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Experimental In Vivo Models of Bacterial Shiga Toxin-Associated Hemolytic Uremic Syndrome
1Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea, 2Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea, 3Department of Biomolecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), Daejeon 34113, Republic of Korea
J. Microbiol. Biotechnol. 2018; 28(9): 1413-1425
Published September 28, 2018 https://doi.org/10.4014/jmb.1803.03012
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Shiga Toxins and Hemolytic Uremic Syndrome
Shiga toxins (Stxs) are ribosome-inactivating proteins expressed by several species of pathogenic bacteria that colonize the gastrointestinal tract. They are responsible for a condition known as hemorrhagic colitis or bloody diarrhea. Stxs bind specifically to the neutral membrane glycolipid globotriaosylceramide (Gb3) receptor expressed on the cell surface of various host cells [1, 2]. After binding to Gb3, Stxs are internalized via endocytosis and trafficked to the endoplasmic reticulum (ER) via retrograde transport through the
Genomic analyses show that
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Table 1 . Serotypes of Shiga toxin-producing
Escherichia coli .Sero–pathotype1 Serotype Frequency of Association with Disease Involvement in outbreaks Association with HUS and HC2 A O55:H7, O157:H7, O157:NM, High Common + B O26:H11, O103:H2, O111:H8, O111:NM, O121:H19, O145:NM, Moderate Uncommon + C O5:NM, O91:H21, O104:H21, O113:H21, O121:NM, O165:H25 and others Low Rare + D O7:H4, O69:H11, O80:NM, O84:H2, O98:NM ,O103:H25, O113:H4, O117:H7, 119:H25, O132:NM, O146:H21, O165:NM, O171:H2, O172:NM, O174:H8 and others Low Rare - E O6:H34, O8:H19, O39:H49, O46:H38, O76:H7, O84:NM, O88:H25, O98:H25, O113:NM, O136:H12, O136:NM, O143:H31, O153:H31, O156:NM, O163:NM, O177:NM and others Not implicated Not implicated - 1Adapted from Gyles
et al ., 2007 [27].2HUS = hemolytic uremic syndrome; HC = hemorrhagic colitis.
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Table 2 . Characteristics of the Shiga toxin family.
Organism Toxin Amino acid homology* (%) Disease progression Receptor Shigella Dysenteriae Stx - D → HC → HUS Gb3 STEC Stx1 - D → HC → HUS Gb3 Stx1c (A)97; (B) 95 D → HC → HUS Gb3 Stx1d (A)93; (B) 92 D Gb3 Stx2 - D → HC → HUS Gb3 Stx2c (A)100; (B) 97 D → HC → HUS Gb3 Stx2c2 (A)100; (B) 97 D Gb3 Stx2d (A)99; (B) 97 D Gb3 Stx2d activatable (A)99; (B) 97 D → HC → HUS Gb3 Stx2e (A)93; (B) 84 D → piglet edema Gb3 & Gb4 Stx2f (A)63; (B) 57 D Gb3 1Adapted from Johannes
et al ., 2010 [30].2D = diarrhea; HUS = hemolytic uremic syndrome; HC = hemorrhagic colitis.
Bacteremia is rare in patients with EHEC infection, but the bacteria-secreted Stxs are widely known as the principal contributors of organ damage. Recently, a 10-month-old pediatric HUS patient with stool positive confirmation of the Shiga toxin-producing O104
While many studies have been performed in in vitro models to delineate the pathogenesis of Stxs [7, 50], far fewer in vivo studies with animal models showing pathophysiological features of Shiga toxicosis have been conducted. Furthermore, how the innate immune responses to Stx mediate damage to the colon and the development of potentially fatal complications such as HUS remains to be fully explored. In this review, we will summarize the experimental in vivo models that have been used to study Stx-induced pathogenesis, the knowledge that has been gained from these studies, and the current progress in the development of therapeutic interventions against Stx.
In Vivo Studies on Stx-Induced HUS Pathogenesis
Various animal models have been developed for the in vivo study of STEC pathogenesis. In many models, the animals are infected by an STEC, which travels to the intestine and starts to secrete the Stx. The toxin then passes through the intestinal mucosa and enters the bloodstream, where it mainly binds to neutrophils (Fig. 1). The toxin then travels to the target organs (Fig. 2). Other models take advantage of the fact that the Stxs reach the target organs via the circulation: they are induced by intravenous or intraperitoneal injection of the Stx alone. Since the main target organ of circulating Stxs is the kidney, many animal models that recapitulate the pathogenic outcomes in the kidney or intestine that are seen in humans have been developed. Moreover, since HUS-related CNS associates with serious consequences, animal models of this complication have also been developed. An up-to-date list of Stx studies in animal models of HUS is presented in Table 3.
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Table 3 . Animal models of Stx injection or STEC infection.
Animal Inoculated material Concentration Route Organ Outcome Ref Mouse Purified Stx2 0.5 and 50 ng/20 g Intraperitoneal Brain N, D [71] Mouse VTEC/VT2 5 × 1010 cfu/1 to 4 ng Oral/Intraperitoneal Brain N, D [68] Mouse Purified Stx1 and Stx2a Stx2a at 7 ng/ml and Stx1 at 1500 ng/ml Intraperitoneal Kidney, Brain N, R [120] Mouse STEC 1010 CFU Oral Intestine I, C [51] Mouse STEC 1010 CFU Oral Intestine R, C [52] Mouse STEC 5 × 103 CFU Oral Intestine I, D, C [53] Mouse STEC 109-10 CFU Oral Kidney, Intestine R, N, C [54] Mouse STEC 102-7 CFU Oral Kidney, Intestine R, I, C [55] Mouse STEC 105-9 CFU Oral Intestine I, D, C [57] Mouse Purified Stx1 and Stx2 400 ng and 1 ng Intraperitoneal and intravenous Kidney R, D [58] Mouse Purified Stx2 1 to 5 ng/20 g Intravenous Kidney R, D [35] Mouse STEC 107-8 CFU Oral Kidney, Intestine, Brain R, N, I [56] Mouse Purified Stx2 225 ng/kg Intraperitoneal Kidney R, D [42] Mouse Purified Stx2 50 ng/kg Intravenous Kidney R, D [70] Mouse Purified Stx2 5 to 0.44 ng/mouse Intravenous Brain N, D [69] Mouse Stx 0.075 ng/g Intravenous Kidney R, D [121] Mouse Purified Stx2 0.15 ng/g Intravenous . D [122] Mouse Purified Stx2 100 ng per mouse Intraperitoneal Kidney R [123] Rat Culture supernatant from the recombinant (sStx2)E. coli Approximately 20 μg/kg Intraperitoneal Kidney, Intestine R, I [72] Rat Culture supernatant from the recombinant (sStx2)E. coli 1 ml/100 g (b.w.) Stx2 (400 ng of Stx2/ml) Intraperitoneal Brain N [76] Rat Purified Stx2 6 μl of Stx2 (1 ng/μl) Intracerebroventricular Brain N [74] Rat Purified Stx2 10 pg/g Intravenous Brain N [124] Rat Purified Stx2 6 μl of Stx2 (1 ng/μl) Intracerebroventricular Brain N [73] Monkey STEC 1011 CFU Oral Intestine I [80] Baboon Purified Stx1 2.0 μg/kg Intravenous Kidney, Brain R, I [81] Baboon Purified Stx1 50 to 200 ng/kg Intravenous Kidney, Intestine, Brain R, N, I [47] Baboon Purified Stx1 and Stx2 Stx1 at 10, 50, or 100 ng/kg or Stx2 at 10, 50 ng/kg Intravenous Kidney R [83] Baboon Purified Stx1 and Stx2 100 ng/kg Intravenous Kidney R, T [82] Baboon Purified Stx1 and Stx2 Stx1 at 10, 50, or 100 ng/kg or Stx2 at 10, 50 ng/kg Intravenous Kidney R, D [84] Calf STEC 1010 CFU Oral Intestine I, C [85] Calf EHEC O104:H4 1010 CFU Oral Intestine I, C [86] Calf STEC 3 × 1010 cfu Intramuscular Intestine I [125] Chick STEC 105 CFU Oral Intestine I [126] Chick STEC 1.6 × 109 Oral Intestine I [93] Chick STEC 108 CFU Oral Intestine I [92] Piglet STEC 1011 CFU Oral . E [88] Piglet STEC 3 × 109 CFU Oral Brain N, D [89] Piglet Stx2e 5 to 500 ng/kg Intravenous . E [87] Piglet STEC 1010 CFU Oral Intestine I [90] Canine purified Stx1 and Stx2 0.03 to 0.05 μg/kg Intravenous . D, T [95] Rabbit O153/O157:H7 5 × 108 / 9 × 108 cfu Oral Kidney, Intestine R, D, D* [96] Rabbit Purified Stx2 0.1 to 4.0 μg/kg Intravenous Brain N [99] Rabbit VT2 5 mg/kg Intravenous Brain N, I [98] Rabbit VT1 4 μg/rabbit Intravenous Kidney, Intestine C [127] Rabbit Purified Stx2 1200 ng/kg Intravenous Kidney, Intestine R, I, T [97] R, Renal damage; N, Neurological manifestations; I, Intestinal pathology; D, Death; C, Colonization; T, Thrombosis; D*, Diarrhea; E, Edema; b.w., body weight.
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Fig. 1. Summary of Shiga toxin-induced HUS pathogenesis. A & E lesions, attaching and effacing lesions; CNS, central nervous system; ER, endoplasmic reticulum; HUS, hemolytic uremic syndrome; Stx, Shiga toxin.
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Fig. 2. Pathways of Shiga toxin infection and its action in target organs. CNS, central nervous system; HUS, hemolytic uremic syndrome.
Rodents
Rodents such as the rat and the mouse are the most commonly used animals for preclinical research because the maintenance costs are relatively low and the animals are easy to handle. They are also very useful for studies on STEC pathogenesis because the clinical outcomes of STEC infection in rodents are similar to those in humans; namely, renal failure, dehydration, diarrhea, and death. The first mouse model of STEC pathogenesis was generated by treating mice with an antibiotic (streptomycin or mitomycin C) that reduced the normal flora in the intestine and then feeding them with an STEC [51-53]. The antibiotic treatment allowed estimation of the ability of STEC to colonize the intestine in the absence of competition from the intestinal flora. Such competition was also eliminated by using germ-free mice [54, 55]. Notably, when germ-free mice were both injected with TNF-α and inoculated with STEC, the mice developed systemic disease, including neuronal damage and inflammation in the CNS as well as glomerular lesions; these signs were even readily observed when a low STEC dose was used [54]. However, the fact that the antibiotic-treated and germ-free mouse models lack gut flora limits their usefulness for studying the natural disease outcomes of STEC infection. Therefore, several researchers developed mouse models of STEC pathogenesis in the context of a normal bowel flora [56, 57]. These physiologically more relevant models include that of Mohawk
Generally, it is accepted that Stxs are a major cause of not only HUS but also CNS damage. HUS occurs after the Stx enters the systemic circulation, resulting in CNS damage in severe cases. Patients with STEC-induced HUS are more likely to progress to CNS dysfunction. These CNS alterations are a major cause of child mortality after acute illness [23, 59-62]. Moreover, the mortality rate associated with HUS with CNS dysfunction is 2–3 times higher than that associated with HUS alone [21, 23, 59, 63-65]. In terms of the pathogenic mechanisms by which Stxs induce CNS disturbances in children, HUS with neurologic involvement can lead to visual system impairment, including blindness [65, 66]; indeed, a recent study shows that Stxs induce apoptosis and ER stress in the retinal pigment epithelium, which plays an important role in maintaining proper visual function [67]. Moreover, experiments in mice with oral STEC infection-induced encephalopathy [68] show that Stxs weaken the blood-brain barrier (BBB) by damaging blood vessels. Recent reports suggest that injection of Stx2 into murine blood vessels damages the striatum, leading to motor deficits and neurovascular injury [69]. In addition, several studies show that mice injected intraperitoneally with 0.025–2.5 μg/kg Stxs develop nervous system symptoms such as hind limb paralysis, lethargy, shivering, abnormal gait, and seizures [68, 70, 71].
In rat models, intraperitoneal injection of culture supernatant from STEC results in histopathological outcomes in the kidney, including acute glomerular necrosis and microvascular thrombosis, which are also seen in STEC-infected humans [72]. STEC-induced CNS damage was also generated in rats by intracerebroventricular administration of purified-Stx2 (6 ng/mice): confocal microscopy revealed neuronal death and glial cell damage [73, 74]. A clue to how Stx induces CNS damage was initially provided by Rensmeester and Hulsman: they showed that the CNS edema and neurological symptoms of patients after epileptic seizures associate with changes in brain aquaporin (AQP) expression [75]. Two other studies showed that exposure of rats and mice to Stx reduced AQP4 expression around blood vessels in the brain [76, 77]. Moreover, heat shock protein 70 (Hsp70), a chaperone protein, interacts with the stress sensor protein IRE1α to protect host cells from ER stress [78]. In vitro experiments showed that Stx2 treatment induces apoptosis and decreases proliferation of B92 and primary rat glial cells by reducing expression of Hsp70 [79].
Non-Human Primates
Despite some ethical and financial problems, primates are still the most suitable animal model for research on infectious diseases because their immune system is very similar to that of humans. Therefore, several studies have assessed the toxic effects of Stxs in primate models. Kang
Another primate model study was conducted by Taylor
Other Animal Models
It is believed that cattle are the most important sources of the STEC that cause foodborne diseases such as HUS in humans. Since cattle infected with STEC do not exhibit any serious disease symptoms, researchers have used cattle to study how to reduce STEC colonization in STEC host animals [85, 86]. Dean-Nystrom
In the 1990s, a HUS-like disease in dogs with renal failure was reported. This led to the development of canine models of HUS [94, 95]. Raife
Conclusions and Future Perspectives
The experiments with various in vivo models, including mice, piglets, and rabbits, have not only provided important insights into the pathological consequences of Stx, they have also revealed potential pathways that could be targeted by therapies for HUS. These animal models are particularly important because even though non-human primate models probably best reproduce the serious clinical outcomes seen in humans (especially the toxin-induced nephrotoxicity), studies with these models are inevitably limited by the scarcity of monkeys and high costs.
While this review focused on in vivo models, it should be mentioned that the in vivo model findings have often been initiated, supported, and extended by in vitro analyses of the effects of Stx on susceptible cell types, including human monocytes/macrophages, endothelial cells, renal epithelial cells, and neuronal cells [50, 100]. Several interesting in vitro models have also been described recently. Thus, Karve
Diarrhea-associated HUS (D+HUS) is a leading cause of pediatric acute renal failure [104, 105]. Despite the many studies with HUS animal models, a therapeutic vaccine that effectively ameliorates D+HUS is not yet available. Instead of vaccine development, toxin-neutralizing therapeutics using several anti-Stxs antibodies were tested in animal models like piglets or rodents, and these antibody treatments effectively rescued the Stx-intoxicated animals from severe mortality [35, 106-108]. In addition, peptide-based neutralizer that directly binds to Stx2 was identified to inhibit Stx binding to Gb3 receptor and successfully protected rodent models from Stx-caused lethality [109-111]. As therapeutic targeting for retrograde trafficking to the Golgi apparatus or the ER of the Stxs, small molecule compounds such as Retro-1, Retro-2 and Exo2 were developed and found to be protective for STEC-infected mice from Stx-induced toxicity [112, 113]. Metal cofactor manganese was found to stimulate degradation of the endosome-to-Golgi transport protein Gpp130 and protect it from Stx1, but not Stx2 in mice [114].
Although many HUS animal model studies show that Stxs upregulate various stress-activated kinase pathways, including p38 MAPK, JNK, ERK, MK2, and ZAK, and that these induce the production of pro-inflammatory cytokines that mediate the tissue damage caused by these toxins [115-117], it has been difficult to find the downstream substrates of these kinase pathways that can be targeted with sufficiently high specificity by a therapy against D+HUS. Further research aiming to identify these targets is warranted. ZAK-deficient mice were protected from gastrointestinal lysine toxicity by reducing CXCL1 production following depurination of the sarcin-ricin loop [118]. Treatment of rabbit ZAK kinase inhibitor imatinib also reduced the number of neutrophils penetrating STEC-infected colon tissues [119]. Other potential targets for treatments that prevent or ameliorate the Stx-induced acute renal damage in HUS may be the signaling molecules that mediate STEC-induced inflammation, apoptosis, autophagy, and ER stress responses. Further studies in both in vitro and in vivo models that evaluate inhibitors targeting these signaling molecules are needed. Moreover, studies that further elucidate the pro-inflammatory cytokine-mediated signaling mechanisms that are activated by Stxs will be critical for development of effective therapies against the emerging infectious diseases caused by
Acknowledgments
This work was supported by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program and by the Bio & Medical Technology Development Program of the National Research Foundation (NRF), funded by the Korean government (MSIP) (NRF-2015M3A9E6028953 and 2016M3A9B6918675). This work was also supported by the Basic Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT & Future Planning (grant number: 2017R1C1B1005137).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Related articles in JMB
Article
Review
J. Microbiol. Biotechnol. 2018; 28(9): 1413-1425
Published online September 28, 2018 https://doi.org/10.4014/jmb.1803.03012
Copyright © The Korean Society for Microbiology and Biotechnology.
Experimental In Vivo Models of Bacterial Shiga Toxin-Associated Hemolytic Uremic Syndrome
Yu-Jin Jeong 1, Sung-Kyun Park 1, Sung-Jin Yoon 2, Young Jun Park 2, 3 and Moo-Seung Lee 1, 3*
1Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea, 2Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea, 3Department of Biomolecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), Daejeon 34113, Republic of Korea
Abstract
Shiga toxins (Stxs) are the main virulence factors expressed by the pathogenic Stx-producing
bacteria, namely, Shigella dysenteriae serotype 1 and certain Escherichia coli strains. These
bacteria cause widespread outbreaks of bloody diarrhea (hemorrhagic colitis) that in severe
cases can progress to life-threatening systemic complications, including hemolytic uremic
syndrome (HUS) characterized by the acute onset of microangiopathic hemolytic anemia and
kidney dysfunction. Shiga toxicosis has a distinct pathogenesis and animal models of Stxassociated
HUS have allowed us to investigate this. Since these models will also be useful for
developing effective countermeasures to Stx-associated HUS, it is important to have clinically
relevant animal models of this disease. Multiple studies over the last few decades have shown
that mice injected with purified Stxs develop some of the pathophysiological features seen in
HUS patients infected with the Stx-producing bacteria. These features are also efficiently
recapitulated in a non-human primate model (baboons). In addition, rats, calves, chicks,
piglets, and rabbits have been used as models to study symptoms of HUS that are
characteristic of each animal. These models have been very useful for testing hypotheses about
how Stx induces HUS and its neurological sequelae. In this review, we describe in detail the
current knowledge about the most well-studied in vivo models of Stx-induced HUS; namely,
those in mice, piglets, non-human primates, and rabbits. The aim of this review is to show how
each human clinical outcome-mimicking animal model can serve as an experimental tool to
promote our understanding of Stx-induced pathogenesis.
Keywords: Shiga toxin, HUS, animal models, STEC
Shiga Toxins and Hemolytic Uremic Syndrome
Shiga toxins (Stxs) are ribosome-inactivating proteins expressed by several species of pathogenic bacteria that colonize the gastrointestinal tract. They are responsible for a condition known as hemorrhagic colitis or bloody diarrhea. Stxs bind specifically to the neutral membrane glycolipid globotriaosylceramide (Gb3) receptor expressed on the cell surface of various host cells [1, 2]. After binding to Gb3, Stxs are internalized via endocytosis and trafficked to the endoplasmic reticulum (ER) via retrograde transport through the
Genomic analyses show that
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Table 1 . Serotypes of Shiga toxin-producing
Escherichia coli ..Sero–pathotype1 Serotype Frequency of Association with Disease Involvement in outbreaks Association with HUS and HC2 A O55:H7, O157:H7, O157:NM, High Common + B O26:H11, O103:H2, O111:H8, O111:NM, O121:H19, O145:NM, Moderate Uncommon + C O5:NM, O91:H21, O104:H21, O113:H21, O121:NM, O165:H25 and others Low Rare + D O7:H4, O69:H11, O80:NM, O84:H2, O98:NM ,O103:H25, O113:H4, O117:H7, 119:H25, O132:NM, O146:H21, O165:NM, O171:H2, O172:NM, O174:H8 and others Low Rare - E O6:H34, O8:H19, O39:H49, O46:H38, O76:H7, O84:NM, O88:H25, O98:H25, O113:NM, O136:H12, O136:NM, O143:H31, O153:H31, O156:NM, O163:NM, O177:NM and others Not implicated Not implicated - 1Adapted from Gyles
et al ., 2007 [27]..2HUS = hemolytic uremic syndrome; HC = hemorrhagic colitis..
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Table 2 . Characteristics of the Shiga toxin family..
Organism Toxin Amino acid homology* (%) Disease progression Receptor Shigella Dysenteriae Stx - D → HC → HUS Gb3 STEC Stx1 - D → HC → HUS Gb3 Stx1c (A)97; (B) 95 D → HC → HUS Gb3 Stx1d (A)93; (B) 92 D Gb3 Stx2 - D → HC → HUS Gb3 Stx2c (A)100; (B) 97 D → HC → HUS Gb3 Stx2c2 (A)100; (B) 97 D Gb3 Stx2d (A)99; (B) 97 D Gb3 Stx2d activatable (A)99; (B) 97 D → HC → HUS Gb3 Stx2e (A)93; (B) 84 D → piglet edema Gb3 & Gb4 Stx2f (A)63; (B) 57 D Gb3 1Adapted from Johannes
et al ., 2010 [30]..2D = diarrhea; HUS = hemolytic uremic syndrome; HC = hemorrhagic colitis..
Bacteremia is rare in patients with EHEC infection, but the bacteria-secreted Stxs are widely known as the principal contributors of organ damage. Recently, a 10-month-old pediatric HUS patient with stool positive confirmation of the Shiga toxin-producing O104
While many studies have been performed in in vitro models to delineate the pathogenesis of Stxs [7, 50], far fewer in vivo studies with animal models showing pathophysiological features of Shiga toxicosis have been conducted. Furthermore, how the innate immune responses to Stx mediate damage to the colon and the development of potentially fatal complications such as HUS remains to be fully explored. In this review, we will summarize the experimental in vivo models that have been used to study Stx-induced pathogenesis, the knowledge that has been gained from these studies, and the current progress in the development of therapeutic interventions against Stx.
In Vivo Studies on Stx-Induced HUS Pathogenesis
Various animal models have been developed for the in vivo study of STEC pathogenesis. In many models, the animals are infected by an STEC, which travels to the intestine and starts to secrete the Stx. The toxin then passes through the intestinal mucosa and enters the bloodstream, where it mainly binds to neutrophils (Fig. 1). The toxin then travels to the target organs (Fig. 2). Other models take advantage of the fact that the Stxs reach the target organs via the circulation: they are induced by intravenous or intraperitoneal injection of the Stx alone. Since the main target organ of circulating Stxs is the kidney, many animal models that recapitulate the pathogenic outcomes in the kidney or intestine that are seen in humans have been developed. Moreover, since HUS-related CNS associates with serious consequences, animal models of this complication have also been developed. An up-to-date list of Stx studies in animal models of HUS is presented in Table 3.
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Table 3 . Animal models of Stx injection or STEC infection..
Animal Inoculated material Concentration Route Organ Outcome Ref Mouse Purified Stx2 0.5 and 50 ng/20 g Intraperitoneal Brain N, D [71] Mouse VTEC/VT2 5 × 1010 cfu/1 to 4 ng Oral/Intraperitoneal Brain N, D [68] Mouse Purified Stx1 and Stx2a Stx2a at 7 ng/ml and Stx1 at 1500 ng/ml Intraperitoneal Kidney, Brain N, R [120] Mouse STEC 1010 CFU Oral Intestine I, C [51] Mouse STEC 1010 CFU Oral Intestine R, C [52] Mouse STEC 5 × 103 CFU Oral Intestine I, D, C [53] Mouse STEC 109-10 CFU Oral Kidney, Intestine R, N, C [54] Mouse STEC 102-7 CFU Oral Kidney, Intestine R, I, C [55] Mouse STEC 105-9 CFU Oral Intestine I, D, C [57] Mouse Purified Stx1 and Stx2 400 ng and 1 ng Intraperitoneal and intravenous Kidney R, D [58] Mouse Purified Stx2 1 to 5 ng/20 g Intravenous Kidney R, D [35] Mouse STEC 107-8 CFU Oral Kidney, Intestine, Brain R, N, I [56] Mouse Purified Stx2 225 ng/kg Intraperitoneal Kidney R, D [42] Mouse Purified Stx2 50 ng/kg Intravenous Kidney R, D [70] Mouse Purified Stx2 5 to 0.44 ng/mouse Intravenous Brain N, D [69] Mouse Stx 0.075 ng/g Intravenous Kidney R, D [121] Mouse Purified Stx2 0.15 ng/g Intravenous . D [122] Mouse Purified Stx2 100 ng per mouse Intraperitoneal Kidney R [123] Rat Culture supernatant from the recombinant (sStx2)E. coli Approximately 20 μg/kg Intraperitoneal Kidney, Intestine R, I [72] Rat Culture supernatant from the recombinant (sStx2)E. coli 1 ml/100 g (b.w.) Stx2 (400 ng of Stx2/ml) Intraperitoneal Brain N [76] Rat Purified Stx2 6 μl of Stx2 (1 ng/μl) Intracerebroventricular Brain N [74] Rat Purified Stx2 10 pg/g Intravenous Brain N [124] Rat Purified Stx2 6 μl of Stx2 (1 ng/μl) Intracerebroventricular Brain N [73] Monkey STEC 1011 CFU Oral Intestine I [80] Baboon Purified Stx1 2.0 μg/kg Intravenous Kidney, Brain R, I [81] Baboon Purified Stx1 50 to 200 ng/kg Intravenous Kidney, Intestine, Brain R, N, I [47] Baboon Purified Stx1 and Stx2 Stx1 at 10, 50, or 100 ng/kg or Stx2 at 10, 50 ng/kg Intravenous Kidney R [83] Baboon Purified Stx1 and Stx2 100 ng/kg Intravenous Kidney R, T [82] Baboon Purified Stx1 and Stx2 Stx1 at 10, 50, or 100 ng/kg or Stx2 at 10, 50 ng/kg Intravenous Kidney R, D [84] Calf STEC 1010 CFU Oral Intestine I, C [85] Calf EHEC O104:H4 1010 CFU Oral Intestine I, C [86] Calf STEC 3 × 1010 cfu Intramuscular Intestine I [125] Chick STEC 105 CFU Oral Intestine I [126] Chick STEC 1.6 × 109 Oral Intestine I [93] Chick STEC 108 CFU Oral Intestine I [92] Piglet STEC 1011 CFU Oral . E [88] Piglet STEC 3 × 109 CFU Oral Brain N, D [89] Piglet Stx2e 5 to 500 ng/kg Intravenous . E [87] Piglet STEC 1010 CFU Oral Intestine I [90] Canine purified Stx1 and Stx2 0.03 to 0.05 μg/kg Intravenous . D, T [95] Rabbit O153/O157:H7 5 × 108 / 9 × 108 cfu Oral Kidney, Intestine R, D, D* [96] Rabbit Purified Stx2 0.1 to 4.0 μg/kg Intravenous Brain N [99] Rabbit VT2 5 mg/kg Intravenous Brain N, I [98] Rabbit VT1 4 μg/rabbit Intravenous Kidney, Intestine C [127] Rabbit Purified Stx2 1200 ng/kg Intravenous Kidney, Intestine R, I, T [97] R, Renal damage; N, Neurological manifestations; I, Intestinal pathology; D, Death; C, Colonization; T, Thrombosis; D*, Diarrhea; E, Edema; b.w., body weight..
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Figure 1. Summary of Shiga toxin-induced HUS pathogenesis. A & E lesions, attaching and effacing lesions; CNS, central nervous system; ER, endoplasmic reticulum; HUS, hemolytic uremic syndrome; Stx, Shiga toxin.
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Figure 2. Pathways of Shiga toxin infection and its action in target organs. CNS, central nervous system; HUS, hemolytic uremic syndrome.
Rodents
Rodents such as the rat and the mouse are the most commonly used animals for preclinical research because the maintenance costs are relatively low and the animals are easy to handle. They are also very useful for studies on STEC pathogenesis because the clinical outcomes of STEC infection in rodents are similar to those in humans; namely, renal failure, dehydration, diarrhea, and death. The first mouse model of STEC pathogenesis was generated by treating mice with an antibiotic (streptomycin or mitomycin C) that reduced the normal flora in the intestine and then feeding them with an STEC [51-53]. The antibiotic treatment allowed estimation of the ability of STEC to colonize the intestine in the absence of competition from the intestinal flora. Such competition was also eliminated by using germ-free mice [54, 55]. Notably, when germ-free mice were both injected with TNF-α and inoculated with STEC, the mice developed systemic disease, including neuronal damage and inflammation in the CNS as well as glomerular lesions; these signs were even readily observed when a low STEC dose was used [54]. However, the fact that the antibiotic-treated and germ-free mouse models lack gut flora limits their usefulness for studying the natural disease outcomes of STEC infection. Therefore, several researchers developed mouse models of STEC pathogenesis in the context of a normal bowel flora [56, 57]. These physiologically more relevant models include that of Mohawk
Generally, it is accepted that Stxs are a major cause of not only HUS but also CNS damage. HUS occurs after the Stx enters the systemic circulation, resulting in CNS damage in severe cases. Patients with STEC-induced HUS are more likely to progress to CNS dysfunction. These CNS alterations are a major cause of child mortality after acute illness [23, 59-62]. Moreover, the mortality rate associated with HUS with CNS dysfunction is 2–3 times higher than that associated with HUS alone [21, 23, 59, 63-65]. In terms of the pathogenic mechanisms by which Stxs induce CNS disturbances in children, HUS with neurologic involvement can lead to visual system impairment, including blindness [65, 66]; indeed, a recent study shows that Stxs induce apoptosis and ER stress in the retinal pigment epithelium, which plays an important role in maintaining proper visual function [67]. Moreover, experiments in mice with oral STEC infection-induced encephalopathy [68] show that Stxs weaken the blood-brain barrier (BBB) by damaging blood vessels. Recent reports suggest that injection of Stx2 into murine blood vessels damages the striatum, leading to motor deficits and neurovascular injury [69]. In addition, several studies show that mice injected intraperitoneally with 0.025–2.5 μg/kg Stxs develop nervous system symptoms such as hind limb paralysis, lethargy, shivering, abnormal gait, and seizures [68, 70, 71].
In rat models, intraperitoneal injection of culture supernatant from STEC results in histopathological outcomes in the kidney, including acute glomerular necrosis and microvascular thrombosis, which are also seen in STEC-infected humans [72]. STEC-induced CNS damage was also generated in rats by intracerebroventricular administration of purified-Stx2 (6 ng/mice): confocal microscopy revealed neuronal death and glial cell damage [73, 74]. A clue to how Stx induces CNS damage was initially provided by Rensmeester and Hulsman: they showed that the CNS edema and neurological symptoms of patients after epileptic seizures associate with changes in brain aquaporin (AQP) expression [75]. Two other studies showed that exposure of rats and mice to Stx reduced AQP4 expression around blood vessels in the brain [76, 77]. Moreover, heat shock protein 70 (Hsp70), a chaperone protein, interacts with the stress sensor protein IRE1α to protect host cells from ER stress [78]. In vitro experiments showed that Stx2 treatment induces apoptosis and decreases proliferation of B92 and primary rat glial cells by reducing expression of Hsp70 [79].
Non-Human Primates
Despite some ethical and financial problems, primates are still the most suitable animal model for research on infectious diseases because their immune system is very similar to that of humans. Therefore, several studies have assessed the toxic effects of Stxs in primate models. Kang
Another primate model study was conducted by Taylor
Other Animal Models
It is believed that cattle are the most important sources of the STEC that cause foodborne diseases such as HUS in humans. Since cattle infected with STEC do not exhibit any serious disease symptoms, researchers have used cattle to study how to reduce STEC colonization in STEC host animals [85, 86]. Dean-Nystrom
In the 1990s, a HUS-like disease in dogs with renal failure was reported. This led to the development of canine models of HUS [94, 95]. Raife
Conclusions and Future Perspectives
The experiments with various in vivo models, including mice, piglets, and rabbits, have not only provided important insights into the pathological consequences of Stx, they have also revealed potential pathways that could be targeted by therapies for HUS. These animal models are particularly important because even though non-human primate models probably best reproduce the serious clinical outcomes seen in humans (especially the toxin-induced nephrotoxicity), studies with these models are inevitably limited by the scarcity of monkeys and high costs.
While this review focused on in vivo models, it should be mentioned that the in vivo model findings have often been initiated, supported, and extended by in vitro analyses of the effects of Stx on susceptible cell types, including human monocytes/macrophages, endothelial cells, renal epithelial cells, and neuronal cells [50, 100]. Several interesting in vitro models have also been described recently. Thus, Karve
Diarrhea-associated HUS (D+HUS) is a leading cause of pediatric acute renal failure [104, 105]. Despite the many studies with HUS animal models, a therapeutic vaccine that effectively ameliorates D+HUS is not yet available. Instead of vaccine development, toxin-neutralizing therapeutics using several anti-Stxs antibodies were tested in animal models like piglets or rodents, and these antibody treatments effectively rescued the Stx-intoxicated animals from severe mortality [35, 106-108]. In addition, peptide-based neutralizer that directly binds to Stx2 was identified to inhibit Stx binding to Gb3 receptor and successfully protected rodent models from Stx-caused lethality [109-111]. As therapeutic targeting for retrograde trafficking to the Golgi apparatus or the ER of the Stxs, small molecule compounds such as Retro-1, Retro-2 and Exo2 were developed and found to be protective for STEC-infected mice from Stx-induced toxicity [112, 113]. Metal cofactor manganese was found to stimulate degradation of the endosome-to-Golgi transport protein Gpp130 and protect it from Stx1, but not Stx2 in mice [114].
Although many HUS animal model studies show that Stxs upregulate various stress-activated kinase pathways, including p38 MAPK, JNK, ERK, MK2, and ZAK, and that these induce the production of pro-inflammatory cytokines that mediate the tissue damage caused by these toxins [115-117], it has been difficult to find the downstream substrates of these kinase pathways that can be targeted with sufficiently high specificity by a therapy against D+HUS. Further research aiming to identify these targets is warranted. ZAK-deficient mice were protected from gastrointestinal lysine toxicity by reducing CXCL1 production following depurination of the sarcin-ricin loop [118]. Treatment of rabbit ZAK kinase inhibitor imatinib also reduced the number of neutrophils penetrating STEC-infected colon tissues [119]. Other potential targets for treatments that prevent or ameliorate the Stx-induced acute renal damage in HUS may be the signaling molecules that mediate STEC-induced inflammation, apoptosis, autophagy, and ER stress responses. Further studies in both in vitro and in vivo models that evaluate inhibitors targeting these signaling molecules are needed. Moreover, studies that further elucidate the pro-inflammatory cytokine-mediated signaling mechanisms that are activated by Stxs will be critical for development of effective therapies against the emerging infectious diseases caused by
Acknowledgments
This work was supported by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program and by the Bio & Medical Technology Development Program of the National Research Foundation (NRF), funded by the Korean government (MSIP) (NRF-2015M3A9E6028953 and 2016M3A9B6918675). This work was also supported by the Basic Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT & Future Planning (grant number: 2017R1C1B1005137).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.
Fig 2.
-
Table 1 . Serotypes of Shiga toxin-producing
Escherichia coli ..Sero–pathotype1 Serotype Frequency of Association with Disease Involvement in outbreaks Association with HUS and HC2 A O55:H7, O157:H7, O157:NM, High Common + B O26:H11, O103:H2, O111:H8, O111:NM, O121:H19, O145:NM, Moderate Uncommon + C O5:NM, O91:H21, O104:H21, O113:H21, O121:NM, O165:H25 and others Low Rare + D O7:H4, O69:H11, O80:NM, O84:H2, O98:NM ,O103:H25, O113:H4, O117:H7, 119:H25, O132:NM, O146:H21, O165:NM, O171:H2, O172:NM, O174:H8 and others Low Rare - E O6:H34, O8:H19, O39:H49, O46:H38, O76:H7, O84:NM, O88:H25, O98:H25, O113:NM, O136:H12, O136:NM, O143:H31, O153:H31, O156:NM, O163:NM, O177:NM and others Not implicated Not implicated - 1Adapted from Gyles
et al ., 2007 [27]..2HUS = hemolytic uremic syndrome; HC = hemorrhagic colitis..
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Table 2 . Characteristics of the Shiga toxin family..
Organism Toxin Amino acid homology* (%) Disease progression Receptor Shigella Dysenteriae Stx - D → HC → HUS Gb3 STEC Stx1 - D → HC → HUS Gb3 Stx1c (A)97; (B) 95 D → HC → HUS Gb3 Stx1d (A)93; (B) 92 D Gb3 Stx2 - D → HC → HUS Gb3 Stx2c (A)100; (B) 97 D → HC → HUS Gb3 Stx2c2 (A)100; (B) 97 D Gb3 Stx2d (A)99; (B) 97 D Gb3 Stx2d activatable (A)99; (B) 97 D → HC → HUS Gb3 Stx2e (A)93; (B) 84 D → piglet edema Gb3 & Gb4 Stx2f (A)63; (B) 57 D Gb3 1Adapted from Johannes
et al ., 2010 [30]..2D = diarrhea; HUS = hemolytic uremic syndrome; HC = hemorrhagic colitis..
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Table 3 . Animal models of Stx injection or STEC infection..
Animal Inoculated material Concentration Route Organ Outcome Ref Mouse Purified Stx2 0.5 and 50 ng/20 g Intraperitoneal Brain N, D [71] Mouse VTEC/VT2 5 × 1010 cfu/1 to 4 ng Oral/Intraperitoneal Brain N, D [68] Mouse Purified Stx1 and Stx2a Stx2a at 7 ng/ml and Stx1 at 1500 ng/ml Intraperitoneal Kidney, Brain N, R [120] Mouse STEC 1010 CFU Oral Intestine I, C [51] Mouse STEC 1010 CFU Oral Intestine R, C [52] Mouse STEC 5 × 103 CFU Oral Intestine I, D, C [53] Mouse STEC 109-10 CFU Oral Kidney, Intestine R, N, C [54] Mouse STEC 102-7 CFU Oral Kidney, Intestine R, I, C [55] Mouse STEC 105-9 CFU Oral Intestine I, D, C [57] Mouse Purified Stx1 and Stx2 400 ng and 1 ng Intraperitoneal and intravenous Kidney R, D [58] Mouse Purified Stx2 1 to 5 ng/20 g Intravenous Kidney R, D [35] Mouse STEC 107-8 CFU Oral Kidney, Intestine, Brain R, N, I [56] Mouse Purified Stx2 225 ng/kg Intraperitoneal Kidney R, D [42] Mouse Purified Stx2 50 ng/kg Intravenous Kidney R, D [70] Mouse Purified Stx2 5 to 0.44 ng/mouse Intravenous Brain N, D [69] Mouse Stx 0.075 ng/g Intravenous Kidney R, D [121] Mouse Purified Stx2 0.15 ng/g Intravenous . D [122] Mouse Purified Stx2 100 ng per mouse Intraperitoneal Kidney R [123] Rat Culture supernatant from the recombinant (sStx2)E. coli Approximately 20 μg/kg Intraperitoneal Kidney, Intestine R, I [72] Rat Culture supernatant from the recombinant (sStx2)E. coli 1 ml/100 g (b.w.) Stx2 (400 ng of Stx2/ml) Intraperitoneal Brain N [76] Rat Purified Stx2 6 μl of Stx2 (1 ng/μl) Intracerebroventricular Brain N [74] Rat Purified Stx2 10 pg/g Intravenous Brain N [124] Rat Purified Stx2 6 μl of Stx2 (1 ng/μl) Intracerebroventricular Brain N [73] Monkey STEC 1011 CFU Oral Intestine I [80] Baboon Purified Stx1 2.0 μg/kg Intravenous Kidney, Brain R, I [81] Baboon Purified Stx1 50 to 200 ng/kg Intravenous Kidney, Intestine, Brain R, N, I [47] Baboon Purified Stx1 and Stx2 Stx1 at 10, 50, or 100 ng/kg or Stx2 at 10, 50 ng/kg Intravenous Kidney R [83] Baboon Purified Stx1 and Stx2 100 ng/kg Intravenous Kidney R, T [82] Baboon Purified Stx1 and Stx2 Stx1 at 10, 50, or 100 ng/kg or Stx2 at 10, 50 ng/kg Intravenous Kidney R, D [84] Calf STEC 1010 CFU Oral Intestine I, C [85] Calf EHEC O104:H4 1010 CFU Oral Intestine I, C [86] Calf STEC 3 × 1010 cfu Intramuscular Intestine I [125] Chick STEC 105 CFU Oral Intestine I [126] Chick STEC 1.6 × 109 Oral Intestine I [93] Chick STEC 108 CFU Oral Intestine I [92] Piglet STEC 1011 CFU Oral . E [88] Piglet STEC 3 × 109 CFU Oral Brain N, D [89] Piglet Stx2e 5 to 500 ng/kg Intravenous . E [87] Piglet STEC 1010 CFU Oral Intestine I [90] Canine purified Stx1 and Stx2 0.03 to 0.05 μg/kg Intravenous . D, T [95] Rabbit O153/O157:H7 5 × 108 / 9 × 108 cfu Oral Kidney, Intestine R, D, D* [96] Rabbit Purified Stx2 0.1 to 4.0 μg/kg Intravenous Brain N [99] Rabbit VT2 5 mg/kg Intravenous Brain N, I [98] Rabbit VT1 4 μg/rabbit Intravenous Kidney, Intestine C [127] Rabbit Purified Stx2 1200 ng/kg Intravenous Kidney, Intestine R, I, T [97] R, Renal damage; N, Neurological manifestations; I, Intestinal pathology; D, Death; C, Colonization; T, Thrombosis; D*, Diarrhea; E, Edema; b.w., body weight..
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