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
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Editor: D. L. Burns
VOL. 75, 2007M. HAEMOLYTICA LKT TRAFFICKING BY LIPID RAFTS 4727