scholarly journals Development and Application of a Mouse Intestinal Loop Model To Study theIn VivoAction of Clostridium perfringens Enterotoxin

2011 ◽  
Vol 79 (8) ◽  
pp. 3020-3027 ◽  
Author(s):  
Justin A. Caserta ◽  
Susan L. Robertson ◽  
Juliann Saputo ◽  
Archana Shrestha ◽  
Bruce A. McClane ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Cell Damage ◽  
Gastrointestinal Symptoms ◽  
Food Poisoning ◽  
The United States ◽  
Intestinal Lumen ◽  
Intestinal Loop ◽  
Loop Model ◽  
Internal Organs ◽  
Content Type

ABSTRACTClostridium perfringensenterotoxin (CPE) is responsible for causing the gastrointestinal symptoms ofC. perfringenstype A food poisoning, the second most commonly identified bacterial food-borne illness in the United States. CPE is produced by sporulatingC. perfringenscells in the small intestinal lumen, where it then causes epithelial cell damage and villous blunting that leads to diarrhea and cramping. Those effects are typically self-limiting; however, severe outbreaks of this food poisoning, particularly two occurring in psychiatric institutions, have involved deaths. Since animal models are currently limited for the study of the CPE action, a mouse ligated intestinal loop model was developed. With this model, significant lethality was observed after 2 h in loops receiving an inoculum of 100 or 200 μg of CPE but not using a 50-μg toxin inoculum. A correlation was noted between the overall intestinal histological damage and lethality in mice. Serum analysis revealed a dose-dependent increase in serum CPE and potassium levels. CPE binding to the liver and kidney was detected, along with elevated levels of potassium in the serum. These data suggest that CPE can be absorbed from the intestine into the circulation, followed by the binding of the toxin to internal organs to induce potassium leakage, which can cause death. Finally, CPE pore complexes similar to those formed in tissue culture cells were detected in the intestine and liver, suggesting that (i) CPE actions are similarin vivoandin vitroand (ii) CPE-induced potassium release into blood may result from CPE pore formation in internal organs such as the liver.

scholarly journals NanH Is Produced by Sporulating Cultures of Clostridium perfringens Type F Food Poisoning Strains and Enhances the Cytotoxicity of C. perfringens Enterotoxin

mSphere ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Jihong Li ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Virulence Factor ◽  
Mother Cell ◽  
Food Poisoning ◽  
Mature Spore ◽  
The United States ◽  
Content Type ◽  
Intestinal Pathology ◽  
Mother Cells

ABSTRACT Clostridium perfringens type F food poisoning (FP) strains cause one of the most common foodborne illnesses. This FP develops when type F FP strains sporulate in the intestines and produce C. perfringens enterotoxin (CPE), which is responsible for the diarrhea and abdominal cramps of this disease. While C. perfringens can produce up to three different sialidases, the current study surveyed FP strains, which confirmed the results of a previous study that they consistently carry the nanH sialidase gene, often as their only sialidase gene. NanH production was found to be associated with sporulating cultures of the surveyed type F FP strains, including SM101 (a transformable derivative of a FP strain). The sporulation-associated regulation of NanH production by strain SM101 growing in modified Duncan-Strong medium (MDS) was shown to involve Spo0A, but it did not require the completion of sporulation. NanH production was not necessary for either the growth or sporulation of SM101 when cultured in MDS. In those MDS cultures, NanH accumulated in the sporulating mother cell until it was released coincidently with CPE. Since CPE becomes extracellular when mother cells lyse to release their mature spores, this indicates that mother cell lysis is also important for NanH release. The copresence of NanH and CPE in supernatants from lysed sporulating cultures was shown to enhance CPE cytotoxicity for Caco-2 cells. This enhancement was attributable to NanH increasing CPE binding and could be replicated with purified recombinant NanH. These in vitro findings suggest that NanH may be an accessory virulence factor during type F FP. IMPORTANCE Clostridium perfringens type F strains cause the second most common bacterial foodborne illness in the United States. C. perfringens enterotoxin (CPE) is responsible for the diarrhea and cramping symptoms of this food poisoning (FP). Previous studies showed that NanI sialidase can enhance CPE activity in vitro. While many type F FP strains do not produce NanI, they do consistently make NanH sialidase. This study shows that, like CPE, NanH is produced by sporulating type F FP strains and then released extracellularly when their sporulating cells lyse to release their mature spore. NanH was shown to enhance CPE cytotoxicity in vitro by increasing CPE binding to cultured Caco-2 cells. This enhancement could be important because many type F FP strains produce less CPE than necessary (in a purified form) to cause intestinal pathology in animal models. Therefore, NanH represents a potential accessory virulence factor for type F FP.

scholarly journals Evidence thatClostridium perfringensEnterotoxin-Induced Intestinal Damage and Enterotoxemic Death in Mice Can Occur Independently of Intestinal Caspase-3 Activation

2018 ◽  
Vol 86 (7) ◽  
Author(s):  
John C. Freedman ◽  
Mauricio A. Navarro ◽  
Eleonora Morrell ◽  
Juliann Beingesser ◽  
Archana Shrestha ◽  
...  
Keyword(s):  
Tissue Damage ◽  
Caspase 3 ◽  
Gastrointestinal Symptoms ◽  
Intestinal Loop ◽  
Intestinal Damage ◽  
Loop Model ◽  
Dependent Manner ◽  
Intestinal Tissue ◽  
Content Type

ABSTRACTClostridium perfringensenterotoxin (CPE) is responsible for the gastrointestinal symptoms ofC. perfringenstype A food poisoning and some cases of nonfoodborne gastrointestinal diseases, such as antibiotic-associated diarrhea. In the presence of certain predisposing medical conditions, this toxin can also be absorbed from the intestines to cause enterotoxemic death. CPE actionin vivoinvolves intestinal damage, which begins at the villus tips. The cause of this CPE-induced intestinal damage is unknown, but CPE can induce caspase-3-mediated apoptosis in cultured enterocyte-like Caco-2 cells. Therefore, the current study evaluated whether CPE activates caspase-3 in the intestines and, if so, whether this effect is required for the development of intestinal tissue damage or enterotoxemic lethality. Using a mouse ligated small intestinal loop model, CPE was shown to cause intestinal caspase-3 activation in a dose- and time-dependent manner. Most of this caspase-3 activation occurred in epithelial cells shed from villus tips. However, CPE-induced caspase-3 activation occurred after the onset of tissue damage. Furthermore, inhibition of intestinal caspase-3 activity did not affect the onset of intestinal tissue damage. Similarly, inhibition of intestinal caspase-3 activity did not reduce CPE-induced enterotoxemic lethality in these mice. Collectively, these results demonstrate that caspase-3 activation occurs in the CPE-treated intestine but that this effect is not necessary for the development of CPE-induced intestinal tissue damage or enterotoxemic lethality.

scholarly journals Potential Therapeutic Effects of Mepacrine againstClostridium perfringensEnterotoxin in a Mouse Model of Enterotoxemia

2019 ◽  
Vol 87 (4) ◽  
Author(s):  
Mauricio A. Navarro ◽  
Archana Shrestha ◽  
John C. Freedman ◽  
Juliann Beingesser ◽  
Bruce A. McClane ◽  
...  
Keyword(s):  
Pore Formation ◽  
Food Poisoning ◽  
Gastrointestinal Diseases ◽  
Intestinal Loop ◽  
Therapeutic Effects ◽  
Intestinal Damage ◽  
Loop Model ◽  
Content Type

ABSTRACTClostridium perfringensenterotoxin (CPE) is a pore-forming toxin that causes the symptoms of common bacterial food poisoning and several non-foodborne human gastrointestinal diseases, including antibiotic-associated diarrhea and sporadic diarrhea. In some cases, CPE-mediated disease can be very severe or fatal due to the involvement of enterotoxemia. Therefore, the development of potential therapeutics against CPE action during enterotoxemia is warranted. Mepacrine, an acridine derivative drug with broad-spectrum effects on pores and channels in mammalian membranes, has been used to treat protozoal intestinal infections in human patients. A previous study showed that the presence of mepacrine inhibits CPE-induced pore formation and activity in enterocyte-like Caco-2 cells, reducing the cytotoxicity caused by this toxinin vitro. Whether mepacrine is similarly protective against CPE actionin vivohas not been tested. When the current study evaluated whether mepacrine protects against CPE-induced death and intestinal damage using a murine ligated intestinal loop model, mepacrine protected mice from the enterotoxemic lethality caused by CPE. This protection was accompanied by a reduction in the severity of intestinal lesions induced by the toxin. Mepacrine did not reduce CPE pore formation in the intestine but inhibited absorption of the toxin into the blood of some mice. Protection from enterotoxemic death correlated with the ability of this drug to reduce CPE-induced hyperpotassemia. Thesein vivofindings, coupled with previousin vitrostudies, support mepacrine as a potential therapeutic against CPE-mediated enterotoxemic disease.

scholarly journals The Potential Therapeutic Agent Mepacrine Protects Caco-2 Cells against Clostridium perfringens Enterotoxin Action

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
John C. Freedman ◽  
Matthew R. Hendricks ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Pore Formation ◽  
Therapeutic Potential ◽  
Food Poisoning ◽  
Host Cells ◽  
Artificial Membranes ◽  
Content Type ◽  
Gi Symptoms ◽  
Human Enterocyte ◽  
Bacterial Food

ABSTRACT Clostridium perfringens enterotoxin (CPE) causes the gastrointestinal (GI) symptoms of a common bacterial food poisoning and several nonfoodborne human GI diseases. A previous study showed that, via an undetermined mechanism, the presence of mepacrine blocks CPE-induced electrophysiologic activity in artificial membranes. The current study now demonstrates that mepacrine also inhibits CPE-induced cytotoxicity in human enterocyte-like Caco-2 cells and that mepacrine does not directly inactivate CPE. Instead, this drug reduces both CPE pore formation and CPE pore activity in Caco-2 cells. These results suggest mepacrine as a therapeutic candidate for treating CPE-mediated GI diseases. Clostridium perfringens enterotoxin (CPE) causes the diarrhea associated with a common bacterial food poisoning and many antibiotic-associated diarrhea cases. The severity of some CPE-mediated disease cases warrants the development of potential therapeutics. A previous study showed that the presence of mepacrine inhibited CPE-induced electrophysiology effects in artificial lipid bilayers lacking CPE receptors. However, that study did not assess whether mepacrine inactivates CPE or, instead, inhibits a step in CPE action. Furthermore, CPE action in host cells is complex, involving the toxin binding to receptors, receptor-bound CPE oligomerizing into a prepore on the membrane surface, and β-hairpins in the CPE prepore inserting into the membrane to form a pore that induces cell death. Therefore, the current study evaluated the ability of mepacrine to protect cells from CPE. This drug was found to reduce CPE-induced cytotoxicity in Caco-2 cells. This protection did not involve mepacrine inactivation of CPE, indicating that mepacrine affects one or more steps in CPE action. Western blotting then demonstrated that mepacrine decreases CPE pore levels in Caco-2 cells. This mepacrine-induced reduction in CPE pore levels did not involve CPE binding inhibition but rather an increase in CPE monomer dissociation due to mepacrine interactions with Caco-2 membranes. In addition, mepacrine was also shown to inhibit CPE pores when already present in Caco-2 cells. These in vitro studies, which identified two mepacrine-sensitive steps in CPE-induced cytotoxicity, add support to further testing of the therapeutic potential of mepacrine against CPE-mediated disease. IMPORTANCE Clostridium perfringens enterotoxin (CPE) causes the gastrointestinal (GI) symptoms of a common bacterial food poisoning and several nonfoodborne human GI diseases. A previous study showed that, via an undetermined mechanism, the presence of mepacrine blocks CPE-induced electrophysiologic activity in artificial membranes. The current study now demonstrates that mepacrine also inhibits CPE-induced cytotoxicity in human enterocyte-like Caco-2 cells and that mepacrine does not directly inactivate CPE. Instead, this drug reduces both CPE pore formation and CPE pore activity in Caco-2 cells. These results suggest mepacrine as a therapeutic candidate for treating CPE-mediated GI diseases.

scholarly journals Reverse Genetics System for Severe Fever with Thrombocytopenia Syndrome Virus

2014 ◽  
Vol 89 (6) ◽  
pp. 3026-3037 ◽  
Author(s):  
Benjamin Brennan ◽  
Ping Li ◽  
Shuo Zhang ◽  
Aqian Li ◽  
Mifang Liang ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Far East ◽  
Gastrointestinal Symptoms ◽  
Case Fatality ◽  
The United States ◽  
Cdna Clones ◽  
Wild Type ◽  
Content Type ◽  
Recombinant Viruses ◽  
Severe Fever

ABSTRACTSevere fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen that was first reported in China in 2009. Phylogenetic analysis of the viral genome showed thatSFTS virusrepresents a new lineage within thePhlebovirusgenus, distinct from the existing sandfly fever and Uukuniemi virus groups, in the familyBunyaviridae. SFTS disease is characterized by gastrointestinal symptoms, chills, joint pain, myalgia, thrombocytopenia, leukocytopenia, and some hemorrhagic manifestations with a case fatality rate of about 2 to 15%. Here we report the development of reverse genetics systems to study STFSV replication and pathogenesis. We developed and optimized functional T7 polymerase-based M- and S-segment minigenome assays, which revealed errors in the published terminal sequences of the S segment of the Hubei 29 strain of SFTSV. We then generated recombinant viruses from cloned cDNAs prepared to the antigenomic RNAs both of the minimally passaged virus (HB29) and of a cell culture-adapted strain designated HB29pp. The growth properties, pattern of viral protein synthesis, and subcellular localization of viral N and NSs proteins of wild-type HB29pp (wtHB29pp) and recombinant HB29pp viruses were indistinguishable. We also show that the viruses fail to shut off host cell polypeptide production. The robust reverse genetics system described will be a valuable tool for the design of therapeutics and the development of killed and attenuated vaccines against this important emerging pathogen.IMPORTANCESFTSV and related tick-borne phleboviruses such as Heartland virus are emerging viruses shown to cause severe disease in humans in the Far East and the United States, respectively. Study of these novel pathogens would be facilitated by technology to manipulate these viruses in a laboratory setting using reverse genetics. Here, we report the generation of infectious SFTSV from cDNA clones and demonstrate that the behavior of recombinant viruses is similar to that of the wild type. This advance will allow for further dissection of the roles of each of the viral proteins in the context of virus infection, as well as help in the development of antiviral drugs and protective vaccines.

Food Poisoning Due to Clostridium perfringens in the United States

1983 ◽  
Vol 147 (1) ◽  
pp. 167-170 ◽  
Author(s):  
W. X. Shandera ◽  
C. O. Tacket ◽  
P. A. Blake
Keyword(s):  
United States ◽  
Clostridium Perfringens ◽  
Food Poisoning ◽  
The United States

scholarly journals Toxin-neutralizing antibodies protect against Clostridium perfringens-induced necrosis in an intestinal loop model for bovine necrohemorrhagic enteritis

2016 ◽  
Vol 12 (1) ◽  
Author(s):  
Evy Goossens ◽  
Stefanie Verherstraeten ◽  
Bonnie R. Valgaeren ◽  
Bart Pardon ◽  
Leen Timbermont ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Neutralizing Antibodies ◽  
Intestinal Loop ◽  
Loop Model

scholarly journals A rapid qualitative assay for detection of Clostridium perfringens in canned food products

2017 ◽  
Vol 64 (2) ◽  
Author(s):  
Gayatri Ashwinkumar Dave
Keyword(s):  
Phospholipase C ◽  
Clostridium Perfringens ◽  
Colorimetric Assay ◽  
Food Poisoning ◽  
The United States ◽  
Necrotic Enteritis ◽  
Yellow Colour ◽  
Negative Results ◽  
Reporter Assays ◽  
Food Borne Infections

Clostridium perfringens (MTCC 1349) is a Gram-positive, anaerobic, endospore forming, and rod-shaped bacterium. This bacterium produces a variety of toxins under strict anaerobic environment. C. perfringens can grow at temperatures ranging between 20°C and 50°C. It is the major causetive agent for gas gangrene, cellulitis, septicemia, necrotic enteritis and food poisoning, which are common toxin induced conditions noted in human and animals. C. perfringens can produce produce four major types of toxins that are used for the classification of strains, classified under type A–E. Across the globe many countries, including the United States, are affected by C. perfringens food poisonings where it is ranked as one of the most common causes of food borne infections. To date, no direct one step assay for the detection of C. perfringens has been developed and only few methods are known for accurate detection of C. perfringens. Long detection and incubation time is the major consideration of these reporter assays. The prensent study proposes a rapid and reliable colorimetric assay for the detection of C. perfringens. In principale, this assay detects the para nitrophenyl (yellow colour end product) liberated due to the hydrolysis of paranitrophenyl phosphetidyl choline (PNPC) through phospholipase C (lecithinase). Constitutive secretion of phospholipase C is a charactristic feature of C. perfringens. This assay detects the presence of the extracellular lecithinse through the PNPC impragnated impregnated probe. The probe is impregnated with peranitrophenyl phosphotidyl choline ester, which is colourless substrate used by lecithinase. The designed assay is specific towards PNPC and detectes very small quantites of lecithinase under conditions used. The reaction is substrate specific, no cross reaction was observed upon incubation with other substrates. In addition, this assay gave negative results with other clostridium strains, no cross reactions were observed with other experimental strains like C. tetani, C. botulinum, C. acetobutyricum, Bacillus subtilis, and Escherichia coli. This assay is extramly rapid and provides reliable and reproducible results within one hour of incubation at 37°C.

scholarly journals A Synthetic Peptide Corresponding to the Extracellular Loop 2 Region of Claudin-4 Protects against Clostridium perfringens EnterotoxinIn VitroandIn Vivo

2014 ◽  
Vol 82 (11) ◽  
pp. 4778-4788 ◽  
Author(s):  
Archana Shrestha ◽  
Susan L. Robertson ◽  
Jorge Garcia ◽  
Juliann Beingasser ◽  
Bruce A. McClane ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Synthetic Peptide ◽  
Inhibitory Effect ◽  
Intestinal Loop ◽  
Extracellular Loop ◽  
Content Type ◽  
Potential Therapeutic Application ◽  
Small Intestinal

ABSTRACTClostridium perfringensenterotoxin (CPE) action starts when the toxin binds to claudin receptors. Claudins contain two extracellular loop domains, with the second loop (ECL-2) being slightly smaller than the first. CPE has been shown to bind to ECL-2 in receptor claudins. We recently demonstrated that Caco-2 cells (a naturally CPE-sensitive enterocyte-like cell line) can be protected from CPE-induced cytotoxicity by preincubating the enterotoxin with soluble full-length recombinant claudin-4 (rclaudin-4), which is a CPE receptor, but not with recombinant nonreceptor claudins, such asrclaudin-1. The current study evaluated whether a synthetic peptide corresponding to the claudin-4 ECL-2 sequence can similarly inhibit CPE actionin vitroandin vivo. Significant protection of Caco-2 cells was also observed using eitherrclaudin-4 or the claudin-4 ECL-2 peptide in both a preincubation assay and a coincubation assay. This inhibitory effect was specific, sincerclaudin-1 and a synthetic peptide based on the claudin-1 ECL-2 offered no protection to Caco-2 cells. However, the claudin-4 ECL-2 peptide was unable to neutralize cytotoxicity if CPE had already bound to Caco-2 cells. When the study was repeatedin vivousing a rabbit small intestinal loop assay, preincubation or coincubation of CPE with the claudin-4 ECL-2 peptide significantly and specifically inhibited the development of CPE-induced luminal fluid accumulation and histologic lesions in rabbit small intestinal loops. No similarin vivoprotection from CPE was afforded by the claudin-1 ECL-2 peptide. These results suggest that claudin-4 ECL-2 peptides should be further investigated for their potential therapeutic application against CPE-associated disease.

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