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 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, 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 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 Comparative Effects of Osmotic, Sodium Nitrite-Induced, and pH-Induced Stress on Growth and Survival of Clostridium perfringens Type A Isolates Carrying Chromosomal or Plasmid-Borne Enterotoxin Genes

2006 ◽  
Vol 72 (12) ◽  
pp. 7620-7625 ◽  
Author(s):  
Jihong Li ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Food Poisoning ◽  
Type A ◽  
Food Preservation ◽  
Vegetative Cells ◽  
Growth And Survival ◽  
Resistance Phenotype ◽  
Enhanced Resistance ◽  
Enterotoxin Genes ◽  
High And Low Temperatures

ABSTRACT About 1 to 2% of Clostridium perfringens isolates carry the enterotoxin gene (cpe) necessary for causing C. perfringens type A food poisoning. While the cpe gene can be either chromosomal or plasmid borne, food poisoning isolates usually carry a chromosomal cpe gene. Previous studies have linked this association between chromosomal cpe isolates (i.e., C-cpe isolates) and food poisoning, at least in part, to both the spores and vegetative cells of C-cpe isolates being particularly resistant to high and low temperatures. The current study now reveals that the resistance phenotype of C-cpe isolates extends beyond temperature resistance to also include, for both vegetative cells and spores, enhanced resistance to osmotic stress (from NaCl) and nitrites. However, by omitting one outlier isolate, no significant differences in pH sensitivity were detected between the spores or vegetative cells of C-cpe isolates versus isolates carrying a plasmid-borne cpe gene. These results indicate that both vegetative cells and spores of C-cpe isolates are unusually resistant to several food preservation approaches in addition to temperature extremes. The broad-spectrum nature of the C-cpe resistance phenotype suggests these bacteria may employ multiple mechanisms to persist and grow in foods prior to their transmission to humans.

scholarly journals Role of Sialic Acid inBrachyspira hyodysenteriaeAdhesion to Pig Colonic Mucins

2019 ◽  
Vol 87 (7) ◽  
Author(s):  
Macarena P. Quintana-Hayashi ◽  
Vignesh Venkatakrishnan ◽  
Freddy Haesebrouck ◽  
Sara Lindén
Keyword(s):  
Sialic Acid ◽  
Bacterial Growth ◽  
Mucus Layer ◽  
Mucin Secretion ◽  
Content Type ◽  
Brachyspira Hyodysenteriae ◽  
Intestinal Mucus ◽  
Bacterial Binding ◽  
Free Sialic Acid

ABSTRACTInfection withBrachyspira hyodysenteriaeresults in mucoid hemorrhagic diarrhea. This pathogen is associated with the colonic mucus layer, mainly composed of mucins. Infection regulates mucinO-glycosylation in the colon and increases mucin secretion as well asB. hyodysenteriaebinding sites on mucins. Here, we analyzed potential mucin epitopes forB. hyodysenteriaeadhesion in the colon, as well as the effect of colonic mucins on bacterial growth. Associations betweenB. hyodysenteriaebinding to pig colonic mucins and mucin glycan data showed thatB. hyodysenteriaebinding was associated with the presence ofN-glycolylneuraminic acid (NeuGc) on mucins. The role of sialic acid inB. hyodysenteriaeadhesion was analyzed after the removal of sialic acid residues on the mucins by enzymatic treatment with sialidase A, which decreased bacterial binding to the mucins. The effect of pig colonic mucins onB. hyodysenteriaegrowth was determined in carbohydrate-free medium.B. hyodysenteriaegrowth increased in the presence of mucins from two out of five infected pigs, suggesting utilization of mucins as a carbon source for growth. Additionally, bacterial growth was enhanced by free sialic acid andN-acetylglucosamine. The results highlight a role of sialic acid as an adhesion epitope forB. hyodysenteriaeinteraction with colonic mucins. Furthermore, the mucin response and glycosylation changes exerted in the colon duringB. hyodysenteriaeinfection result in a potentially favorable environment for pathogen growth in the intestinal mucus layer.

scholarly journals Trypanosoma cruzi trans-sialidase and neuraminidase activities can be mediated by the same enzymes.

1992 ◽  
Vol 175 (2) ◽  
pp. 567-575 ◽  
Author(s):  
S Schenkman ◽  
L Pontes de Carvalho ◽  
V Nussenzweig
Keyword(s):  
Sialic Acid ◽  
Trypanosoma Cruzi ◽  
Transfer Reaction ◽  
Surface Membrane ◽  
Host Cells ◽  
Hydrolysis Of ◽  
Predominant Direction ◽  
Molecular Sieving ◽  
Free Sialic Acid ◽  
Single Enzyme

Trans-sialidase and neuraminidase activities have been detected on the surface membrane of trypomastigotes of Trypanosoma cruzi, and both have been implicated in the parasite's invasion of host cells. We show here that these enzymes are structurally related. They are recognized by two independently derived monoclonal antibodies, are anchored to the membrane by glycosylphosphatidylinositol, copurify by ion exchange, molecular sieving, and hydrophobic chromatography, have maximal activities between pH 6.5 and 7.5, and are inactivated by heating at 56 degrees C. Furthermore, the neuraminidase and trans-sialidase reactions are coupled. An increase of the concentration of acceptors of the transfer reaction decreases the amount of free sialic acid released through the neuraminidase reaction. We conclude that a single enzyme can catalyze the transfer or the hydrolysis of macromolecular-bound sialic acid. The predominant direction of the reaction will depend on the availability of appropriate oligosaccharide acceptors of sialic acid.

scholarly journals Sialic acid facilitates binding and cytotoxic activity of the pore-forming Clostridium perfringens NetF toxin to host cells

PLoS ONE ◽  
2018 ◽  
Vol 13 (11) ◽  
pp. e0206815 ◽  
Author(s):  
Iman Mehdizadeh Gohari ◽  
Eric K. Brefo-Mensah ◽  
Michael Palmer ◽  
Patrick Boerlin ◽  
John F. Prescott
Keyword(s):  
Sialic Acid ◽  
Cytotoxic Activity ◽  
Clostridium Perfringens ◽  
Host Cells

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 NanR Regulates Sporulation and Enterotoxin Production byClostridium perfringensType F Strain F4969

2018 ◽  
Vol 86 (10) ◽  
Author(s):  
Eric Mi ◽  
Jihong Li ◽  
Bruce A. McClane
Keyword(s):  
Sialic Acid ◽  
Clostridium Perfringens ◽  
Vegetative Growth ◽  
Foodborne Illness ◽  
Null Mutant ◽  
Wild Type ◽  
Gastrointestinal Infections ◽  
Content Type ◽  
Antibiotic Associated Diarrhea ◽  
Uptake And Metabolism

ABSTRACTClostridium perfringenstype F strains, which produceC. perfringensenterotoxin (CPE), are a major cause of gastrointestinal infections, including the second most prevalent bacterial foodborne illness and 5 to 10% cases of antibiotic-associated diarrhea. Virulence of type F strains is primarily ascribable to CPE, which is synthesized only during sporulation. Many type F strains also produce NanI sialidase and carry ananoperon that likely facilitates uptake and metabolism of sialic acid liberated from glycoconjugates by NanI. During vegetative growth of type F strain F4969, NanR can regulate expression ofnanI. Given their importance for type F disease, the current study investigated whether NanR can also influence sporulation and CPE production when F4969 or isogenic derivatives are cultured in modified Duncan-Strong sporulation (MDS) medium. An isogenic F4969nanRnull mutant displayed much less sporulation and CPE production but more NanI production than wild-type F4969, indicating that NanR positively regulates sporulation and CPE production but represses NanI production in MDS. Results for thenanRmutant also demonstrated that NanR regulates expression of thenanoperon. AnanI nanRdouble null mutant mirrored the outcome of thenanRnull mutant strain but with a stronger inhibition of sporulation and CPE production, even after overnight incubation. Coupled with results using ananInull mutant, which had no impairment of sporulation or CPE production, NanR appears to carefully modulate the availability of NanI,nanoperon-encoded proteins and sialic acid to provide sufficient nutrients to sustain sporulation and CPE production when F4969 is cultured in MDS medium.

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