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 NanI Sialidase Can Support the Growth and Survival of Clostridium perfringens Strain F4969 in the Presence of Sialyated Host Macromolecules (Mucin) or Caco-2 Cells

2017 ◽  
Vol 86 (2) ◽  
Author(s):  
Jihong Li ◽  
Bruce A. McClane
Keyword(s):  
Sialic Acid ◽  
Clostridium Perfringens ◽  
Growth Promotion ◽  
Food Poisoning ◽  
Host Cells ◽  
Content Type ◽  
Growth And Survival ◽  
Gi Infections ◽  
Human Enterocyte ◽  
Free Sialic Acid

ABSTRACT Enterotoxin-producing Clostridium perfringens type A strains cause human gastrointestinal (GI) infections, including a very common food poisoning and 5 to 10% of all cases of antibiotic-associated diarrhea. This bacterium can utilize free sialic acid for growth, but most sialic acids in the GI tract are sequestered on macromolecules, such as the mucin proteins of mucus or glycoconjugates in host cells. However, many C. perfringens strains produce sialidases that might promote growth and survival by generating free sialic acid from those sialyated host macromolecules or by exposing underlying carbohydrates or proteins for digestion by other enzymes. The current study tested that possibility and found that the C. perfringens nonfoodborne human GI disease strain F4969 can use either a mucin preparation or Caco-2 cells, which are human enterocyte-like cells, to support its growth and survival. An isogenic nanI null mutant and complemented strain were used to show that this enhanced growth and survival using mucin or Caco-2 cells involved NanI, which is the major exosialidase of F4969 and many other C. perfringens strains. Experiments also suggested that, at least in part, this growth promotion involves utilization of NanI-generated sialic acid. In addition, a sialidase inhibitor named siastatin B reduced the growth and survival of F4969 growing with either the mucin preparation or Caco-2 cells. These findings suggest that, when produced, NanI may be a significant contributor to C. perfringens human GI infections by promoting the intestinal growth and survival of this bacterium. They also suggest the possibility that sialidase inhibitors might inhibit C. perfringens infections.

scholarly journals NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718

2015 ◽  
Vol 197 (20) ◽  
pp. 3339-3353 ◽  
Author(s):  
Jihong Li ◽  
John C. Freedman ◽  
Bruce A. McClane
Keyword(s):  
Sialic Acid ◽  
Clostridium Perfringens ◽  
Host Cells ◽  
Start Codon ◽  
Null Mutant ◽  
Wild Type ◽  
Mobility Shift ◽  
Content Type ◽  
Type D ◽  
Epsilon Toxin

ABSTRACTClostridium perfringenstype D strains are usually associated with diseases of livestock, and their virulence requires the production of epsilon toxin (ETX). We previously showed (J. Li, S. Sayeed, S. Robertson, J. Chen, and B. A. McClane, PLoS Pathog 7:e1002429, 2011,http://dx.doi.org/10.1371/journal.ppat.1002429) that BMC202, ananInull mutant of type D strain CN3718, produces less ETX than wild-type CN3718 does. The current study proved that the lower ETX production by strain BMC202 is due tonanIgene disruption, since both genetic and physical (NanI or sialic acid) complementation increased ETX production by BMC202. Furthermore, a sialidase inhibitor that interfered with NanI activity also reduced ETX production by wild-type CN3718. The NanI effect on ETX production was shown to involve reductions incodYandccpAgene transcription levels in BMC202 versus wild-type CN3718. Similar to CodY, CcpA was found to positively control ETX production. A doublecodYccpAnull mutant produced even less ETX than acodYorccpAsingle null mutant. CcpA bound directly to sequences upstream of theetxorcodYstart codon, and bioinformatics identified putative CcpA-bindingcresites immediately upstream of both thecodYandetxstart codons, suggesting possible direct CcpA regulatory effects. AccpAmutation also decreasedcodYtranscription, suggesting that CcpA effects on ETX production can be both direct and indirect, including effects oncodYtranscription. Collectively, these results suggest that NanI, CcpA, and CodY work together to regulate ETX production, with NanI-generated sialic acid from the intestines possibly signaling type D strains to upregulate their ETX production and induce disease.IMPORTANCEClostridium perfringensNanI was previously shown to increase ETX binding to, and cytotoxicity for, MDCK host cells. The current study demonstrates that NanI also regulates ETX production via increased transcription of genes encoding the CodY and CcpA global regulators. Results obtained using singleccpAorcodYnull mutants and accpAcodYdouble null mutant showed thatcodYandccpAregulate ETX production independently of one another but thatccpAalso affectscodYtranscription. Electrophoretic mobility shift assays and bioinformatic analyses suggest that both CodY and CcpA may directly regulateetxtranscription. Collectively, results of this study suggest that sialic acid generated by NanI from intestinal sources signals ETX-producingC. perfringensstrains, via CcpA and CodY, to upregulate ETX production and cause disease.

scholarly journals Evaluating the Involvement of Alternative Sigma Factors SigF and SigG in Clostridium perfringens Sporulation and Enterotoxin Synthesis

2010 ◽  
Vol 78 (10) ◽  
pp. 4286-4293 ◽  
Author(s):  
Jihong Li ◽  
Bruce A. McClane
Keyword(s):  
Bacillus Subtilis ◽  
Clostridium Perfringens ◽  
Food Poisoning ◽  
Sigma Factors ◽  
Wild Type ◽  
Alternative Sigma Factors ◽  
Food Borne ◽  
Food Borne Illness ◽  
Bacterial Food ◽  
Null Mutants

ABSTRACT Clostridium perfringens type A food poisoning is the second most commonly identified bacterial food-borne illness. Sporulation contributes to this disease in two ways: (i) most food-poisoning strains form exceptionally resistant spores to facilitate their survival of food-associated stresses, and (ii) the enterotoxin (CPE) responsible for the symptoms of this food poisoning is synthesized only during sporulation. In Bacillus subtilis, four alternative sigma factors mediate sporulation. The same four sigma factors are encoded by C. perfringens genomes, and two (SigE and SigK) have previously been shown to be necessary for sporulation and CPE production by SM101, a transformable derivative of a C. perfringens food-poisoning strain (K. H. Harry, R. Zhou, L. Kroos, and S. B. Melville, J. Bacteriol. 2009, 191:2728-2742). However, the importance of SigF and SigG for C. perfringens sporulation or CPE production had not yet been assessed. In the current study, after confirming that sporulating wild-type SM101 cultures produce SigF (from a tricistronic operon) and SigG, we prepared isogenic sigF- or sigG-null mutants. Whereas SM101 formed heat-resistant, phase-refractile spores, spore formation was blocked in the sigF- and sigG-null mutants. Complementation fully restored sporulation by both mutants. By use of these mutants and complementing strains, CPE production was shown to be SigF dependent but SigG independent. This finding apparently involved regulation of the production of SigE and SigK, which Harry et al. showed to be necessary for CPE synthesis, by SigF. By combining these findings with those previous results, it is now apparent that all four alternative sigma factors are necessary for C. perfringens sporulation, but only SigE, SigF, and SigK are needed for CPE synthesis.

scholarly journals Cyclic Di-GMP Binding by an Assembly ATPase (PilB2) and Control of Type IV Pilin Polymerization in the Gram-Positive Pathogen Clostridium perfringens

2017 ◽  
Vol 199 (10) ◽  
Author(s):  
William A. Hendrick ◽  
Mona W. Orr ◽  
Samantha R. Murray ◽  
Vincent T. Lee ◽  
Stephen B. Melville
Keyword(s):  
Clostridium Perfringens ◽  
Biochemical Characterization ◽  
Core Protein ◽  
Type Iv Pili ◽  
Host Cells ◽  
Dependent Manner ◽  
Gram Positive ◽  
Content Type ◽  
Type Iv

ABSTRACT The Gram-positive pathogen Clostridium perfringens possesses type IV pili (TFP), which are extracellular fibers that are polymerized from a pool of pilin monomers in the cytoplasmic membrane. Two proteins that are essential for pilus functions are an assembly ATPase (PilB) and an inner membrane core protein (PilC). Two homologues each of PilB and PilC are present in C. perfringens, called PilB1/PilB2 and PilC1/PilC2, respectively, along with four pilin proteins, PilA1 to PilA4. The gene encoding PilA2, which is considered the major pilin based on previous studies, is immediately downstream of the pilB2 and pilC2 genes. Purified PilB2 had ATPase activity, bound zinc, formed hexamers even in the absence of ATP, and bound the second messenger molecule cyclic di-GMP (c-di-GMP). Circular dichroism spectroscopy of purified PilC2 indicated that it retained its predicted degree of alpha-helical secondary structure. Even though no direct interactions between PilB2 and PilC2 could be detected in vivo or in vitro even in the presence of c-di-GMP, high levels of expression of a diguanylate cyclase from C. perfringens (CPE1788) stimulated polymerization of PilA2 in a PilB2- and PilC2-dependent manner. These results suggest that PilB2 activity is controlled by c-di-GMP levels in vivo but that PilB2-PilC2 interactions are either transitory or of low affinity, in contrast to results reported previously from in vivo studies of the PilB1/PilC1 pair in which PilC1 was needed for polar localization of PilB1. This is the first biochemical characterization of a c-di-GMP-dependent assembly ATPase from a Gram-positive bacterium. IMPORTANCE Type IV pili (TFP) are protein fibers involved in important bacterial functions, including motility, adherence to surfaces and host cells, and natural transformation. All clostridia whose genomes have been sequenced show evidence of the presence of TFP. The genetically tractable species Clostridium perfringens was used to study proteins involved in polymerizing the pilin, PilA2, into a pilus. The assembly ATPase PilB2 and its cognate membrane protein partner, PilC2, were purified. PilB2 bound the intracellular signal molecule c-di-GMP. Increased levels of intracellular c-di-GMP led to increased polymerization of PilA2, indicating that Gram-positive bacteria use this molecule to regulate pilus synthesis. These findings provide valuable information for understanding how pathogenic clostridia regulate TFP to cause human diseases.

scholarly journals Structural and Functional Analysis of the Pore-Forming Toxin NetB from Clostridium perfringens

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Xu-Xia Yan ◽  
Corrine J. Porter ◽  
Simon P. Hardy ◽  
David Steer ◽  
A. Ian Smith ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Clostridium Perfringens ◽  
Pore Formation ◽  
Random Mutagenesis ◽  
Planar Lipid Bilayers ◽  
Necrotic Enteritis ◽  
Channel Conductance ◽  
Content Type ◽  
Alpha Hemolysin ◽  
Insight Into

ABSTRACT Clostridium perfringens is an anaerobic bacterium that causes numerous important human and animal diseases, primarily as a result of its ability to produce many different protein toxins. In chickens, C. perfringens causes necrotic enteritis, a disease of economic importance to the worldwide poultry industry. The secreted pore-forming toxin NetB is a key virulence factor in the pathogenesis of avian necrotic enteritis and is similar to alpha-hemolysin, a β-barrel pore-forming toxin from Staphylococcus aureus. To address the molecular mechanisms underlying NetB-mediated tissue damage, we determined the crystal structure of the monomeric form of NetB to 1.8 Å. Structural comparisons with other members of the alpha-hemolysin family revealed significant differences in the conformation of the membrane binding domain. These data suggested that NetB may recognize different membrane receptors or use a different mechanism for membrane-protein interactions. Consistent with this idea, electrophysiological experiments with planar lipid bilayers revealed that NetB formed pores with much larger single-channel conductance than alpha-hemolysin. Channel conductance varied with phospholipid net charge. Furthermore, NetB differed in its ion selectivity, preferring cations over anions. Using hemolysis as a screen, we carried out a random-mutagenesis study that identified several residues that are critical for NetB-induced cell lysis. Mapping of these residues onto the crystal structure revealed that they were clustered in regions predicted to be required for oligomerization or membrane binding. Together these data provide an insight into the mechanism of NetB-mediated pore formation and will contribute to our understanding of the mode of action of this important toxin. IMPORTANCE Necrotic enteritis is an economically important disease of the worldwide poultry industry and is mediated by Clostridium perfringens strains that produce NetB, a β-pore-forming toxin. We carried out structural and functional studies of NetB to provide a mechanistic insight into its mode of action and to assist in the development of a necrotic enteritis vaccine. We determined the structure of the monomeric form of NetB to 1.8 Å, used both site-directed and random mutagenesis to identify key residues that are required for its biological activity, and analyzed pore formation by NetB and its substitution-containing derivatives in planar lipid bilayers.

scholarly journals Pseudomonas aeruginosa GroEL Stimulates Production of PTX3 by Activating the NF-κB Pathway and Simultaneously Downregulating MicroRNA-9

2016 ◽  
Vol 85 (3) ◽  
Author(s):  
Heesung Shin ◽  
Jisu Jeon ◽  
Jung-Hoon Lee ◽  
Shouguang Jin ◽  
Un-Hwan Ha
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Infections ◽  
Therapeutic Potential ◽  
Host Cells ◽  
Soluble Form ◽  
Heat Shock Protein 60 ◽  
Toll Like Receptor 4 ◽  
Content Type ◽  
Host Immune Responses ◽  
Recognition Receptor

ABSTRACT As one of the first lines of host defense, monocytes play important roles in clearing infected microbes. The defensive response is triggered by recognition of diverse microbial moieties, including released factors, which modulate host immune responses to establish a harsh environment for clinically important bacterial pathogens. In this study, we found that the expression of PTX3, a soluble form of pattern recognition receptor, was induced by infection with live Pseudomonas aeruginosa or treatment of cells with its supernatant. P. aeruginosa GroEL, a homolog of heat shock protein 60, was identified as one of the factors responsible for inducing the expression of PTX3 in host cells. GroEL induced PTX3 expression by activating the Toll-like receptor 4 (TLR4)-dependent pathway via nuclear factor-kappa B (NF-κB), while simultaneously inhibiting expression of microRNA-9, which targets the PTX3 transcript. Finally, by acting as an opsonin, GroEL-induced PTX3 promoted the association and phagocytosis of Staphylococcus aureus into macrophages. These data suggest that the host defensive environment is supported by the production of PTX3 in response to GroEL, which thus has therapeutic potential for clearance of bacterial infections.

scholarly journals RIP1, RIP3, and MLKL Contribute to Cell Death Caused by Clostridium perfringens Enterotoxin

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Archana Shrestha ◽  
Iman Mehdizadeh Gohari ◽  
Bruce A. McClane
Keyword(s):  
Cell Death ◽  
Clostridium Perfringens ◽  
Vero Cells ◽  
Kinase Domain ◽  
Content Type ◽  
Mixed Lineage Kinase ◽  
Human Enterocyte ◽  
Induced Apoptosis ◽  
Apoptosis And Necrosis ◽  
Calpain Inhibitors

ABSTRACT Clostridium perfringens type F strains cause gastrointestinal disease when they produce a pore-forming toxin named C. perfringens enterotoxin (CPE). In human enterocyte-like Caco-2 cells, low CPE concentrations cause caspase-3-dependent apoptosis, while high CPE concentrations cause necrosis. Since necrosis or apoptosis sometimes involves receptor-interacting serine/threonine-protein kinase-1 or 3 (RIP1 or RIP3), this study examined whether those kinases are important for CPE-induced apoptosis or necrosis. Highly specific RIP1 or RIP3 inhibitors reduced both CPE-induced apoptosis and necrosis in Caco-2 cells. Those findings suggested that the form of necrosis induced by treating Caco-2 cells with high CPE concentrations involves necroptosis, which was confirmed when high, but not low, CPE concentrations were shown to induce oligomerization of mixed-lineage kinase domain-like pseudokinase (MLKL), a key late step in necroptosis. Furthermore, an MLKL oligomerization inhibitor reduced cell death caused by high, but not low, CPE concentrations. Supporting RIP1 and RIP3 involvement in CPE-induced necroptosis, inhibitors of those kinases also reduced MLKL oligomerization during treatment with high CPE concentrations. Calpain inhibitors similarly blocked MLKL oligomerization induced by high CPE concentrations, implicating calpain activation as a key intermediate in initiating CPE-induced necroptosis. In two other CPE-sensitive cell lines, i.e., Vero cells and human enterocyte-like T84 cells, low CPE concentrations also caused primarily apoptosis/late apoptosis, while high CPE concentrations mainly induced necroptosis. Collectively, these results establish that high, but not low, CPE concentrations cause necroptosis and suggest that RIP1, RIP3, MLKL, or calpain inhibitors can be explored as potential therapeutics against CPE effects in vivo. IMPORTANCE C. perfringens type F strains are a common cause of food poisoning and antibiotic-associated diarrhea. Type F strain virulence requires production of C. perfringens enterotoxin (CPE). In Caco-2 cells, high CPE concentrations cause necrosis while low enterotoxin concentrations induce apoptosis. The current study determined that receptor-interacting serine/threonine-protein kinases 1 and 3 are involved in both CPE-induced apoptosis and necrosis in Caco-2 cells, while mixed-lineage kinase domain-like pseudokinase (MLKL) oligomerization is involved in CPE-induced necrosis, thereby indicating that this form of CPE-induced cell death involves necroptosis. High CPE concentrations also caused necroptosis in T84 and Vero cells. Calpain activation was identified as a key intermediate for CPE-induced necroptosis. These results suggest inhibitors of RIP1, RIP3, MLKL oligomerization, or calpain are useful therapeutics against CPE-mediated diseases.

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 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 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.

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