shiga toxin as a bacterial defense against a eukaryotic predator, tetrahymena
TRANSCRIPT
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Shiga toxin as a bacterial defense against a 3
eukaryotic predator, Tetrahymena 4
(Running title: Bacterially-produced Shiga toxin kills Tetrahymena) 5
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William Lainhart�, Gino Stolfa� and Gerald. B. Koudelka*, 7
Department of Biological Sciences University at Buffalo, Buffalo, NY 14260 8
� these authors contributed equally to this work 9
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*Corresponding Author 11
Gerald B. Koudelka 12
Department of Biological Sciences 13
University at Buffalo 14
Cooke Hall, North Campus 15
Buffalo, NY 14260 16
716-645-2363x158 18
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Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.00508-09 JB Accepts, published online ahead of print on 5 June 2009
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SUMMARY 21
Bacterially-derived exotoxins kill eukaryotic cells by inactivating factors and/or 22
pathways that are universally conserved among eukaryotic organisms. The genes that 23
encode these exotoxins are commonly found on bacterial viruses (bacteriophages). 24
When studied in the context of mammals, these toxins cause diseases ranging from 25
cholera to diphtheria to enterohemorrhagic diarrhea. Phage-encoded exotoxin genes 26
are widespread in the environment and are found with unexpectedly high frequency in 27
regions lacking the presumed mammalian targets, suggesting mammals are not the 28
primary ‘targets’ of these exotoxins. We suggest that such exotoxins may have evolved 29
for the purpose of bacterial antipredator defense. We show here that Tetrahymena 30
thermophila, a bacterivorous predator, are killed when co-cultured with bacteria bearing 31
a Shiga toxin (Stx)-encoding temperate bacteriophage. In co-cultures with 32
Tetrahymena, the Stx-encoding bacteria display a growth advantage over those that do 33
not produce Stx. Tetrahymena are also killed by purified Stx. Disruption of the gene 34
encoding the StxB subunit or adding an excess of the non-toxic StxB subunit 35
substantially reduced Stx holotoxin toxicity suggesting that this subunit mediates intake 36
and/or trafficking of Stx by Tetrahymena. Bacterially-mediated Tetrahymena killing was 37
blocked by mutations that prevented the bacterial SOS response (recA-) or by enzymes 38
that breakdown H2O2 (catalase), suggesting that the production of H2O2 by 39
Tetrahymena signals its presence to the bacteria, leading to bacteriophage induction 40
and production of Stx. 41
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INTRODUCTION 42
Genes encoding bacterial exotoxins are frequently carried in bacteria by 43
bacteriophages (4, 19). Phage-encoded exotoxins such as cholera, diphtheria, 44
botulinium and Shiga cause disease in mammals (4). Although these toxins do affect 45
humans and other mammals, these phage-encoded exotoxin genes are found at high 46
frequencies in free phages and lysogenic bacteria isolated from environments where 47
the presumed corresponding targets are not prevalent (6). These exotoxins kill 48
eukaryotic cells by means of receptors and pathways that are generally conserved 49
among eukaryotic organisms. These observations have led to the hypothesis that 50
humans and other susceptible mammals are neither the original nor primary “targets” 51
of these toxins (31). 52
If not mammals, then who are the true targets of these ubiquitous exotoxins? 53
One clue may come from a consideration of the bacterial ecology and evolutionary 54
biology. A major source of bacterial mortality is consumption by single-celled 55
eukaryotic predators, such as ciliates and other protozoa (13). The coordinated release 56
of exotoxins, either at the ‘pre-‘ or ‘post-ingestional’ states (31) could comprise one of 57
the bacteria’s major anti-predator defense strategies. Hence humans may be innocent 58
bystanders in the evolutionary battle between protozoans and their bacterial prey. 59
A family of phages strongly related to the well-characterized coliphage λ genes 60
encoding Shiga-like exotoxin (Stx). Stx encoding bacteriophages are associated with a 61
broad range of hosts, including several serotypes of E. coli, Enterobacter cloacae, 62
Shigella flexneri, and Citrobacter freundii. One well-studied Stx-encoding strain is the 63
Escherichia coli O157:H7 strain EDL933. This strain was responsible for the first 64
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reported multi-state US outbreak of Shiga toxin-caused haemorrhagic colitis in 1982 65
(40). Related strains continue to cause serious food- and water-borne infections world-66
wide. 67
Analysis of the genome sequence of EDL933 bacterial strain and the lambdoid 68
bacteriophages liberated from it reveals that the disease-causing Shiga toxin is 69
encoded by each of two lysogenic lambdoid prophages, 933W and 933J (32, 34, 36, 70
38). Lambdoid bacteriophages all share a common developmental program. Upon 71
infection of a bacterial cell, the lambdoid phages choose between two developmental 72
fates. The phage can grow lytically, thereby killing the host. Alternatively in lysogenic 73
growth, the phage chromosome inserts into the host chromosome and is replicated 74
along with it, until a signal that induces lytic growth is perceived by the lysogenized 75
phages. Since transcription of the stx genes is under the control of a promoter that is 76
only active during lytic growth (53), Stx is not produced when the toxin encoding 77
bacteriophages are in the lysogenic state. Inactivation of the respective bacteriophage 78
repressors (53) causes lytic induction of the lysogenic phages and exotoxin production 79
increases substantially upon phage lysogen induction (27, 32, 34, 54). Stx is 80
apparently not exported through any bacterial secretory machinery. Its release from the 81
bacteria depends on phage genes that cause bacterial cell lysis (52, 53). These genes 82
are also only expressed during phage lytic growth. Therefore, the Shiga toxin gene can 83
reside harmlessly within the bacterial host until phage induction causes production and 84
release of Shiga toxin. 85
Stx is a compound A-B5 toxin, consisting of a 32 kDa A subunit and a 86
pentameric 9.7 kDa B subunit. The five B subunits form a disulfide-bonded ring, into 87
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which is inserted the C-terminal end of the A subunit (see (41-43, 45). Stx toxicity in 88
mammalian cells is mediated by the binding of the B subunit of the holo-toxin to its 89
receptor, globotriaosylceramide (Gb3), on the surface of mammalian cells. Holotoxin 90
enters mammalian cells by clathrin-dependent, receptor-mediated endocytosis (42). 91
The B-subunit directs the retrograde transport of the A-subunit from endosomes to the Golgi 92
apparatus and finally to the endoplasmic reticulum (22, 44), where the catalytic A subunit is 93
cleaved from the holotoxin by furin and released into the cytoplasm (56). Once inside 94
cells, the StxA subunit causes cell death by selectively removing the adenine from 95
position 4224 on 22s rRNA, thereby inhibiting protein synthesis. 96
A major source of bacterial mortality is consumption by single-celled protozoan 97
predators. Ciliates, including Tetrahymena thermophila, control bacterial densities in 98
many ecosystems (3). Protozoan bacterivory has been shown to reduce bacterial 99
numbers (47) and is therefore accepted as a pivotal process in microbial “population 100
control”. Earlier investigations have demonstrated the utility of using Tetrahymena 101
species and E. coli as a model predator-prey interaction to studying food web 102
dynamics (23). Tetrahymena cells can be grown using only E. coli as a food source 103
(23). Methods have been described to quantify predation of bacterial cultures by 104
Tetrahymena (11, 49). 105
We propose that the Stx-encoding bacteriophages evolved to reside within the 106
bacteria to function as part of their anti-predator arsenal and that the presence of the 107
toxin gene may confer an evolutionary advantage on the growth and survivability of this 108
bacterial population by killing the predator. We tested this hypothesis by exploring how 109
the presence of an exotoxin-encoding bacteriophage resident within bacteria influences 110
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the growth and survival of the bacterial population and a model unicellular eukaryotic 111
predator, Tetrahymena thermophila. 112
A recent study compared the relative survivorship of Stx+ and Stx- bacteria in co-113
cultures with Tetrahymena pyriformis (49). Consistent with a role of exotoxins in 114
augmenting the fitness of bacterial populations that carry them, these investigators 115
found that the ratio of Stx+:Stx bacteria increased under these conditions. However, the 116
mechanism by which Stx enhances survivability was not clear from this study. Under 117
the conditions of those experiments, the presence of Stx-encoding phages increased 118
the survivability of bacteria in food vacuoles inside T. pyriformis. However, the results 119
also indicated that increased bacterial survivorship was largely independent of Stx 120
expression. This study did not examine the effect of bacterial Stx production on the 121
growth and survivability of the Tetrahymena predator, a factor that could profoundly 122
influence bacterial survival. The focus of the work presented here is the effect of Stx on 123
Tetrahymena viability. 124
We find that when co-cultured with a model predator, Tetrahymena thermophila, 125
Stx-encoding bacteria kills this predator. Stx-encoding strains are less efficiently 126
predated than are strains that do not encode this exotoxin. We also found that 127
Tetrahymena appears to release a factor that signals the bacteria of the presence of a 128
predator and stimulates the production of toxin by the bacteria. The results are 129
consistent with a role for bacterial exotoxins in the bacterial anti-predator arsenal. 130
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RESULTS 131
To determine whether unicellular eukaryotes can be potential targets of 132
exotoxins such as Shiga toxin, we measured the growth of Tetrahymena thermophila 133
when these cells are co-cultured with several bacterial strains. As a first step in our 134
study, T. thermophila, acclimated to feed on non-phage bearing W3110 bacteria, were 135
washed and suspended in medium containing Stx+ or Stx- bacteria. Under these 136
culture conditions, T. thermophila growth requires the presence of bacteria. Consistent 137
with the use of bacteria as food by T. thermophila, when grown in the presence of 138
W3110::λ, a Stx- bacterial strain that is lysogenized with wild-type bacteriophage λ, a 139
phage that does not encode an exotoxin, the number of T. thermophila approximately 140
doubles within 6 hours (Figure 1A). In contrast when T. thermophila are fed the Shiga 141
toxin encoding strain EDL933, the number of T. thermophila cells decreases to <1/2 142
the input number of cells by 6 hours (Figure 1A). Hence, EDL933 does not support T. 143
thermophila growth, instead this bacteria causes T. thermophila death. 144
The killing of T. thermophila seen in the presence of the Stx encoding bacteria 145
(Figure 1A) is not due to a reduction in the amount of their bacterial food source. Figure 146
1B shows that in co-culture with T. thermophila the amount of EDL933 increases over 147
6 hours in co-culture. In contrast, the number of W3110::λ decreases between ~2-fold 148
over the same time period. These observations are consistent with the idea that the 149
presence of Stx-encoding bacteriophages is advantageous to a bacterial population 150
because, when subject to T. thermophila predation, the Stx-encoding bacteria survive 151
better than those that do not encode Stx. 152
As compared to W3110::λ, EDL933 contains approximately 1 Mb of additional 153
DNA, much of which encodes other pathogenicity-related genes in addition to stx. Thus 154
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to more directly test the idea that Shiga toxin encoded by the lysogenized 155
bacteriophage can kill T. thermophila, we determined whether an EDL933 strain 156
bearing complete disruption/deletion of both the stx1 and stx2 gene clusters, 157
EDL933∆stx (18), affects the growth of T. thermophila. Similar to the results presented 158
in Figure 1, when T. thermophila are fed the Shiga toxin encoding strain EDL933, the 159
number of T. thermophila decreases >2-fold over a 6 hour incubation (Figure 2A). In 160
contrast when T. thermophila are co-cultured with the EDL933∆stx strain, the number 161
of T. thermophila increases by ~50% in this time period (Figure 2A). These 162
observations demonstrate that the killing of T. thermophila in co-cultures with EDL933 163
is due to Stx encoded by this bacterial strain. Consistent with the findings in Figure 1, 164
in co-culture with the T. thermophila, the number of EDL933∆stx bacteria decrease >2-165
fold over the 6 hour incubation period, while the number of EDL933 increases slightly 166
(Figure 2B). This finding supports the idea that Shiga toxin can function as part of an 167
antipredator defense strategy. 168
To directly demonstrate that Stx can kill T. thermophila, we determined whether 169
adding purified Stx2 could kill T. thermophila in axenic cultures. We found that adding 170
increasing concentrations of Stx2 progressively decreases the number of T. 171
thermophila cells that survive after a six hour incubation with toxin (Figure 3). Under 172
our conditions, adding 25 ng/mL of purified Stx kills ≥90% of the cells in culture. 173
Similarly, partially purified Stx-containing extracts (33) obtained from mitomycin C-174
induced EDL933 cells also kills T. thermophila (not shown). These findings confirm that 175
Shiga toxin is responsible for EDL933-mediated killing of T. thermophila. 176
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We wished to delineate the factors that regulate the cytotoxic effect of Shiga 177
toxin on T. thermophila. Stx entry into mammalian cells requires the binding of the B 178
subunit of the holo-toxin to its Gb3 receptor on the surface of mammalian cells. We 179
reasoned that if the B-subunit has a role in Stx-dependent T. thermophila killing, adding 180
excess StxB subunit should compete with holotoxin’s cytotoxic effects. Added partially 181
purified B-subunit alone does not exhibit any cytolethal effect on T. thermophila 182
cultures (Figure 4), a finding that is similar to that obtained with mammalian cells (20, 183
29). Adding an increasing weight of the partially purified StxB subunit in the presence 184
of intact Stx progressively inhibits killing of T. thermophila caused by the added purified 185
Stx. The inhibitory effect of added B subunit in Stx-mediated killing saturates between 186
47-63 µg/mL. This finding implies that the added B-subunit competes with binding of 187
holo-Stx to specific receptor/trafficking sites in T. thermophila. The StxB blockade of 188
holo-toxin mediated killing is specific, adding similar quantities of BSA (Figure 4) or 189
boiled B-subunit (not shown) does not inhibit Stx-mediated T. thermophila killing. 190
Figure 4 indicates that in order to interfere with Stx-mediated bacterial killing an 191
excess of StxB over the amount Stx holo-toxin must be added to the cultures. We do 192
not yet know the nature of, or how many Stx holotoxin receptor/trafficking sites there 193
are within T. thermophila. The requirement for ‘excess’ StxB to block killing indicates 194
that there many such sites, although the observation that the StxB inhibitory effect is 195
saturable suggests the number of sites is not infinite. Consistent with our findings, 196
excess B-subunit is also needed to block Stx holotoxin-mediated killing of mammalian 197
cells (20, 30). Addition of sub-stochiometric ratios of Stx holotoxin:Gb3 receptor is 198
sufficient to kill mammalian cells (35), and thus excess StxB subunit is needed to 199
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saturate the receptors to prevent killing. We speculate a similar situation occurs with T. 200
thermophila. Nonetheless, regardless of precise mechanism by which StxB blocks Stx-201
mediated T. thermophila killing, the data in Figure 4 indicate that the B-subunit has a 202
role in mediating cytotoxicity by Stx holotoxin. 203
The results in Figure 4 indicate that B-subunit mediates the cytotoxicity of 204
added, purified Stx. To determine whether B-subunit also plays a role in the cytolethal 205
effect of bacteria bearing Stx-encoding bacteriophages (Figures 1 & 2), we co-cultured 206
T. thermophila with EDL933∆stx bearing either of two plasmids, one encoding Stx2 207
holotoxin and the other encoding only the Stx2A subunit, and measured the growth 208
kinetics T. thermophila and bacteria. When co-cultured with EDL933∆stx bearing the 209
Stx-holotoxin-encoding plasmid (EDL933∆stx/pStxAB), the number T. thermophila 210
decreases ~3-fold over a 6 hour incubation. The efficiency of T. thermophila killing is 211
slightly larger than that seen in co-cultures with EDL933, presumably due to the 212
increased production of Stx holotoxin from the plasmid. Consistent with the results 213
presented in Figures 1 & 2 and our proposal that Stx acts as an antipredator agent, the 214
number of bacterial cells bearing pStxAB increases in the presence of T. thermophila. 215
In contrast, T. thermophila are not killed when co-cultured with EDL933∆stx 216
bearing a plasmid that encodes only the Stx2A subunit (EDL933∆stx/pStxA). Instead 217
this stxA+ strain supports T. thermophila growth to a similar extent as does non-Stx 218
encoding bacteria (see Figure 1). Also, the number of EDL933∆stx/pStxA cells 219
decreases in the presence of T. thermophila, showing that these cells are more 220
efficiently predated than are the stx+ EDL933∆stx/pStxAB cells. Control immunoblots 221
indicate that identical amounts of StxA subunit are produced by the EDL933∆stx/pStxA 222
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and EDL933∆stx/pStxAB strains. These findings indicate that the B-subunit is involved 223
in killing of T. thermophila mediated by Stx encoding bacteria and is essential to its 224
potential role as an antipredator defense molecule. 225
Shiga toxin is not produced by EDL933 as long as the resident toxin-encoding 226
bacteriophages remain in their lysogenic state. Lytic induction of lysogenic 227
bacteriophage occurs upon inactivation of the phage repressor (53). The best-studied 228
mechanism of repressor inactivation involves RecA-stimulated repressor 229
autoproteolysis (9, 37, 46) that occurs during the host’s SOS response to DNA damage. 230
Fuchs et al. (16) showed that 933r, an otherwise isogenic recA mutant of EDL933, has 231
severely reduced the virulence in mice. Hence we used 933r to examine whether the 232
bacterial SOS response plays a similar role in mediating T. thermophila killing. In 233
contrast to the results with wild-type EDL933, when T. thermophila are fed 933r, the 234
number of T. thermophila cells remains unchanged. This finding indicates that recA 235
mutation blocks the bacterially-mediated killing of T. thermophila. 236
The form of RecA that stimulates repressor autocleavage arises as a 237
consequence of DNA damage (15). Such damage can be caused numerous agents, 238
including natural products (e.g., microbially-produced antibiotics) and reactive oxygen 239
species (ROS). We hypothesized that ROS released by T. thermophila (14, 39) may 240
indicate the presence of this predator to EDL933 cells. The ROS would be expected to 241
activate the bacterial SOS response, causing induction of the Stx-encoding prophages 242
in these cells, leading to Stx production and release. 243
We tested this idea by co-culturing EDL933 and T. thermophila for 2 hours in the 244
absence or presence of either superoxide dismutase (SOD) or catalase, enzymes that 245
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degrade superoxide or H2O2, respectively. As found previously, the number of T. 246
thermophila cells decreases over time in the absence of any antioxidant enzymes, 247
while the number of EDL933 cells increases (Figure 5). We obtained identical results 248
with co-cultures containing SOD suggesting this enzyme does not influence toxin 249
production by EDL933. In contrast, the number of T. thermophila cells increased when 250
these cells are co-cultured in the presence of catalase while the number of EDL933 251
cells decreased (Figure 5). Therefore, the EDL933/T. thermophila co-cultures with 252
catalase behaved identically to co-cultures consisting of T. thermophila and the control 253
bacteria, W3110::λ. This finding suggests that catalase blocks the release of Shiga 254
toxin by EDL933 by removing H2O2. This observation is consistent with the finding that 255
the bacterial SOS response mediates enhanced Shiga toxin production by EDL933, 256
leading to increased T. thermophila killing. 257
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DISCUSSION 258
The data presented here clearly show that Shiga toxin, either released from an 259
induced lysogenic bacteriophage or added exogenously, is capable of killing the single-260
celled eukaryote, Tetrahymena thermophila. To our knowledge, this is the first direct 261
demonstration of Shiga toxin lethality in any protozoan or other bacterial predator. Our 262
results also show that the presence of a Shiga toxin encoding gene within bacteria 263
decreases T. thermophila predation efficiency. Our findings are consistent with the 264
results of a previous investigation showing that in the presence of Tetrahymena 265
species, bacterial exotoxins augment the fitness of bacterial populations that carry 266
them (49). Together with these previous findings (49), our results strongly suggest that 267
Stx can function as an antipredator defense and support the hypothesis that this toxin 268
may have originated in bacteria as an antipredator adaptation. 269
There is a growing body of evidence that predation by bacterivorous protozoa 270
can play a large role in shaping the composition of bacterial populations (24, 25, 48, 271
55). The cytotoxic effect of a bacterial exotoxin on a protozoan predator indicates that 272
these molecules can have important ecological functions within natural microbial 273
communities. Therefore, future studies with T. thermophila can provide information 274
about the evolution and function of bacteriotoxins on ciliate predators. 275
Two of our findings suggest that various aspects of the interaction between Shiga 276
toxin encoding bacteria and mammalian cells have an ancient evolutionary origin in the 277
predator-prey interaction between these bacteria and protozoan. First, the finding that 278
EDL933 bacteria appear to sense the presence of the T. thermophila by detecting the 279
presence of reactive oxygen species and respond by inducing the synthesis and 280
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release of Stx may represent a foreshadowing of the situation found in the mammalian 281
response to bacterial infection. Consistent with the findings of others (14, 39), we have 282
observed that T. thermophila releases H2O2 into the media (data not shown). Similarly, 283
reactive oxygen species like H2O2 or superoxide, generated and released by leukocytes 284
and neutrophils activates the SOS response in Shiga toxin encoding E. coli, leading to 285
toxin release (51) and subsequent death of the ‘attacking’ eukaryotic cell. Since H2O2 is 286
present in internal vesicles of Tetrahymena species (14), it is also possible that 287
bacteriophage induction occurs inside the cells after the bacteria are eaten. 288
Second, similar to its role in mammalian cell killing, we found that the B-subunit 289
plays an essential role in Stx-mediated T. thermophila killing. In mammalian systems, 290
Shiga toxin is produced outside of the cell as a consequence of lytic growth of Stx-291
encoding bacteriophage. Stx entry into mammalian cells occurs by receptor mediated 292
endocytosis, which requires the binding of the holotoxin’s B subunit to the neutral 293
glycolipid globotriaosylceramide (Gb3) receptor. Mammalian cells lacking this glycolipid 294
are immune to the toxin (7). Tetrahymena has all of the machinery for clathrin-295
mediated endocytosis (12, 21). Therefore, Tetrahymena could use a clathrin-296
dependent receptor mediated endocytosis mechanism to import Shiga toxin. However, 297
exhaustive studies performed in our labs and the laboratories of Dr. Clifford Lingwood 298
at the University of Toronto (personal comm.) failed to identify any Stx binding lipids in 299
the glycolipid-containing fraction. Moreover, inspection of the T. thermophila genome 300
sequence (8, 10) failed to identify any close homologues of the gene that encodes the 301
mammalian Gb3 synthase enzyme (26). These observations are inconsistent with Stx 302
entry into Tetrahymena occurring by “standard” Gb3 receptor-mediated endocytosis. 303
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Nonetheless our findings showing that excess B subunit blocks the killing of T. 304
thermophila by purified Stx2 holotoxin (Fig. 4) and that expression of the B subunit is 305
required for T. thermophila intoxication suggests that alternative Stx receptors could be 306
present in these cells. 307
Tetrahymena can ingest particulate and soluble nutrients through the oral 308
apparatus and funnel them into their food vacuoles. Hence in the absence of a Shiga 309
toxin receptor, uptake of either released Stx or Stx-encoding bacteria via 310
Tetrahymena’s oral cavity might provide an alternative and/or the sole route for toxin 311
entry into these cells. This ability to capture bacteria through their oral apparatus could 312
provide a novel route for toxin entry into Tetrahymena. If this idea is correct, it suggests 313
that B-subunit affects Shiga toxin cytotoxicity in Tetrahymena by influencing 314
intracellular trafficking. 315
Considering that the lysogenic bacterial host must die to release the Shiga toxin, 316
the fitness benefits of this antipredator defense mechanism cannot accrue to the 317
individual organism but to the overall bacterial population-a population that would 318
include cells that are not lysogenic for the toxin-encoding bacteriophage. At first glance 319
this strategy may seem overly ‘altruistic’. However, the genes encoding this exotoxin 320
are found on mobile, temperate bacteriophage. Due to the high propensity to grow 321
lytically (28), the phage has limited ability to lysogenize naïve hosts. The propensity of 322
the phage to lytically infect, and thereby kill, the naïve hosts would restrict the benefit of 323
Shiga toxin antipredator activities on the fitness of this segment of the bacterial 324
population. It should be noted that lytic growth in the naïve hosts would increase the 325
amount of Stx produced, amplifying the killing capacity of the original sacrificed cell. The 326
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ability of non-lysogens to enhance Stx production by a bacterial population has already 327
been demonstrated (17). 328
Hence, the presence of toxins genes, in this case Shiga toxin, on the temperate 329
phages and linkage of toxin expression to lytic growth may provide substantial 330
advantages to bacterial lysogens that ‘choose’ to harbor these phage. Utilization of an 331
exotoxin encoded on an inducible phage is then cost-effective defensive strategy for 332
the bacterial population. These lysogenic populations sacrifice a few cells to produce a 333
toxin that kills its major predator and produce infectious phage that have the potential 334
to eliminate bacterial competitors (53). 335
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Experimental Procedures 336
Strains, chemicals. 337
EDL933 was obtained from the ATCC and 933r, an isogenic recA mutant of EDL933 338
(16) was obtained from Jörg Hacker, Universität Würzburg. EDL933W∆stx, an EDL933 339
variant bearing a complete deletion of all stx genes (18) was obtained from Christine 340
Miller, Institut National de la Recherche Argonomique. The W3110::λ lysogen was 341
constructed using wild-type λ bacteriophage as described (2). Tetrahymena 342
thermophila (CU427.4), was obtained from the Tetrahymena Stock Center (Cornell 343
University). 344
Plasmid encoding Stx2 holotoxin (pStxAB) was constructed by amplifying DNA 345
encoding the entire stxAB region of bacteriophage 933W (38) from genomic DNA of 346
the EDL933 and inserting this DNA into the plasmid pET17b (EMB Biosciences). The 347
plasmid encoding only Stx2A (pStx2A) was constructed by disrupting the stx2B gene in 348
pStx2AB. This disruption was accomplished by inserting the coding sequence of the 349
chloramphenicol resistance (cmr) gene, isolated from pACYC184 by PCR, into the 350
unique PflMI restriction site in the stxB gene. As assessed by immunodot analysis (50), 351
both these plasmids direct the low level expression of holotoxin or Stx2A subunit when 352
transformed into bacterial cells lacking the T7 RNA polymerase gene. 353
Catalase and superoxide dismutase were obtained from Worthington 354
Biochemicals. Mitomycin C was obtained from Sigma. Purified Stx2 holotoxin and anti-355
Shiga toxin antibodies were purchased from Toxin Technologies. 356
Effect of co-culture on Tetrahymena and bacterial viability 357
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Tetrahymena and bacterial cells needed for the co-culture experiments were 358
prepared as follows. Cultures of the specified bacteria were to grown saturation at 37°C 359
in M9 + 0.08% glucose. Cells in these cultures were harvested, washed twice with M9 + 360
0.08% sodium citrate and suspended in the same media. Tetrahymena were diluted 5-361
fold from saturated liquid cultures and grown for two days at 30°C in protease peptone 362
plus FeCl2. These cells were harvested, washed twice with 10 mM Tris-HCl (pH 7.4) 363
and suspended in M9 + 0.08% sodium citrate in a volume sufficient to give 104 cells/mL. 364
To each washed Tetrahymena culture, or control cultures containing no Tetrahymena, 365
either 108 of washed bacterial cells/mL or an equal volume of media were added. The 366
co-cultures were maintained at 30°C. Where indicated, 4 µg/mL of either superoxide 367
dismutase or catalase were added to each culture. 368
At various times after initiating co-culture and two aliquots were removed from 369
each. One aliquot was used to determine the amount of Tetrahymena present in the 370
culture. Tetrahymena counts were obtained by counting the number of Lugol stained 371
cells or by trypan blue exclusion, as visualized in a hemocytometer. Tetrahymena that 372
are killed by exposure to Stx or Stx-expressing bacteria were not visible by either 373
method, presumably because they have lysed. The number of bacteria was determined 374
plating the culture and counting the number of colony forming units (CFU). Each 375
measurement was performed in triplicate and the data averaged. Each experiment was 376
repeated a minimum of three times. The data presented represent the average of the 377
three, or greater, replicates. 378
Purification of stxB 379
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StxB was purified from JM105:pSBC54 (Ampr) (1) as described (5). The 380
concentrated protein was stored at -80°C in 10mM PBS supplemented with 15% 381
glycerol. 382
Effect of holo-Shiga toxin on Tetrahymena viability. 383
Tetrahymena were grown in protease peptone to a density of 106 cells/mL. 384
These cells were diluted 100-fold in fresh medium and incubated for up to 6 hrs at 385
30°C in the absence or presence of 1) 1-1000 ng/mL of purified Stx2A or 2) 25 ng/mL 386
Stx2A in the presence of increasing concentrations of purified stxB subunit. As controls 387
for the effect of adding protein to the growth media, Tetrahymena were separately 388
incubated with increasing concentrations of heat-denatured toxin or an equivalent 389
weight of BSA. The number of viable Tetrahymena was determined at various times 390
after initiating incubation. Each measurement was performed in triplicate and the data 391
averaged. Each experiment was repeated a minimum of three times. The data 392
presented represent the average of the three, or greater, replicates. 393
Statistical Methods 394
Error bars presented in the figures represent standard deviation of the means of 395
multiple (>3) replicate experiments. T-tests were used to test the significance between 396
the mean of the measured initial and amounts of bacteria and/or Tetrahymena 397
subsequent to treatment in each experiment. 398
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399
Acknowedgements 400
The authors wish to thank the members of our laboratories for valuable discussions. We 401
also acknowledge the contributions of Dr. Todd Hennessey, for invaluable contributions 402
of material, assistance and advice. GBK acknowledges the support of the College of 403
Arts and Sciences, University at Buffalo. 404
405
406
407
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597
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599
Figure Legends 600
601
Figure 1. Relative amount of (A) Tetrahymena and (B) E. coli cells surviving co-culture. 602
Co-cultures and cell counts were performed as described in Experimental Procedures. 603
Cell numbers are expressed as fold change in number of cells remaining in culture at 604
increasing lengths of time after beginning incubation. At the start of incubation, bacteria 605
and Tetrahymena cells were present at 108 and 105 cells/mL, respectively. Error bars 606
represent standard deviation of ≥3 independent experiments, with each experiment 607
being comprised of a minimum of 3 individual measurements. (* p <.01; ** p < .001) 608
Figure 2. Role of Stx in Shiga toxin encoding bacteria-mediated killing of Tetrahymena 609
(A) and inhibition of Tetrahymena predation of bacteria (B). Tetrahymena were co-610
cultured with EDL933 or EDL933∆stx (18), a strain bearing complete disruptions in both 611
the stx1 and stx2 gene clusters. Co-cultures and cell counts were performed as 612
described in Experimental Procedures. Cell numbers are expressed as fold change in 613
number of cells remaining in culture at 6 hours relative to the number of cells present at 614
the start of the co-culture. Input cell numbers, experimental design and error analysis 615
were as described in the legend to Figure 1. Differences between stx+ and stx- are 616
significant at p <.005 or greater 617
Figure 3. Effect of purified Stx2 on survival of axenically growing Tetrahymena. The 618
indicated amount of purified Stx2 holoxtoxin was added to 105 cells/mL of axenically 619
growing Tetrahymena. The number of live Tetrahymena cells was determined after 6 620
hrs. Data is presented as percent of input Tetrahymena cells killed during a 6 hr 621
incubation with indicated amount Stx2. 622
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Figure 4. StxB-mediated inhibition of Stx2A holotoxin-dependent Tetrahymena killing. 623
Tetrahymena (105 cells/mL) were axenically grown in the presence of 65 µg/mL of 624
purified StxB (gray bars) subunit or BSA (black bars) alone or 25 ng/mL of Stx2 625
holotoxin and increasing concentrations of either StxB or BSA. The number of live 626
Tetrahymena cells was determined after 6 hours. Data is presented as the number of 627
live Tetrahymena cells after incubation relative to the number of live Tetrahymena at the 628
start of incubation. Error bars represent standard deviation of ≥3 independent 629
experiments, with each experiment being comprised of a minimum of 3 individual 630
measurements 631
Figure 5. 632
Effect of anti-oxidant enzymes on the survival of EDL933 and Tetrahymena in co-633
cultures. Co-cultures containing 105 Tetrahymena cells/mL and 108 were grown in the 634
absence or presence of 4 µg/mL of either superoxide dismutase or catalase, as 635
indicated. Tetrahymena cell counts are given as the number of live cells remaining at 636
end of the 6 hr incubation relative to amount of cells present at start of incubation. 637
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