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 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 NanI Sialidase Is an Important Contributor toClostridium perfringensType F Strain F4969 Intestinal Colonization in Mice

2018 ◽  
Vol 86 (12) ◽  
Author(s):  
Mauricio A. Navarro ◽  
Jihong Li ◽  
Bruce A. McClane ◽  
Eleonora Morrell ◽  
Juliann Beingesser ◽  
...  
Keyword(s):  
Small Intestine ◽  
Food Poisoning ◽  
Extracellular Enzyme ◽  
Gastrointestinal Diseases ◽  
Intestinal Colonization ◽  
Null Mutant ◽  
Content Type ◽  
Intestinal Segments

ABSTRACTClostridium perfringenstype F (formerly enterotoxigenicC. perfringenstype A) strains produce an enterotoxin (CPE) to cause acute cases of food poisoning and chronic nonfoodborne human gastrointestinal diseases (NFD), e.g., antibiotic-associated diarrhea (AAD). NFD strains also produce NanI sialidase, an extracellular enzyme that releases sialic acids from sialyated host macromolecules. Recentin vitrostudies suggested that NanI may contribute to NFD strain intestinal colonization by enhancing the adherence of such strains to intestinal cells and promoting their bacterial growth using generated sialic acid as an energy source. The current study tested this hypothesis by developing a mouse intestinal colonization model involving clindamycin pretreatment to produce conditions mimicking those during AAD. In this model, the type F NFD strain F4969 persisted for at least 4 days in the small intestine, cecum, and colon. When clindamycin-pretreated mice were challenged by oral gavage with equivalent numbers of F4969 bacteria or its isogenicnanInull mutant, significantly lower numbers of thenanImutant were recovered from all intestinal segments, and it was completely cleared from the small intestine by day 4. Complementation of the mutant to restore NanI production also promoted colonization. When the samenanInull mutant strain was coinoculated into the mouse model together with ananI-producing strain, the numbers of this mutant were restored to wild-type F4969 levels in all intestinal segments. This result suggests that sialidases produced by other bacteria might also provide some support forC. perfringensintestinal colonization. Collectively, thesein vivofindings identify NanI to be the first known significant contributor to chronic intestinal colonization by NFD strains.

scholarly journals Dissecting the Structure-Function Relationship of a Fungicidal Peptide Derived from the Constant Region of Human Immunoglobulins

2016 ◽  
Vol 60 (4) ◽  
pp. 2435-2442 ◽  
Author(s):  
Tecla Ciociola ◽  
Thelma A. Pertinhez ◽  
Laura Giovati ◽  
Martina Sperindè ◽  
Walter Magliani ◽  
...  
Keyword(s):  
Self Assembly ◽  
Galleria Mellonella ◽  
Critical Role ◽  
Yeast Cells ◽  
Therapeutic Effects ◽  
Constant Region ◽  
Content Type ◽  
Complementarity Determining Regions

ABSTRACTSynthetic peptides encompassing sequences related to the complementarity-determining regions of antibodies or derived from their constant region (Fc peptides) were proven to exert differential antimicrobial, antiviral, antitumor, and/or immunomodulatory activitiesin vitroand/orin vivo, regardless of the specificity and isotype of the parental antibody. Alanine substitution derivatives of these peptides exhibited unaltered, increased, or decreased candidacidal activitiesin vitro. The bioactive IgG-derived Fc N10K peptide (NQVSLTCLVK) spontaneously self-assembles, a feature previously recognized as relevant for the therapeutic activity of another antibody-derived peptide. We evaluated the contribution of each residue to the peptide self-assembling capability by circular-dichroism spectroscopy. The interaction of the N10K peptide and its derivatives withCandida albicanscells was studied by confocal, transmission, and scanning electron microscopy. The apoptosis and autophagy induction profiles in yeast cells treated with the peptides were evaluated by flow cytometry, and the therapeutic efficacy against candidal infection was studied in aGalleria mellonellamodel. Overall, the results indicate a critical role for some residues in the self-assembly process and a correlation of that capability with the candidacidal activities of the peptidesin vitroand their therapeutic effectsin vivo.

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 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.

scholarly journals Role of Host Xanthine Oxidase in Infection Due to Enteropathogenic and Shiga-Toxigenic Escherichia coli

2013 ◽  
Vol 81 (4) ◽  
pp. 1129-1139 ◽  
Author(s):  
John K. Crane ◽  
Tonniele M. Naeher ◽  
Jacqueline E. Broome ◽  
Edgar C. Boedeker
Keyword(s):  
Escherichia Coli ◽  
Xanthine Oxidase ◽  
Host Defense ◽  
Pathogenic Bacteria ◽  
Intestinal Cell ◽  
Intestinal Loop ◽  
Loop Model ◽  
Content Type ◽  
E Coli

ABSTRACTXanthine oxidase (XO), also known as xanthine oxidoreductase, has long been considered an important host defense molecule in the intestine and in breastfed infants. Here, we present evidence that XO is released from and active in intestinal tissues and fluids in response to infection with enteropathogenicEscherichia coli(EPEC) and Shiga-toxigenicE. coli(STEC), also known as enterohemorrhagicE. coli(EHEC). XO is released into intestinal fluids in EPEC and STEC infection in a rabbit animal model. XO activity results in the generation of surprisingly high concentrations of uric acid in both cultured cell and animal models of infection. Hydrogen peroxide (H2O2) generated by XO activity triggered a chloride secretory response in intestinal cell monolayers within minutes but decreased transepithelial electrical resistance at 6 to 22 h. H2O2generated by XO activity was effective at killing laboratory strains ofE. coli, commensal microbiotas, and anaerobes, but wild-type EPEC and STEC strains were 100 to 1,000 times more resistant to killing or growth inhibition by this pathway. Instead of killing pathogenic bacteria, physiologic concentrations of XO increased virulence by inducing the production of Shiga toxins from STEC strains.In vivo, exogenous XO plus the substrate hypoxanthine did not protect and instead worsened the outcome of STEC infection in the rabbit ligated intestinal loop model of infection. XO released during EPEC and STEC infection may serve as a virulence-inducing signal to the pathogen and not solely as a protective host defense.

scholarly journals Alanine Scanning Mutagenesis of the MEDI4893 (Suvratoxumab) Epitope Reduces Alpha Toxin Lytic Activity In Vitro and Staphylococcus aureus Fitness in Infection Models

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
C. Tkaczyk ◽  
E. Semenova ◽  
Y. Y. Shi ◽  
K. Rosenthal ◽  
V. Oganesyan ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Pore Formation ◽  
Severe Disease ◽  
Treatment Strategies ◽  
Lytic Activity ◽  
Alpha Toxin ◽  
Alanine Scanning Mutagenesis ◽  
Content Type

ABSTRACT Alpha toxin (AT) is a cytolytic pore-forming toxin that plays a key role in Staphylococcus aureus pathogenesis; consequently, extensive research was undertaken to understand the AT mechanism of action and its utility as a target for novel prophylaxis and treatment strategies against S. aureus infections. MEDI4893 (suvratoxumab) is a human anti-AT IgG1 monoclonal antibody (MAb) that targets AT and is currently in phase 2 clinical development. As shown previously, the MEDI4893-binding epitope on AT is comprised of the highly conserved amino acid regions 177 to 200 and 261 to 271, suggesting these amino acids are important for AT function. To test this hypothesis and gain insight into the effect of mutations in the epitope on AT neutralization by MEDI4893, nine MEDI4893 contact residues in AT were individually mutated to alanine. Consistent with our hypothesis, 8 out of 9 mutants exhibited >2-fold loss in lytic activity resulting from a defect in cell binding and pore formation. MEDI4893 binding affinity was reduced >2-fold (2- to 27-fold) for 7 out of 9 mutants, and no binding was detected for the W187A mutant. MEDI4893 effectively neutralized all of the lytic mutants in vitro and in vivo. When the defective mutants were introduced into an S. aureus clinical isolate, the mutant-expressing strains exhibited less severe disease in mouse models and were effectively neutralized by MEDI4893. These results indicate the MEDI4893 epitope is highly conserved due in part to its role in AT pore formation and bacterial fitness, thereby decreasing the likelihood for the emergence of MAb-resistant variants.

Limited efficacy of gene transfer in herpes simplex virus-thymidine kinase/ganciclovir gene therapy for brain tumors

2005 ◽  
Vol 102 (2) ◽  
pp. 328-335 ◽  
Author(s):  
Piotr Hadaczek ◽  
Hanna Mirek ◽  
Mitchel S. Berger ◽  
Krystof Bankiewicz
Keyword(s):  
Gene Therapy ◽  
Brain Tumor ◽  
Gene Transfer ◽  
Thymidine Kinase ◽  
Therapeutic Effects ◽  
Content Type ◽  
Over Time ◽  
Fine Print

Object. Low efficacy of gene transfer, transient gene expression, and toxicity of viral vectors are the major hurdles in successful anticancer gene therapy. The authors conducted in vitro (U87MG cell line) and in vivo (xenograft, tumor-bearing rodent model) studies to address the stability of transduction by using the adenoassociated virus serotype-2 (AAV2)—thymidine kinase (TK) vector over time. Methods. Standard methods for cell growth and a ganciclovir (GCV) cytotoxicity assay were applied. The AAV2-TK was infused into implanted tumors in athymic rats via convection-enhanced delivery (CED). Thymidine kinase expression was evaluated through immunohistochemical analysis, and the distribution volumes of the transduced tumors were calculated. Twenty-four hours following the viral infusions, animals were treated with GCV (50 mg/kg intraperitoneally every day for 10 days; six rats) or phosphate-buffered saline (six rats). A rapid decrease in TK expression over time was observed both in vitro and in vivo. A large volume of the tumor (up to 39%) was transduced with AAV2-TK following CED. Administration of GCV resulted in limited therapeutic effects (survival of 25.8 compared with 21.3 days). Conclusions. Rapid elimination of TK expression from dividing tumor cells and focal transduction of the brain tumor were most likely responsible for the limited bystander effect in this approach. Immediate administration of GCV is crucial to assure maximal efficacy in the elimination of cancer cells. In addition, the complete or diffused transduction of a brain tumor with TK may be required for its total eradication.

scholarly journals Identification of an Important Orphan Histidine Kinase for the Initiation of Sporulation and Enterotoxin Production byClostridium perfringensType F Strain SM101

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
John C. Freedman ◽  
Jihong Li ◽  
Eric Mi ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Histidine Kinase ◽  
Insertional Mutagenesis ◽  
Food Poisoning ◽  
Gastrointestinal Diseases ◽  
Kinase Domain ◽  
Block Type ◽  
Null Mutant ◽  
Content Type

ABSTRACTClostridium perfringenstype F strains cause a common human foodborne illness and many cases of nonfoodborne human gastrointestinal diseases. Sporulation plays two critical roles during type F enteric disease. First, it produces broadly resistant spores that facilitate type F strain survival in the food and nosocomial environments. Second, production ofC. perfringensenterotoxin (CPE), the toxin responsible for causing the enteric symptoms of type F diseases, is restricted to cells in the process of sporulation. While later steps in the regulation ofC. perfringenssporulation have been discerned, the process leading to phosphorylation of Spo0A, the master early regulator of sporulation and consequent CPE production, has remained unknown. Using an insertional mutagenesis approach, the current study identified the orphan histidine kinase CPR0195 as an important factor regulatingC. perfringenssporulation and CPE production. Specifically, a CPR0195 null mutant of type F strain SM101 made 103-fold fewer spores than its wild-type parent and produced no detectable CPE. In contrast, a null mutant of another putativeC. perfringensorphan histidine kinase (CPR1055) did not significantly affect sporulation or CPE production. Studies using aspoIIAoperon promoter-driven reporter plasmid indicated that CPR0195 functions early during sporulation, i.e., prior to production of sporulation-associated sigma factors. Furthermore,in vitrostudies showed that the CPR0195 kinase domain can autophosphorylate and phosphorylate Spo0A. These results support the idea of CPR0195 as an important kinase that initiatesC. perfringenssporulation by directly phosphorylating Spo0A. This kinase could represent a novel therapeutic target to blockC. perfringenssporulation and CPE production during type F disease.IMPORTANCEClostridium perfringenstype F enteric diseases, which include a very common form of food poisoning and many cases of antibiotic-associated diarrhea, develop when type F strains sporulate and produceC. perfringensenterotoxin (CPE) in the intestines. Spores are also important for transmission of type F disease. Despite the importance of sporulation for type F disease and the evidence thatC. perfringenssporulation begins with phosphorylation of the Spo0A transcriptional regulator, the kinase phosphorylating Spo0A to initiate sporulation and CPE production had not been ascertained. In response, the current report now provides identification of an orphan histidine kinase named CPR0195 that can directly phosphorylate Spo0A. Results using a CPR0195 null mutant indicate that this kinase is very important for initiatingC. perfringenssporulation and CPE production. Therefore, the CPR0195 kinase represents a potential target to block type F disease by interfering with intestinalC. perfringenssporulation and CPE production.

scholarly journals Pathogenicity of Salmonella Strains Isolated from Egg Shells and the Layer Farm Environment in Australia

2014 ◽  
Vol 81 (1) ◽  
pp. 405-414 ◽  
Author(s):  
Andrea R. McWhorter ◽  
Dianne Davos ◽  
K. K. Chousalkar
Keyword(s):  
Virulence Genes ◽  
Solid Phase ◽  
Gastrointestinal Disease ◽  
Clinical Signs ◽  
Food Poisoning ◽  
Model Systems ◽  
Content Type ◽  
Invasive Capacity

ABSTRACTIn Australia, the egg industry is periodically implicated during outbreaks ofSalmonellafood poisoning.Salmonella entericaserovar Typhimurium and other nontyphoidalSalmonellaspp., in particular, are a major concern for Australian public health. Several definitive types ofSalmonellaTyphimurium strains, but primarilySalmonellaTyphimurium definitive type 9 (DT9), have been frequently reported during egg-related food poisoning outbreaks in Australia. The aim of the present study was to generate a pathogenicity profile of nontyphoidalSalmonellaisolates obtained from Australian egg farms. To achieve this, we assessed the capacity ofSalmonellaisolates to cause gastrointestinal disease using bothin vitroandin vivomodel systems. Data fromin vitroexperiments demonstrated that the invasion capacity ofSalmonellaserovars cultured to stationary phase (liquid phase) in LB medium was between 90- and 300-fold higher than bacterial suspensions in normal saline (cultured in solid phase). During thein vivoinfection trial, clinical signs of infection and mortality were observed only for mice infected with either 103or 105CFU ofS. Typhimurium DT9. No mortality was observed for mice infected withSalmonellaserovars with medium or low invasive capacity in Caco-2 cells. Pathogenicity gene profiles were also generated for all serovars included in this study. The majority of serovars tested were positive for selected virulence genes. No relationship between the presence or absence of virulence genes by PCR and eitherin vitroinvasive capacity orin vivopathogenicity was detected. Our data expand the knowledge of strain-to-strain variation in the pathogenicity of Australian egg industry-relatedSalmonellaspp.

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