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An essential role of invasin for colonization and persistence 1
of Yersinia enterocolitica in its natural reservoir host, the pig 2
3
Julia Schaake1+, Anna Drees2+, Petra Grüning2+, Frank Uliczka1, Fabio Pisano1, 4
Tanja Thiermann1, Alexandra von Altrock3, Frauke Seehusen4, Peter Valentin-5
Weigand2*, Petra Dersch1* 6
1Department of Molecular Infection Biology, Helmholtz-Centre for Infection Research, 7 38124 Braunschweig, Germany 8 2Institute for Microbiology, University of Veterinary Medicine Hannover, 30173 9 Hannover, Germany 10 3Clinic for Swine, Small Ruminants, Forensic Medicine and Ambulatory Services, 11 University of Veterinary Medicine Hannover, 30173 Hannover, Germany 12 4Institute for Pathology, University of Veterinary Medicine Hannover, 30173 13 Hannover, Germany 14 15
*Corresponding authors: 16
Petra Dersch Peter Valentin-Weigand 17
Dept. of Molecular Infection Biology, Institute for Microbiology 18 Helmholtz Center of Infection Research University of Veterinary Medicine 19
Hannover 20
38124 Braunschweig 30173 Hannover 21
Germany Germany 22
Phone: +49-531-6181-5700 Phone: +49-511-953 7362 23
FAX: +49-531-6181-5709 FAX: +49-511-953 7697 24
e-mail: [email protected] e-mail: [email protected] 25
26
+These authors contributed equally to this study. 27
Running title: Colonization of pigs by Y. enterocolitica 28
Keywords: Y. enterocolitica, invasion, colonization, persistence, pig model 29
IAI Accepts, published online ahead of print on 16 December 2013Infect. Immun. doi:10.1128/IAI.01001-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.
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Abstract 30
In this study an oral minipig infection model was established to investigate the 31
pathogenicity of Yersinia enterocolitica bioserotype 4/O:3 (YeO:3). YeO:3 strains are 32
highly prevalent in pigs, which are usually symptomless carriers, and they represent the 33
most common cause of human yersiniosis. To assess the pathogenic potential of the 34
YeO:3 serotype, we compared the colonization properties of YeO:3 with YeO:8, a highly 35
mouse-virulent Y. enterocolitica serotype, in minipigs and mice. We found that YeO:3 is 36
a significantly better colonizer of swine than YeO:8. Coinfection studies with YeO:3 37
mutant strains demonstrated that small variations within the YeO:3 genome leading to 38
higher amounts of the primary adhesion factor invasin (InvA) improved colonization 39
and/or survival of this serotype in swine, but had only a minor effect on the colonization 40
of mice. We further demonstrated that a deletion of the invA gene abolished long-term 41
colonization in the pigs. Our results indicate a primary role for invasin in naturally 42
occurring Y. enterocolitica O:3 infections in pigs and revealed a higher adaptation of 43
YeO:3 compared to YeO:8 strains to their natural pig reservoir host. 44
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Introduction 45
The Gram-negative enteropathogenic bacterium Y. enterocolitica is a fecal-oral zoonotic 46
pathogen, which is associated with a number of different enteric diseases, collectively 47
called yersiniosis. Symptoms include enteritis, enterocolitis, severe diarrhoea, mesen-48
teric lymphadenitis, pseudoappendicitis, hepatic and splenic abscesses. The diseases 49
are normally self-limiting. However, postinfectious extraintestinal sequelae including 50
reactive arthritis, erythema nodosum and thyroiditis are also common (1-3). 51
Yersiniosis is a frequent bacterial enteric disease in Europe (16.5 cases per million) 52
with a predilection for young children (4-7). The most frequently isolated Y. entero7, l. 53
colitica strains which are harmful to humans and animals are classified into the bio-54
serotypes 1B/O:8, 2/O:5,27, 2/O:9, 3/O:3 and 4/O:3 (Bottone, 1999). Bioserotype 55
1B/O:8 strains (YeO:8) are highly virulent for mice and most studies on Y. enterocolitica 56
pathogenesis were performed using this strain type, in particular Y. enterocolitica 57
8081v. However, by far most human yersiniosis in Europe, Canada, China and Japan is 58
caused by bioserotype 4/O:3 strains which have a low pathogenicity in mouse models 59
(1, 4, 8). Y. enterocolitica O:8 infections are more common in North America, but food-60
borne outbreaks of serotype O:3 strains have emerged in recent years and have 61
replaced O:8 as the predominant Y. enterocolitica serotype (9-13). 62
Y. enterocolitica can colonize a broad range of domestic (e.g. sheep, cattle, goats 63
and poultry) and wild animals (e.g. boars, wild rodents), but the most important reservoir 64
are pigs which are of major importance for transmission to humans (10, 14). In 65
particular YeO:3 strains - the most frequent source of human infections - can be 66
routinely isolated from pigs which are usually symptomless carriers (1, 15, 16). Pigs can 67
carry these pathogens for long time periods in the oropharynx (tonsils) and the intestinal 68
tract without any clinical signs, leading to a prevalence of 35%-70% in fattening pig 69
herds (17-19). As a consequence, outbreaks of yersiniosis are mostly associated with 70
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the consumption of raw or undercooked pork or pork products (e.g. chitterlings) (18, 20, 71
21). 72
The high prevalence of YeO:3 strains in pigs suggest bioserotype- and/or host-73
specific colonization properties. However, up to date little is known about bacterial 74
components and pathogen-host cell interactions of YeO:3 isolates which determine their 75
adaption to the intestine of pigs and pathogenicity for humans. Analysis of YeO:8 strains 76
demonstrated that this serotype initiates infections by tight binding to the intestinal 77
mucosa which is frequently followed by the transmigration of the epithelial layer of the 78
ileum, resulting in the colonization of the underlying lymphoid tissues (Peyer's patches). 79
Subsequently, Y. enterocolitica can spread via the lymph and/or blood into the 80
mesenteric lymph nodes or to extraintestinal sites such as liver and spleen (22). Several 81
virulence factors of YeO:8 strains were identified to promote colonization of host tissues. 82
Among them are the surface-exposed outer membrane proteins invasin (InvA) and 83
YadA. They promote efficient adhesion to and invasion into intestinal cells via direct or 84
indirect binding of β1-integrin receptors (23-26). In YeO:8 strains, InvA and YadA 85
constitute independent colonization factors which are differentially expressed and 86
appear to act during different stages of the infection. The invasin protein is 87
predominantly synthesized at moderate temperatures (15-28°C), whereas only low 88
amounts of invasin were detectable at 37°C (27). In mice, the LD50 values of the YeO:8 89
wild-type 8081v and the isogenic invA mutant were essentially identical but the 90
colonization of the lymphoid tissues was delayed (23). This suggested that InvA might 91
prime the bacteria to support efficient and rapid transcytosis of the intestinal epithelium 92
during the very early stages of infection. The YadA adhesin is a multifunctional bacterial 93
invasin that also protects bacteria from complement lysis and phagocytosis. YadA is 94
exclusively expressed at 37°C and acts together with a type III secretion system which 95
is responsible for the injection of antiphagocytic effectors proteins (Yops) into phago-96
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cytes to modulate the host immune response during later stages of the infection (25, 26, 97
28). 98
To gain more information about the pathogenic properties of the more frequent Y. 99
enterocolitica O:3 serotypes, we previously compared the colonization mechanisms of 100
different human and animal YeO:3 isolates with that of the well-characterized YeO:8 101
8081v strain and found that Y. enterocolitica O:3 strains have unique cell adhesion and 102
invasion properties (29, 30). These differences are mainly attributable to significant 103
variations of the interplay and expression profile of the same repertoire of virulence 104
factors in response to temperature. YeO:3 strains, but not YeO:8 strains, were able to 105
invade human cells at 37°C due to highly activated and nearly constitutive expression of 106
invasin caused by i) an additional promoter provided by an IS1667 element inserted in 107
the invA promoter region, and ii) a P98S substitution in the invA transcriptional activator 108
protein RovA which renders the regulator less susceptible to proteolysis by the Lon 109
protease (29, 30). In addition, efficient cell attachment by expression of YadA and 110
down-regulation of the O-antigen synthesis are required to allow InvA-mediated entry in 111
cultured human epithelial cells at body temperature. Most likely, interaction of the 112
somewhat longer YadA molecules promotes initial host cell attachment to extracellular 113
matrix (ECM) bound to β1-integrins which facilitates subsequent high-affinity and direct 114
binding of the shorter invasin molecules to β1-integrin receptors. Why this distinct 115
colonization mechanism is restricted to pig-associated YeO:3 strains remained unclear. 116
In this study we established a novel experimental oral infection model for Yersinia in 117
minipigs and demonstrate that the small variations in the expression profile of 118
pathogenicity factors in YeO:3 strains provoke a fine-tuned readjustment of virulence-119
associated processes which confers better colonization and/or survival of this serotype 120
in swine. 121
122
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Materials and Methods 123 124 Ethics statement 125
All animal work was performed in strict accordance with the German regulations of the 126
Society for Laboratory Animal Science (GV-SOLAS) and the European Health 127
Recommendations of the Federation of Laboratory Animal Science Associations 128
(FELASA). The protocol was approved by the Niedersaechsisches Landesamt für 129
Verbraucherschutz und Lebensmittelsicherheit: animal licensing committee permission 130
no. 33.9.42502-04-055/09 (mice), 33.12-42502-04-10/0173 and 33.9-42502-04-11/0462 131
(pigs). All efforts were made to minimize suffering of the animals. 132
133
Bacterial strains, media and growth conditions 134
The following strains were used in this study: YeO:8 strain 8081v (bioserotype 1B/O:8, 135
patient isolate, wildtype), YeO:3 strains Y1 (bioserotype 4/O:3, patient isolate, wildtype), 136
YE13 (Y1, ∆rovA, ProvAO:3::rovAS98, ClmR, KanR), YE14 (Y1, ∆rovA, ProvAO:3::rovAP98, 137
ClmR, KanR), YE15 (Y1, PinvA∆IS1667), and YE21 (Y1, ∆invA, KanR) (23, 29). Overnight 138
cultures of Y. enterocolitica were grown at 25°C in Luria-Bertani (LB) broth. The 139
antibiotics used for bacterial selection were: carbenicillin 100 µg/ml, kanamycin 50 140
µg/ml and gentamicin 50 µg/ml. For infection experiments, bacteria were grown at 25°C, 141
washed and diluted in phosphate buffered saline (PBS) prior to infection. 142
143
Mouse infections 144
Bacteria used for oral infection were grown overnight in LB medium at 25°C, washed 145
and resuspended in PBS. 6-8 weeks old female BALB/c mice were purchased from 146
Janvier (St. Beverthin Cedex, France). Two independent groups of 5 mice were orally 147
infected with Y. enterocolitica strains in single infection and co-infection experiments 148
using a ball-tipped feeding needle. For single infection experiments 5 x 108 bacteria of 149
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the Y. enterocolitica strains 8081v or Y1 were administered orogastrically. In co-infec-150
tion experiments, mice were infected with a mixture of equal numbers of 5 x 108 151
bacteria of Y. enterocolitica strains Y1 and YE14, Y1 and YE21, or YE13 and YE15, 152
respectively. 153
Three days after infection, mice were euthanized by CO2. Jejunum, ileum, Peyer’s 154
patches, cecum, colon, mesenteric lymph nodes, liver and spleen were isolated. The 155
isolated tissues were rinsed with sterile PBS and incubated with 50 µg/ml gentamicin in 156
order to kill bacteria on the luminal surface. After 30 min, gentamicin was removed by 157
extensive washing with PBS for three times. Subsequently, all organs were weighed 158
and homogenized in sterile PBS at 30,000 rpm for 30 sec using a Polytron PT 2100 159
homogenizer (Kinematica, Switzerland). Bacterial numbers were determined by plating 160
independent serial dilutions of the homogenates on LB plates with and without 161
antibiotics. The colony forming units (cfu) were counted and are given as cfu per g 162
organ/tissue. The competitive index relative to wildtype strain Y1 was calculated as 163
described (31). 164
165
Minipig infections 166
Bacteria used for oral infection were grown overnight in LB medium at 25°C, washed 167
and resuspended in PBS. Five to seven weeks old Mini-Lewe minipigs were purchased 168
from the Forschungsgut Ruthe of the University of Veterinary Medicine Hannover. 169
Minipigs were separated into independent groups and sampled by tonsil and rectal 170
swabs 7 days prior to infection. The analyses of blood samples, tonsil and rectal swabs 171
confirmed that the minipigs were free of Y. enterocolitica prior to infection. For single 172
infections approximately 109 bacteria of the Y. enterocolitica strains 8081v or Y1 were 173
used for oral infection. In co-infection experiments, minipigs were orally infected with an 174
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equal mixture of 109 bacteria of Y. enterocolitica strains Y1 and YE14, Y1 and YE21 or 175
YE13 and YE15. 176
Post infection blood samples were taken once, rectal swabs twice a week. At 177
indicated times post infection, minipigs were narcotized by Azaperon (Stresnil®) and 178
Ketamin (Ursotamin®) before final blood sampling and then euthanized by Pento-179
barbital (Release®). Tonsils, jejunum, ileum, cecum, colon, mesenteric lymph nodes, 180
liver, kidneys and spleen were isolated. The tissues were rinsed with sterile PBS and 181
incubated with 50 µg/ml gentamicin in order to kill bacteria on the luminal surface. After 182
30 min, gentamicin was removed by extensive washing with PBS for three times. 183
Subsequently, all organs were weighed and homogenized in sterile PBS at 30,000 rpm 184
for 30 sec using a Polytron PT 2100 homogenizer (Kinematica, Switzerland). Bacterial 185
numbers were determined by plating two independent serial dilutions of the 186
homogenates on cefsulodin-irgasan-novobiocin (CIN) agar plates with and without 187
antibiotics. The colony forming units (cfu) were counted and are given as cfu per g 188
organ/tissue. The competitive index relative to wildtype strain Y1 was calculated as 189
described (31). 190
191
Qualitative detection of bacteria in tissues and rectal swabs of the minipigs via 192
cold enrichment 193
Rectal and tonsil swabs were stored in 10 ml PBS at 4°C, streaked on CIN agar plates 194
at day 7, 14 and 21 and incubated at 30°C for up to 48 h. Organ samples were 195
homogenized in sterile PBS as described above and 200 µl of the homogenate were 196
added to 10 ml sterile PBS. This solution was stored at 4°C, streaked on CIN agar 197
plates after 7, 14 and 21 days and then incubated at 30°C for up to 48 h. 198
199
Detection of specific antibodies in pig sera 200
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Blood samples were taken weekly before and after oral infection of piglets by puncture 201
of the cranial vena cava. Serum was extracted using Serum Monovettes® (Sarstedt, 202
Germany). Serological examinations were performed using a commercial microtiter 203
plate based enzyme immunoassay (PIGTYPE® YOPSCREEN ELISA system, Labor 204
Diagnostik Leipzig, Germany), based on recombinant Yersinia outer proteins (Yops). 205
These antigens are expressed only by pathogenic Yersinia strains. The optical density 206
(OD) was measured in a spectrophotometer and an OD value of 20% was used as cut-207
off value. 208
209
Histological analysis 210
Samples of the euthanized minipigs (ileum and mesenteric lymph nodes (MLNs)) were 211
collected and fixed in 10% non-buffered formalin. Following a fixation period of 24 h 212
samples were embedded according to standard procedures. The tissue processing was 213
performed automatically (Shandon Path Centre® Tissue Processor, Thermo, USA). One 214
slide of each paraffin block was cut at 1.5-3 μm thickness by a microtome and stained 215
with hematoxylin eosin (H&E). The degree of inflammation in the lamina propria of the 216
ileum was assessed semi-quantitatively by using the following scoring system: 0 = no 217
inflammation; 0.5 = minimal inflammation; 1 = very mild inflammation; 1.5 mild 218
inflammation; 2 = moderate inflammation; 2.5 = moderate to severe inflammation; 3 = 219
severe inflammation. The statistical analysis of the data was performed by using the 220
statistics program SPSS (Superior Performing Systems, version 20.0). A group-wise 221
comparison by conducting a Mann-Whitney-U test was performed concerning the semi-222
quantitative parameters general inflammation, infiltration of neutrophils and histiocytes. 223
A p value of ≤ 0.05 is considered as a statistically significant change. 224
225
226
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Results 227
Establishment of a minipig infection model for Y. enterocolitica 228
To investigate interactions of different Y. enterocolitica wild-type and mutant strains with 229
their natural and most important animal reservoir we established an oral infection model 230
in miniature pigs (minipigs). Minipigs were chosen due to their advantages in handling 231
and housing and their increasing popularity as laboratory animals as they develop 232
human-sized organs between 6 and 8 months of age (32). First, minipigs were 233
challenged with oral doses of 108, 109 and 1010 colony-forming units (cfu) of the YeO:3 234
wild-type strain Y1, a recent patient isolate which showed host cell interaction properties 235
identical to other characterized human and porcine isolates (29, 30). All pigs appeared 236
healthy and did not show any clinical signs of infection, i. e. increased temperature, 237
diarrhea or loss of appetite. The efficiency of the infection was monitored by reisolation 238
of the bacteria from tonsils, different gut sections (jejunum, ileum, cecum and colon), 239
MLNs and organs (liver, spleen and kidney) seven days post infection. Treatment of the 240
infected sections allowed particularly isolation of bacteria which had been internalized 241
into the tissue. As shown in Fig. 1A, a comparable amount of the bacteria was isolated 242
from tonsils and the gut sections in all three different groups. We were able to detect 243
yersiniae in the MLNs, liver and kidney almost exclusively only in those animals which 244
had been infected with the highest dose of bacteria (Fig. 1A). 245
In agreement with these results, all infected animals shed bacteria in the feces at day 246
7 post infection. In further experiments we analyzed the time course of infection using 247
an oral dose of 109 cfu per piglet in order to generate an infection with a low risk of 248
provoking a clinical apparent disease. Results showed that the bacteria were shed in 249
feces from day 1 up to day 21 post infection. Using cold enrichment yersiniae could be 250
reisolated from the different intestinal and extraintestinal organs from day 3 up to day 14 251
post infection. Reisolation of the bacteria was still possible from the intestinal sections of 252
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all infected animals at day 21 post infection. However, the organ burden was signi-253
ficantly lower as compared to day 3 and 7, respectively. No severe pathological lesions 254
could be observed in any of the infected animals (Fig. S1, Table S1). In addition, blood 255
samples from piglets were taken weekly before and after oral infection with 109 cfu of 256
the serotype O:3 strain Y1. Sera were tested for antibodies against Yersinia outer 257
proteins (Yops). Infected pigs showed a clear seroconversion between day 7 and day 258
14 post infection, whereas antibody titers of the control animals remained negative (Fig. 259
1B). A complete blood count (red blood cell counts, leucocytes, differential cell count, 260
platelets) showed no or mild signs of inflammation in some pigs of the YeO:3 and YeO:8 261
infected groups, but also in the control groups (Table S1). Minimal to moderate inflam-262
mation of the lamina propria and submucosa of the small and large intestine is fre-263
quently observed in uninfected minipigs (33). In summary, we could demonstrate that 264
YeO:3 colonizes and persists in the lymphatic tissues of the tonsils and in the intestinal 265
tract of the minipigs, causing infections without severe inflammation and pathological 266
changes, which is in accordance with natural infection in productive livestock. 267
268
YeO:3 colonization of minipigs differs significantly from YeO:8 269
The newly established oral minipig model and the well-established BALB/c mouse 270
model were used to assess and compare colonization and pathogenic properties of 271
YeO:3 strain Y1 and YeO:8 strain 8081v. Colonization of the tonsils and different 272
sections of the intestinal tract (jejunum, ileum, cecum and colon) was monitored by 273
scoring the bacterial loads in the different tissue specimens. The YeO:8 strain could 274
only be isolated from the tonsils of a single infected minipig, but it was not detectable in 275
any of the intestinal sections at day 7 post infection. In strong contrast, high numbers of 276
YeO:3 bacteria (103-106 cfu/g organ) were reisolated from all tested intestinal sections 277
of all infected minipigs which remained symptomless as in our previous experiments 278
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(Fig. 2). Histological examinations revealed detection of bacterial colonies in intestinal 279
wall sections only of YeO:3, but not of YeO:8 infected piglets (Fig. S1). Overall, no or 280
only very mild signs of inflammation and symptoms of gut-associated lymphoid tissue 281
(GALT) hyperplasia, like increased lymphoid tissue in the jejunum and colon wall, were 282
observed, and occasionally very mild epithelial necrosis is evident sometimes with atro-283
phy/shortening of the villi. However, these findings were frequently also seen in un-284
infected control pigs (Fig. S1, Table S1) (33). 285
Detection by cold enrichment further demonstrated that the serotype O:3 strain could 286
be reisolated from extraintestinal organs, such as the MLNs, spleen and kidney of the 287
minipigs 7 days post infection, whereas YeO:8 was only found in the tonsils and ileum 288
of 30% of the infected minipigs (Fig. S2). Weekly taken blood samples tested for 289
presence of anti-Yop antibodies showed a seroconversion in pigs infected with YeO:3 290
between day 7 and 14 post infection, whereas titers of the YeO:8 infected piglets and 291
control animals remained negative (Fig. 3). 292
Strikingly, this difference in the colonization pattern was not observed in the BALB/c 293
infection model. Comparable amounts of YeO:3 and YeO:8 were isolated from all tested 294
intestinal tissues of the mice, and an even higher number of YeO:8 was detectable in 295
the mouse Peyer’s patches compared to YeO:3 (Fig. 2B). Mice infected with YeO:8 296
showed severe signs of illness (weight loss, diarrhea, piloerection and lethargy). In 297
contrast, mice infected with YeO:3 remained clinically healthy. 298
To further investigate how long the different wild-type yersiniae were shed by the pigs 299
via feces, rectal swabs were taken and cultivated using cold enrichment. The results 300
showed that 50% of the minipigs infected with YeO:3 shed bacteria with their feces from 301
day 1 and 100% from day 7 up to day 21 post infection. In contrast, only 25% of the Y. 302
enterocolitica serotype O:8 infected animals shed the bacteria at day 1 and no yersiniae 303
were reisolated from feces of the YeO:8 infected group of pigs from day 10 to 21 (Fig. 304
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S3). Taken together, this strongly indicated that in contrast to YeO:8, YeO:3 is able to 305
efficiently colonize and persist in the tonsils and the intestinal tract of the pigs for much 306
longer time periods. 307
308
Enhanced RovA levels are advantageous for the colonization of the porcine ileum 309
by YeO:3 in pigs 310
Our previous study highlighted important differences in the adhesion properties of 311
YeO:3 strains and other Y. enterocolitica serotypes. One difference concerns the global 312
virulence regulator RovA which is known to regulate the expression of several Yersinia 313
virulence factors including the primary internalization factor invasin (29, 34-36). It was 314
found that a thermal upshift renders the RovA regulatory protein more susceptible to 315
degradation in YeO:8 strains. In contrast, a single proline to serine substitution at posi-316
tion 98 (P98S) present in the majority of all characterized YeO:3 strains, including Y1, 317
increases the stability of RovA without affecting the thermosensing ability of the regu-318
lator (29). As a consequence, considerably higher amounts of RovA are synthesized by 319
the bacteria and this could compensate for the thermo-induced reduction of RovA DNA-320
binding at elevated temperatures. Since YeO:3 is a much better colonizer of intestinal 321
tissues of swine than YeO:8, we tested whether a more temperature-stable RovA 322
variant would be advantageous for persistence of the bacteria in pigs with an overall 323
slightly higher body temperature (38°C-40°C) compared to mice or humans (37°C) and 324
compared this with the effect of the more stable RovA on virulence in mice. We 325
performed co-infection experiments to minimize inherent inter-animal biological varia-326
tions thereby revealing even subtle differences of the colonization and persistence 327
patterns. Mice and minipigs were infected with approximately 5x108 or 109 bacteria, 328
respectively. Each inoculum comprised a mixture of equal numbers of the parental 329
kanamycin (Kan)S YeO:3 wild-type strain Y1 (rovAYeO:3(S98)) and the KanR mutant strain 330
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YE14 (rovAYeO:3(P98)). The latter expresses the RovAYeO:3(P98) mutant protein which is 331
rapidly degraded similar to the RovAYeO:8(P98) variant. At day 7 post infection, gut tissue 332
colonization of the bacteria was monitored by scoring the bacterial load in the different 333
intestinal parts (jejunum, ileum, cecum, colon) of mice and pigs, in the Peyer´s patches 334
(PPs) in mice and in the tonsils of pigs. In both mice and pigs, the bacteria were able to 335
invade into all examined organs (Fig. 4). Comparison of the calculated competitive 336
indices revealed that in general slightly higher numbers of bacterial YeO:3 strain Y1 337
expressing the stable version of RovA (RovAYeO:3(S98)) than of the strain YE14 were 338
isolated from the infected tissues of the pigs (Fig. 4A). In contrast, identical or even 339
more bacteria of the YeO:3 strain YE14 expressing the less stable version of RovA 340
(RovAYeO:3(P98)) were isolated from the infected mouse tissues (Fig. 4B-D). However, 341
these results were only statistically significant for the infected ileum of the pigs, 342
indicating that a stable RovA variant might cause a small, but not a major competitive 343
advantage for the colonization of pigs. 344
345
Presence of the IS1667 inserted into the invA promoter region of YeO:3 strains 346
increases colonization of the intestinal tract of pigs 347
Another difference between YeO:3 and YeO:8 is that in YeO:3 the primary inter-348
nalization factor invasin is highly and (nearly) constitutively synthesized, whereas it is 349
strongly repressed at 37°C in YeO:8 (30). Constitutive expression of invasin in YeO:3 350
was acquired by an IS1667 insertion into the rovA regulatory region harboring an IS-351
specific promoter. Its presence allowed invA expression at 37°C even in the absence of 352
RovA (30). To test whether constitutive invA expression would be beneficial for YeO:3, 353
we performed co-infection experiments with the KanR derivative of the YeO:3 wild type 354
strain (YE13) and the KanS mutant derivative YE15 (PinvO:3∆IS) in which the IS1667 355
element was deleted from the invA promoter region. Presence of IS1667 does not seem 356
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to have an impact on the colonization and persistence of YeO:3 in the intestinal tract of 357
mice (Fig. 5B, D). In contrast, significantly lower numbers of the ΔIS1667 mutant were 358
reisolated from different tissue sections of the intestinal tract (Fig. 5A,C). In particular 359
the calculated competitive index illustrated very clearly that YeO:3 IS1667 deletion 360
mutants have a substantial disadvantage invading the different tissues of the porcine 361
intestinal tract, although absence of the IS1667 element had no influence on the coloni-362
zation of the tonsils (Fig. 5C), and did not lead to a reduction of fecal shedding during 363
the first week after infection (Fig. S4A). 364
365
Invasin is important for persistence of Y. enterocolitica O:3 in the intestinal tract 366
of pigs and mice 367
Since constitutive invA expression was found to be beneficial for bacterial colonization 368
of the different tissues of the porcine intestinal tract, we also investigated whether 369
invasin per se is an essential virulence factor for efficient colonization and persistence 370
of YeO:3 within the tissues of the porcine intestinal tract. Equivalent to previous studies 371
in this work co-infection experiments were performed in minipigs and mice with an equal 372
mixture of the KanS YeO:3 wildtype Y1 and the KanR mutant derivative YE21 (∆invA). 373
Tissue colonization of the intestinal tract by the invA mutant strain was drastically 374
reduced in pigs and mice as compared to the wildtype (Fig. 6). About 101-104 fold less 375
bacteria were recovered from the different tissue sections of the intestinal tract of pigs 376
and mice and the Peyer’s patches of mice. Calculation of the competitive indices further 377
demonstrated that the decrease in the efficiency to persist in the intestinal tract was 378
significant (Fig. 6), indicating that invasin is a very important colonization factor for Y. 379
enterocolitica O:3. These findings were also confirmed by the analysis of bacterial 380
shedding, demonstrating that only 30% and 50% of the minipigs shed the invA mutant 381
bacteria at day 3 and 7 post infection compared to 70% and 90% of the minipigs 382
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infected with the wildtype, respectively (Fig. S4B). In contrast, no significant difference 383
of the bacterial load was observed in the tonsils of the pigs. This suggests that other 384
adhesion factors of the pathogen are implicated in the colonization of the lymphatic 385
tissue of the porcine throat. Y. enterocolitica O:3 strains encode additional adhesins (Ail, 386
YadA, PsaA) and other non-characterized putative adhesion factors (e.g. another InvA-387
type protein) (37) which could contribute to this process. 388
389
390
Discussion 391
Y. enterocolitica causes enteric infections in a wide range of domestic and wild animals 392
like dogs, goats, sheep, boars and pigs (21). In many countries, pigs are the 393
predominant reservoir of bioserotype 4/O:3, the most frequent cause of human 394
infections. Since YeO:3 infected pigs are usually asymptomatic carriers and as such 395
represent a substantial disease-causing potential for humans, we developed an oral 396
minipig model to characterize tissue colonization of Y. enterocolitica serotype O:3. As 397
high prevalence of YeO:3 strains in fattening pigs indicated serotype- and host-specific 398
colonization strategies, we compared persistence of a YeO:3 patient isolate with the 399
best-characterized and highly mouse-virulent YeO:8 8081v in minipigs and mice. 400
This study using a novel minipig model for enteric Yersinia infections clearly demon-401
strates that the porcine model is very suitable for the analysis of bacterial colonization 402
and persistence in its natural reservoir. We found that YeO:3, but not YeO:8, is able to 403
successfully colonize and persist in the different sections of the intestinal tract of the 404
minipigs. The course of the YeO:3 infection was symptomless although invasion of 405
intestinal tissue sections and shedding of bacteria were observed during the first three 406
weeks after oral challenge. In contrast, only the tonsils were efficiently colonized by 407
YeO:8 in some of the infected minipigs. These results reflect previous observations in 408
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surveys that mainly serotype O:3 strains were isolated from the oral cavity, intestinal 409
tract and feces of pigs (21, 38, 39). Young pigs generally get infected within the first 410
three weeks after entering contaminated areas (e.g. pens) or by other infected pigs, and 411
they remain intestinal and pharyngeal carriers for long time periods (38). Experimental 412
infections of 10- and 24-week-old pigs of larger breeds with Y. enterocolitica serotype 413
O:3 confirmed this observation and showed that similar to the infected minipigs in this 414
study, large numbers of the bacteria were still shed 2-3 weeks after infection (40). In 415
addition, in our study the minipigs had seroconverted at day 14 which corresponds to 416
what has been described for experimentally infected large breeds (41). 417
Pigs, but also wild rodents and other wild animals have been shown to be reservoirs 418
for Y. enterocolitica O:8. Indistinguishable genotypes have been found among strains 419
isolated from humans and wild rodents, and small rodents were considered to be 420
responsible for human infections and outbreaks in monkeys in Japan (42, 43). In one 421
single study, an experimental oral pig model using a conventional large breed was used 422
to assess the pathogenic potential of a restriction-deficient (R-M+) derivative of strain 423
YeO:8 8081v. Similar to the minipig model in this study, the 8081v derivate persisted 424
mainly in the lymphoid tissues of the tonsils, and no bacteria were detected in the small 425
intestine on day 7 post infection (19). Challenge with a 500-fold higher dose of bacteria 426
also caused asymptomatic infections without major pathological signs in larger breed 427
pigs, and only a very slight enteric catarrhalic inflammation was detectable early during 428
infection (19). However, several histological changes were observed in the brain 429
(meningoencephalitits), lung (pneumonia catarrhalia), liver (lymphohistiocytic nodules) 430
and several other tissues (hyperplasia of lymph follicles in the small intestine and the 431
mesenteric lymph nodes) between days 14-45 post infection (19). 432
Until now it remained unclear which of the different virulence factors and regulators of 433
Y. entercolitica O:3 are required for efficient colonization and persistence in pigs. Here, 434
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we demonstrate that the internalization factor invasin (InvA) is crucial for the establish-435
ment of an intestinal infection in the minipigs. Invasin was shown to promote efficient 436
colonization of all tested gut tissue sections (jejunum, ileum, cecum, colon). A YeO:3 437
invA mutant strain was still able to colonize the tonsils of the minipigs, but its ability to 438
persist in the intestinal tract was strongly reduced. Interestingly, similar to the oral 439
minipig model, also colonization of the different intestinal tissues and the Peyer’s 440
patches was reduced by up to 1000-fold in the oral mouse model, suggesting that 441
invasin plays a primary role in the initiation of the colonization of the intestinal tissue by 442
YeO:3. Invasin is well-known to be necessary for rapid and efficient penetration of the 443
intestinal barrier by Y. entercolitica in mice. However, a defect in the ability of YeO:8 444
invA mutants to colonize Peyer’s patches has only been reported for the very early 445
infection stages (23, 44). During the subsequent establishment of a systemic infection, 446
invasin of YeO:8 seems of secondary importance since the invA mutant started to colo-447
nize the Peyer’s patches after a delay of 3-4 days and was able to reach liver and 448
spleen at essentially the same time frame and efficiency (23) as the respective wildtype 449
strain. The observation that invA expression in YeO:8 is downregulated upon a tem-450
perature shift from 25°C to 37°C (27) supports this assumption and indicates that 451
alternative entry pathways predominantly expressed at body temperature during later 452
stages of the infection promote dissemination to deeper tissues and establishment of a 453
systemic infection. Based on results of this study, invasin seems to be more important 454
for the initiation and establishment of a successful YeO:3 infection than previously 455
observed for YeO:8. 456
In our preceding study, we demonstrated that YeO:3 constitutively expresses high 457
levels of InvA which is acquired through an IS1677 insertion in the invA regulatory 458
region and a more temperature-stable variant of the transcriptional activator of invA, 459
RovA. In this study, we demonstrate that elevated InvA levels caused by these YeO:3 460
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specific variations enhance colonization of the intestinal tissues by YeO:3 in the pigs, 461
but not in mice. This strongly suggests that these differences between YeO:3 and 462
YeO:8 strains confer better survival and/or persistence of YeO:3 in pigs. Whether this is 463
(partially) due to a higher body temperature of pigs (38°C-40°C) remains speculative. 464
The comparison of the colonization of pigs by YeO:3, YeO:3ΔinvA and YeO:8 also 465
indicates that expression of invasin alone does not account for the huge difference 466
between the YeO:3 and YeO:8 strain. Other virulence-associated traits identified in bio-467
serotype 4/O:3 strains which are absent in 1B/O:8 isolates (37) are likely to provide a 468
serious advantage in colonization of pigs. For example, Y. enterocolitica O:3 strain Y11 469
encodes an alternative pattern of putative virulence determinants, i.e. an RtxA-like toxin, 470
beta-fimbriae, a novel type 3 secretion system, a bacteriocin cluster, a four-gene cluster 471
homologous to the aatPABCD operon of enteroaggregative E. coli, which plays a role in 472
virulence by extransporting dispersin, and it harbors the agaVWEF operon supporting 473
the utilization of N-acetyl-galactosamine (GalNAc) (37). This sugar represents the 474
majority of sugars in the mucin of the pig’s small intestine and can be used by the 475
YeO:3 strain Y11 as single carbon source, but not by the O:8/1B strain (37, 45). The 476
ability to utilize GalNAc of the pig gut mucin might represent a crucial fitness factor of Y. 477
enterocolitica O:3 that supports adaptation to their natural host, the pig (37). 478
Among the set of virulence traits that experienced an alteration between YeO:3 and 479
YeO:8 strains, we show that invasin upregulation plays a significant role in host coloni-480
zation and successful persistence. It is well known that invasin promotes direct binding 481
to β1-integrin receptors expressed on M cell in the intestinal epithelium in mice to induce 482
uptake and transcytosis to reach subepithelial layers (24, 46). An overall alignment of 483
the murine and porcine β1-integrin amino acid sequence indicated that both receptors 484
are highly homologous (93.5% amino acid identity), indicating that the identical receptor 485
family is used by the bacteria for colonization in swine. Not much is known about 486
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expression of β1-integrin receptors in porcine tissues. However, some evidence exists 487
that β1-integrins are expressed in the porcine intestine and in isolated intestinal 488
epithelial cells of swine (47, 48). Efficient invasin-mediated engagement of β1-integrin 489
receptors in the intestine could inhibit spreading to other host sites and enable the 490
pathogen to remain in the intestine of the pigs for extended time periods. Adhesion 491
would also allow prolonged bacterial multiplication within the intestine and this would 492
exceed the rate of excretion from the lumen of the gut (49). Low expression of invasin in 493
the YeO:8 strains at 37°C is disadvantageous for the adhesion process and would 494
explain low efficiency of YeO:8 to persist in the porcine intestinal tract. Besides, Y. 495
enterocolitica encodes several other adhesins, such as YadA and Ail (50). Alternative 496
adhesion and invasion strategies were shown to compensate for lack of InvA-mediated 497
cell adhesion by YeO:8 in the mouse infection model and this could account for the 498
observation that LD50 values of both strains are very similar and that lack of invasin 499
causes only subtle differences for systemic infection in mice (23, 44). Interestingly, 500
although the alternative adhesins YadA and Ail of YeO:8 promote bacterial attachment 501
to human epithelial cells independently of invasin (25, 51), none of them seems to be 502
able to promote long-term persistence of YeO:8 in the intestinal tract of pig. Since loss 503
of invasin alone is sufficient to abolish efficient colonization of the porcine intestinal tract 504
by YeO:3, neither YadA nor Ail seem to be able to fully complement this defect. This 505
strongly suggests that in particular inactivation of invasin function might be sufficient to 506
prevent YeO:3 colonization. This knowledge might help to develop future strategies to 507
reduce the high prevalence of YeO:3 strains in fattening pigs/pig herds and, thereby, to 508
lower the risk of transmission of Y. enterocolitica to humans from pigs and pork 509
products. 510
511
Acknowledgements 512
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We thank Dr. Martin Fenner for helpful discussions. We also thank Tatjana Stolz and 513
Sandra Stengel for help with the animal infection experiments. This work was supported 514
by the German Federal Ministry for Research and Education (BMBF) (Consortium FBI-515
Zoo), the ‘Fonds der Chemischen Industrie’, and the German Center for Infection 516
Research (DZIF). Julia Schaake was also supported by the President’s Initiative and 517
Networking Fund of the Helmholtz Association of German Research Centres (HGF) 518
under contract number VH-GS-202. Anna Drees was funded by a fellowship of the 519
Ministry of Science and Culture of Lower Saxony (Georg-Christoph-Lichtenberg 520
Scholarship) within the framework of the PhD program "EWI-Zoonosen" of the 521
Hannover Graduate School for Veterinary Pathobiology, Neuroinfectiology, and 522
Translational Medicine (HGNI)". 523
524
525
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674
675
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Tables 676
677
Table 1: Strains used in this study. 678
Strains Description Source and reference
Ye 8081v bioserotype 1B/O:8, patient isolate, wildtype (27)
Y1 bioserotype 4/O:3, patient isolate, wildtype (29)
YE13 Y1, ProvAO:3::rovAS98, CmR, KnR (29)
YE14 Y1, ProvAO:3::rovAP98, CmR, KnR (29)
YE15 Y1, PinvA∆IS1667 (29)
YE21 Y1, ∆invA, KnR (29)
679
680
681
Figure Legends 682
683
Figure 1: Establishment of a Y. enterocolitica minipig model. 684
(A) Influence of the infectious dose on Y. enterocolitica O:3 colonization in minipigs. 685
Mini-Lewe piglets were orally infected with YeO:3 strain Y1 (wt). The infection inoculum 686
contained 108, 109 or 1010 cfu. The animals were sacrificed at day 7 post infection and 687
the numbers of surviving bacteria in tonsils, jejunum, ileum, cecum, colon, mesenteric 688
lymph nodes, liver, spleen and kidney were determined. (B) Seroconversion induced by 689
Y. enterocolitica YeO:3. Mini-Lewe piglets were orally infected with 109 cfu of Y. entero-690
colitica serotype O:3 strain Y1 or mock infected with PBS. Sera were analyzed using an 691
ELISA system based on recombinant Yersinia outer proteins (Yops). Data are pre-692
sented as means OD %. 693
694
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Figure 2. Y. enterocolitica O:3 and O:8 infection of minipigs and mice. Mini-Lewe 695
piglets were orally infected with 109 YeO:3 strain Y1 (wt) or YeO:8 strain 8081v (wt). 696
Accordingly, BALB/c mice were infected orally with 5 x 108 of the described bacterial 697
strains. Mice were sacrificed at day 3 and minipigs at day 7 post infection. Numbers of 698
surviving bacteria in the tonsils of the minipigs, in the Peyer´s patches of the mice and 699
in jejunum, ileum, cecum and colon of mice and minipigs were determined. Data are 700
presented as a scatter plot of numbers of cfu per gram of organ as determined by 701
counts of viable bacteria after serial plating. Each spot represents the cfu count in the 702
indicated tissue. The levels of statistical significance for differences between test groups 703
were determined by the Mann Whitney test. Stars indicate results that differed 704
significantly from those of Y1 with *(P<0.05), **(P<0.01) or ***(P<0.001). 705
706
Figure 3. Comparison of seroconversion induced by Y. enterocolitica YeO:3 and 707
YeO:8 in minipigs. Mini-Lewe piglets were orally infected with 109 cfu of Y. enterocoli-708
tica serotype O:3 strain Y1, Y. enterocolitica serotype O:8 strain 8081v or mock infected 709
with PBS. Sera were analyzed using an ELISA system based on recombinant Yersinia 710
outer proteins (Yops). Data are presented as means OD %. 711
712
Figure 4. Influence of constitutive RovA expression on Y. enterocolitica O:3 713
infection. Mini-Lewe minipigs were infected orally with 109 bacteria (A, C), and BALB/c 714
mice were co-infected via the orogastric route with 5 x 108 bacteria (B, D). The infection 715
inoculum contained an equal mixture of the YeO:3 strains Y1 (wt, rovAS98) and YE14 716
(rovAP98). Mice were sacrificed on day 3, minipigs on day 7 post infection. Numbers of 717
surviving bacteria in the tonsils of the minipigs, in Peyer´s patches of the mice and in 718
jejunum, ileum, cecum and colon of mice and minipigs were determined. (A, B) Data 719
are presented as a scatter plot of numbers of cfu per gram of organ as determined by 720
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counts of viable bacteria on plates. Each spot represents the cfu count in the indicated 721
tissue. The levels of statistical significance for differences between test groups were 722
determined by the Mann Whitney test. (C, D) Data are graphed as competitive index 723
values for difference in the virulence compared to Y1. Stars indicate results that differed 724
significantly from those of Y1 with *(P<0.05), **(P<0.01) or ***(P<0.001). 725
726
Figure 5. Impact of the insertion element IS1667 in the invA promoter region on Y. 727
enterocolitica O:3 virulence. Co-infections with equal mixtures of YE13 (Y1 KanR) and 728
YE15 (Y1 ∆IS1667) were performed in Mini-Lewe minipigs and BALB/c mice. Mice were 729
infected orogastrically with in total 5 x 108 bacteria, pigs were infected orally with 109 730
bacteria. Mice were sacrificed 3 days and minipigs 7 days post infection. Number of 731
surviving bacteria in the tonsils of the minipigs, in Peyer´s patches of the mice and in 732
jejunum, ileum, cecum and colon of mice and minipigs were determined. (A, B) Data 733
are presented as a scatter plot of numbers of cfu per gram of organ as determined by 734
counts of viable bacteria on plates. Each spot represents the cfu count in the indicated 735
tissue. The levels of statistical significance for differences between test groups were 736
determined by the Mann Whitney test. (C, D) Data are graphed as competitive index 737
values for difference in the virulence compared to Y13. Stars indicate results that 738
differed significantly from those of Y13 with *(P<0.05), **(P<0.01) or ***(P<0.001). 739
740
Figure 6. Invasin is important for persistence of Y. enterocolitica O:3 in the 741
intestinal tract of pigs and mice. BALB/c mice and Mini-Lewe minipigs were co-742
infected via the oral route with equal mixtures of Y1 (wt) and YE21 (Y1 ∆invA). The total 743
inoculum doses were 5 x 108 bacteria for mice and 109 bacteria for pigs. Mice were 744
sacrificed 3 days and minipigs 7 days post infection. Numbers of surviving bacteria in 745
the tonsils of the minipigs, in Peyer´s patches of the mice and in jejunum, ileum, cecum 746
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31
and colon of mice and minipigs were determined. (A, B) Data are presented as a scatter 747
plot of numbers of cfu per gram of organ as determined by counts of viable bacteria on 748
plates. Each spot represents the cfu count in the indicated tissue. The levels of 749
statistical significance for differences between test groups were determined by the 750
Mann Whitney test. (C, D) Data are graphed as competitive index values for difference 751
in the virulence compared to Y1. Stars indicate results that differed significantly from 752
those of Y1 with *(P<0.05), **(P<0.01) or ***(P<0.001). 753
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