, 332 (2012);
Somshuvra Mukhopadhyay and Adam D. Linstedt
Protects Against Shiga Toxicosis
Manganese Blocks Intracellular Trafficking of Shiga Toxin and
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Manganese Blocks Intracellular
Trafficking of Shiga Toxin and
Protects Against Shiga Toxicosis
Somshuvra Mukhopadhyay and Adam D. Linstedt*
Infections with Shiga toxin (STx)–producing bacteria cause more than a million deaths each
year and have no definitive treatment. To exert its cytotoxic effect, STx invades cells through
retrograde membrane trafficking, escaping the lysosomal degradative pathway. We found
that the widely available metal manganese (Mn2+) blocked endosome-to-Golgi trafficking of
STx and caused its degradation in lysosomes. Mn2+targeted the cycling Golgi protein GPP130,
which STx bound in control cells during sorting into Golgi-directed endosomal tubules that
bypass lysosomes. In tissue culture cells, treatment with Mn2+yielded a protection factor of 3800
against STx-induced cell death. Furthermore, mice injected with nontoxic doses of Mn2+were
completely resistant to a lethal STx challenge. Thus, Mn2+may represent a low-cost therapeutic
agent for the treatment of STx infections.
more than 150 million individuals each year
and cause more than a million deaths (1). There
is no definitive medical treatment. Indeed, treat-
ment with antibiotics is contraindicated because
it increases the risk of STx release and life-
threatening disease (1–3). STx consists of a cat-
alytically toxic A subunit bound to a B subunit
higa toxin (STx)–producing bacteria of
the Shigella genus and enterohemorrhagic
Escherichia coli (EHEC) species infect
that mediates its membrane trafficking from
the cell surface through endosomes, the Golgi
apparatus, and the endoplasmic reticulum (ER),
where the toxin translocates to the cytosol and in-
activates ribosomes (4). Direct trafficking of STx
endosomes and lysosomes, is a crucial step that
allows STx to avoid degradation. Small-molecule
inhibitors targeting this step hold therapeutic prom-
ise, but the STx interactions responsible for its
endosomal sorting remain unclear.
Exposure of cells to 50 to 500 mM manga-
nese (Mn2+) induces degradation of GPP130 (5, 6),
a membrane protein that cycles between the Golgi
and endosomes (7, 8). Because GPP130 plays
a role, albeit undefined, in endosome-to-Golgi
trafficking of STx (9), we asked whether Mn2+
acts as a small-molecule inhibitor of STx. To
investigate the effect of Mn2+on STx trafficking,
we used a fluorescently tagged version of the B
subunit of STx (STxB) that exhibits transport
kinetics identical to the holotoxin (10, 11). Where-
as STxB efficiently trafficked from the cell sur-
face to the Golgi in control cells, cells exposed to
500 mM Mn2+lacked GPP130, and STxB accu-
mulated in peripheral punctate structures resem-
bling endosomes (Fig. 1, A and B). We confirmed
that STxB did not reach the ER (fig. S1) by using
a STxB version with a KDEL tag, which increases
its retention in the ER (12).
The Mn-induced block in STxB trafficking
was specific. There was no difference between
Mn2+-treated and control cells in cholera toxin
B subunit (CTxB) trafficking (Fig. 1C and fig.
S2), which follows the same route as STx to the
Golgi (13). Moreover, Mn2+did not affect epi-
dermal growth factor (EGF) internalization and
degradation (fig. S3) or ER-to-cell-surface traf-
ficking of vesicular stomatitis virus G protein
(fig. S4). Further, Mn2+did not alter the localiza-
tion or trafficking of GP73 and TGN46, endog-
enous proteins that constitutively cycle between
the Golgi and endosomes, or Lamp2, which traf-
fics from the Golgi to lysosomes (fig. S5). Fi-
nally, 500 mM Mn2+did not affect cell viability
The accumulation of STxB in intracellular
punctae implies that Mn2+blocks STxB after
internalization. Indeed, STxB moved to Rab5-
positive early endosomes and, instead of exit-
ing to the Golgi, trafficked to Rab7-positive
Department of Biological Sciences, Carnegie Mellon Univer-
sity, Pittsburgh, PA 15213, USA.
*To whom correspondence should be addressed. E-mail:
Fig. 1. Manganese specifically blocks STxB trafficking. (A) STxB transport
for the indicated times in HeLa cells untreated or pretreated with 500 mM
Mn2+for 4 hours. Scale bar, 10 mm. (B and C) Percentage of cellular STxB
or CTxB in the Golgi at the indicated times postinternalization (mean T SE;
20 cells per point). See fig. S2 for CTxB images. (D) Normalized cell viability
by methylthiazolylphenyl-tetrazolium bromide (MTT) assay. Mn2+was 500 mM
for 12 hours (mean T SE; n = 3 experiments).
20 JANUARY 2012VOL 335
on June 16, 2012
late endosomes (Fig. 2A). Mn2+did not alter
Rab5 and Rab7 distribution (fig. S6). Exit to the
Golgi involves tubules that extend from early
endosomes (14). To determine whether Mn2+
blocks this step, we compared the early endo-
some localization of internalized STxB to EGF,
which is excluded from tubular extensions and
traffics to late endosomes. As expected, STxB
was detected in endosomal tubules in control
cells while EGF was excluded (Fig. 2, B and C,
and fig. S7). STxB tubules contained the retromer
component SNX-1 (fig. S8), confirming that
they were bona fide Golgi-directed transport in-
termediates (14). GPP130 was also detected on
STxB tubules and endosomes when trafficking
of both proteins was synchronized (fig. S9). The
sorting of STxB into endosomal tubules was
abolished in Mn2+-treated cells (Fig. 2, B and C).
The block in tubulation appeared specific because
the sorting of CTxB into endosomal tubules was
not affected (fig. S10). Sorting of STxB into en-
dosomal tubules was also blocked after small inter-
fering RNA–induced depletion of GPP130 (fig.
S11), suggesting that GPP130 is the target of Mn.
The presence of STxB in Rab7-positive en-
dosomes suggests that Mn2+may induce toxin
degradation. Indeed, Mn2+caused a dramatic
loss of STxB (Fig. 2D and fig. S12). Degrada-
tion occurred after a lag and was blocked by
dominant-negative Rab7 (Rab7-T22N), imply-
ing that STxB trafficked to, and was degraded
in, lysosomes (Fig. 2D). Rab7-T22N did not
affect STxB trafficking in control cells (fig. S13)
because STxB normally bypasses late endosomes
(15). Thus, Mn2+diverts STxB to late endosomes
and lysosomes, where it is degraded. This is im-
portant from a therapeutic perspective because
Mn-treated cells will not contain residual toxin
that could escape to the cytosol over time.
To determine whether GPP130 is the Mn2+
target, we used a rescue approach with the goal of
restoring endosome-to-Golgi trafficking of STxB
by expression of an Mn2+-insensitive GPP130 con-
struct. GPP130 contains a short cytosolic domain,
a single transmembrane domain, a coiled-coil
lumenal stem domain, and an acidic C terminus
(fig. S14). The stem contains three targeting de-
terminants: residues 36 to 87 and 176 to 245
confer Golgi localization, and residues 88 to 175
mediate endosome-to-Golgi cycling (16). Deletion
of any of these makes GPP130 Mn2+insensitive
(5). Based on this, we generated GPP1301-175-
GFP (green fluorescent protein) and verified that
it was Mn2+insensitive but retained its ability
to traffic between the Golgi and endosomes
(fig. S15). Expression of this construct restored
the ability of STxB to traffic to the Golgi after
Mn2+(Fig. 3, A and B), indicating that GPP130
was the target of Mn2+. As a control, we gener-
ated GPP130∆88-175-GFP, an Mn2+-insensitive
construct that lacked residues 88 to 175 required
for endosome-to-Golgi cycling. This construct
failed to traffic out of the Golgi (fig. S15) and
also failed to restore STxB trafficking (Fig. 3,
A and B).
Of the known cellular factors involved in
STxB trafficking, all but GPP130 are cytosolic,
raising the possibility that STx evolved to avoid
lumen of early endosomes. Indeed, we observed
robust, direct, and specific binding of STxB to
the GPP130 stem domain exhibiting a dissocia-
tion constant (Kd) of 150 nM (Fig. 3, C and D).
Binding mapped to residues 36 to 87 (Fig. 3, E
and F). Because deleting these residues yielded a
construct, GPP130∆36-87-GFP, that cycled yet was
Mn2+insensitive (fig. S15), we could use the res-
cue assayto test the functional importance of STx
binding to GPP130. GPP130∆36-87-GFP failed to
rescue STxB trafficking to the Golgi (Fig. 3, A
and B), indicating that the GPP130 lumenal stem
domain directly interacts with STxB to mediate
STxB sorting into endosomal tubules (schema-
tized in fig. S16). It is possible that other toxins
similarly coopt proteins in the GPP130 pathway
to escape degradation.
To determine whether Mn2+protects cells
against Shiga toxicity, we first performed a dose-
response analysis in control cells using STx1.
STx1, secreted by EHEC, has a B subunit iden-
tical to STx secreted by Shigella, and the A
subunit differs in only one position (17). As ex-
pected (18), the lethal dose (LD50) of STx1 was
0.05 ng/ml (Fig. 4A). Mn2+protected STx1-treated
HeLa cells (Fig. 4, A and B, and fig. S17A),
and 50% cell death was not evident, even at a
concentration 2000 times as high as the LD50.
The estimated protection factor was 3800 (Fig.
4B). Mn2+by itself did not compromise viabil-
ity (fig. S17).
Finally, we investigated whether Mn2+pro-
tects mice during lethal Shiga toxicity. Intra-
peritoneal injection was used because this model
has a definitive end point (death in 3 to 4 days)
and recapitulates features of STx-induced renal
Fig. 2. Manganese alters sorting at endosome, causing STxB degradation.
(A) STxB localization compared with Rab5-GFP or Rab7-GFP at the indi-
cated times postinternalization in Mn2+-treated cells. Scale bar, 4 mm. Arrows
indicate overlap. (B) Endosomal tubulation of STxB in comparison with EGF at
the indicated times postinternalization. See fig. S7A for full images. Scale
bar, 1 mm. (C) Percentage of endosomes per cell with STxB tubules at the
indicated times (mean T SE; 10 cells per point; 100 to 150 endosomes per
cell). See fig. S7B for EGF tubules. (D) Cellular STxB at the indicated times
normalized to the control at 0 hours postinternalization (mean T SE; 15 cells
per point). See fig. S12 for images.
VOL 335 20 JANUARY 2012
on June 16, 2012
damage evident in humans (19–21). To identify
a test dose for Mn2+, a concentration series was
injected that yielded an apparent LD50of 125
mg per kg of weight (mg/kg) (Fig. 4C). Because
Mn2+is cleared from the system within hours
(22), we also tested daily injections, and doses
up to 50 mg/kg were not toxic and did not change
body weight (Fig. 4D). Thus, as a proof of pro-
tection dose, we used 50 mg/kg Mn2+once
daily beginning 5 days before STx1 exposure,
followed by 25 mg/kg Mn2+once daily after
toxin exposure. Life-threatening complications
of STx infections in humans develop days after
onset of enteric symptoms, providing an oppor-
tunity for treatment after diagnosis. Each mouse
received a single injection of 25 mg/kg STx1.
Mice with no Mn2+treatment became agitated
and restless within 24 hours, lost 5 to 10% of
body weight at 48 to 72 hours (Fig. 4E), and
died at 72 to 96 hours (Fig. 4F). In contrast, all
Mn-treated animals remained healthy and sur-
vived for the duration of the study (Fig. 4, E and
F). Complete protection was also evident with
daily Mn2+injections of 25 mg/kg and 10 mg/kg,
Fig. 3. GPP130 is the cellular Mn2+target and directly binds STxB to mediate
its endosome-to-Golgi trafficking. (A) STxB localization 30 min postinternalization
in Mn-treated cells expressing the indicated constructs. Endogenous GPP130
was detected using an antibody to the acidic domain. Scale bar, 10 mm. (B)
Percentage of cellular STxB in Golgi from (A). Data for control and Mn2+
groups without GPP130 transfection are replotted from Fig. 1B (mean T SE;
15 cells each). (C and D) Quantitation of His-STxB recovery after incubation
with the indicated concentrations of either glutathione S-transferase (GST) or GST-GPP13036-247and the corresponding Coomassie-stained gels. (E and F)
Coomassie-stained gels and quantitation of His-STxB recovery after incubation with 5 mM of the indicated GST constructs (mean T SE; n = 3 experiments).
Fig. 4. Treatment with Mn2+protects against STx1-induced death. (A) Cell
viability by MTT assay after 24 hours exposure to STx1 at the indicated con-
centrations. Mn2+sample was pretreated with 500 mM for 4 hours and then Mn2+
was reduced to 125 mM during STx1 exposure (mean T SE; n = 3 experiments).
(B) LD50with boxed 95% confidence interval from (A). (C) Fraction of mice
surviving injection of Mn2+at the indicated concentration (n = 4 mice per dose).
(D) Body weight change after 96 hours of daily Mn2+injections at the indicated
concentrations (n = 4 mice per dose, except n = 2 for 100 mg/kg). Only animals
with depressed body weight exhibited locomotive or any other abnormalities. (E)
Body weight change of mice injected with STx1 only (n = 6 mice) or STx1 and
Mn2+at the indicated concentration (n = 6 mice for 50 mg/kg Mn2+and 4 for
other doses). Final weight recorded on day of death or on day 8 for survivors.
(F) Survival curves from (E). (G) Histology of kidneys by toluidine blue staining
(scale bar, 100 mm; inset, 4X) and electron microscopy (scale bar, 10 mm).
20 JANUARY 2012VOL 335
on June 16, 2012
but not 1 mg/kg (Fig. 4, E and F). As expected
(21), histologic examination of the kidneys of
STx1-treated mice revealed extensive damage in
the cortical convoluted tubules, whereas animals
protected by Mn2+showed no STx1-induced renal
damage (Fig. 4G). Thus, Mn2+effectively protects
against STx-induced toxicity and death in vivo
even during fulminant systemic toxicosis.
In conclusion, Mn2+may be effective in the
management of STx infections. In contrast to
other experimental strategies (20, 23–25), Mn2+
is an essential nutrient, its toxicology is well
studied (26, 27), and it is already approved for
oral and intravenous use. The low cost and wide
availability of Mn2+make it amenable for use
in developing countries, where >95% of STx
infections occur. Further, it may be possible to
combine Mn2+with antibiotic therapy because
Mn2+may block the toxic effects of STx re-
leased from dying bacteria.
References and Notes
1. T. Ochoa, T. G. Cleary, in Oski’s Pediatrics: Principles
and Practice, J. A. McMillan et al., Eds. (Lippincott
Williams and Wilkins, Philadelphia, 2006), pp. 1116–1121.
2. K. L. Mohawk, A. R. Melton-Celsa, T. Zangari, E. E. Carroll,
A. D. O’Brien, Microb. Pathog. 48, 131 (2010).
3. C. S. Wong, S. Jelacic, R. L. Habeeb, S. L. Watkins,
P. I. Tarr, N. Engl. J. Med. 342, 1930 (2000).
4. M. E. Fraser, M. M. Chernaia, Y. V. Kozlov, M. N. James,
Nat. Struct. Biol. 1, 59 (1994).
5. S. Mukhopadhyay, C. Bachert, D. R. Smith, A. D. Linstedt,
Mol. Biol. Cell 21, 1282 (2010).
6. S. Mukhopadhyay, A. D. Linstedt, Proc. Natl. Acad. Sci. U.S.A.
108, 858 (2011).
7. A. D. Linstedt, A. Mehta, J. Suhan, H. Reggio, H. P. Hauri,
Mol. Biol. Cell 8, 1073 (1997).
8. S. Puri, C. Bachert, C. J. Fimmel, A. D. Linstedt, Traffic 3,
9. R. Natarajan, A. D. Linstedt, Mol. Biol. Cell 15, 4798 (2004).
10. F. Mallard, L. Johannes, Methods Mol. Med. 73, 209 (2003).
11. Materials and methods are available as supporting
material on Science Online.
12. L. Johannes, D. Tenza, C. Antony, B. Goud, J. Biol. Chem.
272, 19554 (1997).
13. K. Sandvig, B. van Deurs, Gene Ther. 12, 865 (2005).
14. V. Popoff et al., J. Cell Sci. 120, 2022 (2007).
15. F. Mallard et al., J. Cell Biol. 143, 973 (1998).
16. C. Bachert, T. H. Lee, A. D. Linstedt, Mol. Biol. Cell 12,
17. M. E. Fraser et al., J. Biol. Chem. 279, 27511 (2004).
18. I. S. Shin et al., BMB Rep 42, 310 (2009).
19. S. Ishikawa et al., Infect. Immun. 71, 3235 (2003).
20. K. L. Mohawk, A. D. O’Brien, J. Biomed. Biotechnol.
2011, 258185 (2011).
21. V. L. Tesh et al., Infect. Immun. 61, 3392 (1993).
22. H. Suzuki, O. Wada, Environ. Res. 26, 521 (1981).
23. J. B. Saenz, T. A. Doggett, D. B. Haslam, Infect. Immun.
75, 4552 (2007).
24. B. Stechmann et al., Cell 141, 231 (2010).
25. S. Fukuda et al., Nature 469, 543 (2011).
26. J. A. Moreno et al., Toxicol. Sci. 112, 394 (2009).
27. M. Aschner, K. M. Erikson, E. Herrero Hernández,
R. Tjalkens, Neuromol. Med. 11, 252 (2009).
Acknowledgments: We thank D. R. Smith, T. H. Lee, J. S. Minden,
J. L. Brodsky, and M. A. Puthenveedu for advice; N. N. Urban
and J. Dry-Henich for help with animal work; Y. Creeger for
fluorescence-activated cell sorting; S. Truschel and B. Ballou for
protein preparation; and J. Suhan for electron microscopy.
Supported by NIH grant R01GM084111 to A.D.L. and an
American Heart Association fellowship to S.M. A patent on the
use of Mn2+to treat Shiga toxicosis is pending. The data
reported in this paper are tabulated in the main text and in
the supporting online material.
Supporting Online Material
Materials and Methods
Figs. S1 to S17
References (28, 29)
28 October 2011; accepted 5 December 2011
Illusions Promote Mating Success in
Laura A. Kelley1and John A. Endler1,2*
Sexual selection studies normally compare signal strengths, but signal components and sensory
processing may interact to create misleading or attention-capturing illusions. Visual illusions
can be produced by altering object and scene geometry in ways that trick the viewer when seen
from a particular direction. Male great bowerbirds actively maintain size-distance gradients of
objects on their bower courts that create forced-perspective illusions for females viewing their
displays from within the bower avenue. We show a significant relationship between mating success
and the female’s view of the gradient; this view explains substantially more variance in mating
success than the strength of the gradients. Illusions may be widespread in other animals because
males of most species display to females with characteristic orientation and distance, providing
excellent conditions for illusions.
ases in females (2) but can be limited by psy-
chophysics (3), cognitive mechanisms (4), and
species recognition (5). Discussions of signal
evolution normally consider only signal intensity
(2–5), but signal components and receiver sen-
sory processes may interact to create misleading
or attention-capturing illusions (6–8) indepen-
dently of signal strength.
Illusions can arise when the two-dimensional
projection of a scene on the retina corresponds to
nimals produce a vast array of sexual
displays (1). Signal evolution can be
driven by males exploiting sensory bi-
a three-dimensional scene that has geometry dif-
ferent from that of the real scene (6). An object
viewed by an observer subtends an angle f on
the observer’s eye, which is dependent on the
object’s size and distance (Fig. 1 and fig. S1).
When objects of similar size increase in distance
from an observer, their f values decrease (Fig.
1A, Fig. 2B, and fig. S1A), and this information
is unconsciously used to infer the size and dis-
tance of objects (6, 9). Forced-perspective illu-
sions occur when the natural relationship between
distance and f is violated (6–9). A scene where
objects decrease in size as distance increases
(negative gradient) results in more rapidly de-
creasing f than normal, making the scene appear
larger than it is (Fig. 2C), a pattern often found in
architecture [see references in (8)]. Conversely, a
(positive gradient) results in f remaining con-
ly than normal, and the scene appears smaller
(Fig. 2A). Additional illusions may result from
object arrangement (6) (fig. S2), from interac-
tions between objects and perspective cues (6),
and when the viewer’s head is moved (7).
Male bowerbirds construct bowers that serve
only to attract females for mating (10). Females
assess potential mates via various traits, includ-
ing the number and type of colored decorations
(11–13), vocal mimicry (14), and male courtship
bowerbirds (Ptilonorhynchus nuchalis) construct
bowers with an avenue 0.6 m in length, opening
collectively called gesso (Fig. 1C), and the male
presents colored objects over the gesso during
display. Females view males displaying over the
the avenue (10); this predetermined viewing ge-
ometry is an essential requirementfor forced per-
spective (7–9). Males arrange gesso objects on
distance from the bower increases (positive gra-
dient; Fig. 1, B and C), creating forced perspec-
tive for the female within the avenue (8). Forced
perspective could be an honest mate choice sig-
nal because males rapidly restore experimental-
ly reversed gradients and vary in their gradient
quality (8). To investigate whether this illusion
influences mate choice, we tested for relation-
ships between mating success and geometry. If
the gradients or their generated perspective are
degree of mating success.
We monitored the mating success and court
gradients in the population in the eucalyptus
woodland at Dreghorn cattle station (20.25°S,
147.73°E) (8). The strength of the gradient at
each court is the slope (b) of the regression of
Sciences, Deakin University, Geelong, VIC 3216, Australia.
Townsville, QLD 4811, Australia.
*To whom correspondence should be addressed. E-mail:
VOL 33520 JANUARY 2012
on June 16, 2012