynamin-2 and clathrin knockdown) failed, due to a high rate
of BL-3 cell death. Thus, we cannot exclude the possibility that
LKT is internalized via both clathrin- and lipid raft-indepen-
dent pathways. Furthermore, recent investigations revealed
that numerous other mechanisms may be involved in protein
trafficking into cells (29). For example, endocytosis in neuronal
cells and lymphocytes occurs via lipid rafts that lack caveolae
(12, 52). In our study, we were unable to demonstrate the
presence of caveolae in BL-3 cells using immunoblotting and
immunofluorescence techniques (data not shown). Although
this does not completely exclude the possibility of caveolae in
BL-3 cells, it seems likely that internalization of LKT is lipid
raft associated but independent of caveolae.
An unexpected observation in this study was that LKT bind-
ing decreased when BL-3 cells were treated with cytochalasin
D or clathrin coat formation was inhibited. As suggested in
recent reports, perhaps actin and related cytoskeletal proteins
FIG. 7. Potassium depletion in BL-3 cells inhibits LKT-mediated
cytotoxicity. BL-3 cells were depleted of K?by incubation in K?-free
buffer for 60 min. Cells were then incubated with LKT, and cytotoxicity
was measured. BL-3 cells treated with heat-inactivated LKT and K?-
depleted BL-3 cells alone served as controls. The data are the means
and standard errors of the means of three separate experiments per-
formed. The asterisk indicates that the P value is ?0.05 for a compar-
ison with K?-depleted BL-3 cells. K?-depleted BL-3 cells demon-
strated a significant reduction in tranferrin and LKT internalization
(data not shown).
FIG. 8. Cytochalasin D treatment inhibits LKT-mediated cytotox-
icity and LKT internalization in BL-3 cells. In panel A, ?-actin was
depleted by treating BL-3 cells with cytochalasin D (Cytochal D) (2
?g/ml) for 60 min. Cells were then incubated with LKT, and cytotox-
icity was assessed as described in the text. The data are as the means
and standard errors of the means of three separate experiments per-
formed. The asterisk indicates that the P value is ?0.05 for a compar-
ison with LKT alone. In panel B, intracellular staining for LKT was
performed using an anti-LKT MAb and Texas Red-conjugated anti-
mouse IgG. FITC-conjugated phalloidin was added to visualize actin
filaments, and BL-3 cells were visualized by fluorescent microscopy.
Cytochalasin D-treated cells showed a reduction in LKT internaliza-
tion (red) and staining for ?-actin (green). Cells were counterstained
with DAPI nuclear stain (blue). BL-3 cells alone or treated with cy-
tochalasin D were used as controls. Bar ? 10 ?m.
VOL. 75, 2007 M. HAEMOLYTICA LKT TRAFFICKING BY LIPID RAFTS4725
were responsible for surface clustering or membrane expres-
sion of LFA-1 (17, 40). Inhibition of actin polymerization re-
duced LKT-mediated cytotoxicity and LKT internalization in
BL-3 cells. This finding indicates that cytoskeletal proteins play
a crucial role in either LKT association with membrane lipid
rafts or internalization of LKT-bearing vesicles into BL-3 cells.
Actin and talin tethered to the cytoplasmic tail of LFA-1 are
responsible for its movement on the cell surface, while ?-actin
is responsible for the internalization of clathrin-coated vesicles
following membrane excision by dynamins (13). Dynamins
have been shown to relax the integrin-stress fiber connection,
which in turn allows focal adhesion components to disassemble
and integrins to be endocytosed or move across the cell mem-
brane (9). Thus, knocking down dynamin would inhibit disas-
sembly of membrane receptor complexes in BL-3 cells, thereby
preventing vesicle endocytosis. Inhibition of actin polymeriza-
tion in BL-3 cells seemed to have an effect on LKT internal-
ization similar to the effect of reduction of dynamin. This
observation is consistent with the hypothesis that actin fibers
serve two apparently disparate functions in the cell: holding
membrane integrins in place and promoting vesicle endocyto-
sis (27, 43).
In summary, the cellular mechanisms by which RTX toxins
cause cell damage are complex and intriguing. Our study indi-
cates that after LKT binds to LFA-1, LKT relocation to lipid
rafts, raft clustering, clathrin pit formation, and dynamin-2-
and actin-dependent internalization of LKT are a series of
dynamic events that ultimately lead to death of BL-3 cells. To
our knowledge, this is the first report of an RTX toxin which
employs lipid rafts and clathrin-coated pits for its internaliza-
tion and cytotoxicity. Future investigations should identify ad-
ditional events that are involved in the cytotoxicity of this
reported group of toxins.
We thank Linda Schuler and Timothy Piazza (Madison, WI) for
providing Eps15 plasmids and S. Srikumaran (Pullman, WA) for gen-
erously providing the anti-LKT MAb (MM601). We thank David J.
McClenahan (Madison, WI) for assisting with flow cytometry and
Dagmara I. Kisiela (Madison, WI) and Erica Behling-Kelly (UT
Southwestern Medical Center) for critical reading of the manuscript.
This work was funded by grants 2004-14841 and 2006-17522 from
the USDA National Research Initiative, by grant WIS04884 from the
Wisconsin Agricultural Experiment Station, and by the Walter and
Martha Renk Endowed Laboratory for Food Safety.
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Editor: D. L. Burns
VOL. 75, 2007M. HAEMOLYTICA LKT TRAFFICKING BY LIPID RAFTS 4727