scholarly journals Native or Proteolytically Activated NanI Sialidase Enhances the Binding and Cytotoxic Activity of Clostridium perfringens Enterotoxin and Beta Toxin

2017 ◽  
Vol 86 (1) ◽  
Author(s):  
James R. Theoret ◽  
Jihong Li ◽  
Mauricio A. Navarro ◽  
Jorge P. Garcia ◽  
Francisco A. Uzal ◽  
...  
Keyword(s):  
Cytotoxic Activity ◽  
Clostridium Perfringens ◽  
Full Length ◽  
Host Cells ◽  
Proteolytic Activation ◽  
Clostridium Perfringens Enterotoxin ◽  
Content Type ◽  
Sialidase Activity ◽  
Epsilon Toxin ◽  
Beta Toxin

ABSTRACTManyClostridium perfringensstrains produce NanI as their major sialidase. Previous studies showed that NanI could potentiateC. perfringensepsilon toxin cytotoxicity by enhancing the binding of this toxin to host cells. The present study first determined that NanI exerts similar cytotoxicity-enhancing effects onC. perfringensenterotoxin and beta toxin, which are also important toxins forC. perfringensdiseases (enteritis and enterotoxemia) originating in the gastrointestinal (GI) tract. Building upon previous work demonstrating that purified trypsin can activate NanI activity, this study next determined that purified chymotrypsin or mouse intestinal fluids can also activate NanI activity. Amino acid sequencing then showed that this effect involves the N-terminal processing of the NanI protein. Recombinant NanI (rNanI) species corresponding to major chymotrypsin- or small intestinal fluid-generated NanI fragments possessed more sialidase activity than did full-length rNanI, further supporting the proteolytic activation of NanI activity. rNanI species corresponding to proteolysis products also promoted the cytotoxic activity and binding of enterotoxin and beta toxin more strongly than did full-length rNanI. Since enterotoxin and beta toxin are produced in the intestines during human and animal disease, these findings suggest that intestinal proteases may enhance NanI activity, which in turn could further potentiate the activity of intestinally active toxins during disease. Coupling these new results with previous findings demonstrating that NanI is important for the adherence ofC. perfringensto enterocyte-like cells, NanI sialidase is now emerging as a potential auxiliary virulence factor forC. perfringensenteritis and enterotoxemia.

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 Sequencing and Diversity Analyses Reveal Extensive Similarities between Some Epsilon-Toxin-Encoding Plasmids and the pCPF5603 Clostridium perfringens Enterotoxin Plasmid

2008 ◽  
Vol 190 (21) ◽  
pp. 7178-7188 ◽  
Author(s):  
Kazuaki Miyamoto ◽  
Jihong Li ◽  
Sameera Sayeed ◽  
Shigeru Akimoto ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Open Reading Frames ◽  
Toxin Gene ◽  
Type B ◽  
Clostridium Perfringens Enterotoxin ◽  
Type D ◽  
Transfer Genes ◽  
Epsilon Toxin ◽  
Beta2 Toxin ◽  
Beta Toxin

ABSTRACT Clostridium perfringens type B and D isolates produce epsilon-toxin, the third most potent clostridial toxin. The epsilon-toxin gene (etx) is plasmid borne in type D isolates, but etx genetics have been poorly studied in type B isolates. This study reports the first sequencing of any etx plasmid, i.e., pCP8533etx, from type B strain NCTC8533. This etx plasmid is 64.7 kb, carries tcp conjugative transfer genes, and encodes additional potential virulence factors including beta2-toxin, sortase, and collagen adhesin but not beta-toxin. Interestingly, nearly 80% of pCP8533etx open reading frames (ORFs) are also present on pCPF5603, an enterotoxin-encoding plasmid from type A isolate F5603. Pulsed-field gel electrophoresis and overlapping PCR indicated that a pCP8533etx-like etx plasmid is also present in most, if not all, other type B isolates and some beta2-toxin-positive, cpe-negative type D isolates, while other type D isolates carry different etx plasmids. Sequences upstream of the etx gene vary between type B isolates and some type D isolates that do not carry a pCP8533etx-like etx plasmid. However, nearly all type B and D isolates have an etx locus with an upstream IS1151, and those etx loci typically reside near a dcm ORF. These results suggest that pCPF5603 and pCP8533etx evolved from insertion of mobile genetic elements carrying enterotoxin or etx genes, respectively, onto a common progenitor plasmid.

scholarly journals CodY Is a Global Regulator of Virulence-Associated Properties for Clostridium perfringens Type D Strain CN3718

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Jihong Li ◽  
Menglin Ma ◽  
Mahfuzur R. Sarker ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Null Mutant ◽  
Intestinal Infection ◽  
Wild Type ◽  
Global Regulator ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Type D ◽  
Epsilon Toxin ◽  
Virulence Properties

ABSTRACT CodY is known to regulate various virulence properties in several Gram-positive bacteria but has not yet been studied in the important histotoxic and intestinal pathogen Clostridium perfringens. The present study prepared an isogenic codY-null mutant in C. perfringens type D strain CN3718 by insertional mutagenesis using the Targetron system. Western blot analysis indicated that, relative to wild-type CN3718 or a complementing strain, this isogenic codY mutant produces reduced levels of epsilon toxin (ETX). Using supernatants from cultures of the wild-type, codY-null mutant, and complementing strains, CodY regulation of ETX production was shown to have cytotoxic consequences for MDCK cells. The CodY regulatory effect on ETX production was specific, since the codY-null mutant still made wild-type levels of alpha-toxin and perfringolysin O. Sialidase activity measurements and sialidase Western blot analysis of supernatants from CN3718 and its isogenic derivatives showed that CodY represses overall exosialidase activity due to a reduced presence of NanH in culture supernatants. Inactivation of the codY gene significantly decreased the adherence of CN3718 vegetative cells or spores to host Caco-2 cells. Finally, the codY mutant showed increased spore formation under vegetative growth conditions, although germination of these spores was impaired. Overall, these results identify CodY as a global regulator of many C. perfringens virulence-associated properties. Furthermore, they establish that, via CodY, CN3718 coordinately regulates many virulence-associated properties likely needed for intestinal infection. IMPORTANCE Clostridium perfringens is a major human and livestock pathogen because it produces many potent toxins. C. perfringens type D strains cause intestinal infections by producing toxins, especially epsilon toxin (ETX). Previous studies identified CodY as a regulator of certain virulence properties in other Gram-positive bacteria. Our study now demonstrates that CodY is a global regulator of virulence-associated properties for type D strain CN3718. It promotes production of ETX, attachment of CN3718 vegetative cells or spores to host enterocyte-like Caco-2 cells, and spore germination; the last two effects may assist intestinal colonization. In contrast, CodY represses sporulation. These results provide the first evidence that CodY can function as a global regulator of C. perfringens virulence-associated properties and that this strain coordinately regulates its virulence-associated properties using CodY to increase ETX production, host cell attachment, and spore germination but to repress sporulation, as would be optimal during type D intestinal infection.

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 Genotypic and Phenotypic Characterization of Clostridium perfringens Isolates from Darmbrand Cases in Post-World War II Germany

2012 ◽  
Vol 80 (12) ◽  
pp. 4354-4363 ◽  
Author(s):  
Menglin Ma ◽  
Jihong Li ◽  
Bruce A. McClane
Keyword(s):  
World War Ii ◽  
Heat Resistance ◽  
Clostridium Perfringens ◽  
Type A ◽  
World War ◽  
Content Type ◽  
Post World War Ii ◽  
Heat Resistant ◽  
Type C ◽  
Beta Toxin

ABSTRACTClostridium perfringenstype C strains are the only non-type-A isolates that cause human disease. They are responsible for enteritis necroticans, which was termed Darmbrand when occurring in post-World War II Germany. Darmbrand strains were initially classified as type F because of their exceptional heat resistance but later identified as type C strains. Since only limited information exists regarding Darmbrand strains, this study genetically and phenotypically characterized seven 1940s era Darmbrand-associated strains. Results obtained indicated the following. (i) Five of these Darmbrand isolates belong to type C, carry beta-toxin (cpb) and enterotoxin (cpe) genes on large plasmids, and express both beta-toxin and enterotoxin. The other two isolates arecpe-negative type A. (ii) All seven isolates produce highly heat-resistant spores withD100values (the time that a culture must be kept at 100°C to reduce its viability by 90%) of 7 to 40 min. (iii) All of the isolates surveyed produce the same variant small acid-soluble protein 4 (Ssp4) made by type A food poisoning isolates with a chromosomalcpegene that also produce extremely heat-resistant spores. (iv) The Darmbrand isolates share a genetic background with type A chromosomal-cpe-bearing isolates. Finally, it was shown that both thecpeandcpbgenes can be mobilized in Darmbrand isolates. These results suggest thatC. perfringenstype A and C strains that cause human food-borne illness share a spore heat resistance mechanism that likely favors their survival in temperature-abused food. They also suggest possible evolutionary relationships between Darmbrand strains and type A strains carrying a chromosomalcpegene.

scholarly journals The VirS/VirR Two-Component System Regulates the Anaerobic Cytotoxicity, Intestinal Pathogenicity, and Enterotoxemic Lethality of Clostridium perfringens Type C Isolate CN3685

mBio ◽  
2011 ◽  
Vol 2 (1) ◽  
Author(s):  
Menglin Ma ◽  
Jorge Vidal ◽  
Juliann Saputo ◽  
Bruce A. McClane ◽  
Francisco Uzal
Keyword(s):  
Clostridium Perfringens ◽  
Regulatory System ◽  
Gas Gangrene ◽  
Necrotic Enteritis ◽  
Vegetative Cells ◽  
Content Type ◽  
Two Component ◽  
Type C ◽  
Beta Toxin

ABSTRACT Clostridium perfringens vegetative cells cause both histotoxic infections (e.g., gas gangrene) and diseases originating in the intestines (e.g., hemorrhagic necrotizing enteritis or lethal enterotoxemia). Despite their medical and veterinary importance, the molecular pathogenicity of C. perfringens vegetative cells causing diseases of intestinal origin remains poorly understood. However, C. perfringens beta toxin (CPB) was recently shown to be important when vegetative cells of C. perfringens type C strain CN3685 induce hemorrhagic necrotizing enteritis and lethal enterotoxemia. Additionally, the VirS/VirR two-component regulatory system was found to control CPB production by CN3685 vegetative cells during aerobic infection of cultured enterocyte-like Caco-2 cells. Using an isogenic virR null mutant, the current study now reports that the VirS/VirR system also regulates CN3685 cytotoxicity during infection of Caco-2 cells under anaerobic conditions, as found in the intestines. More importantly, the virR mutant lost the ability to cause hemorrhagic necrotic enteritis in rabbit small intestinal loops. Western blot analyses demonstrated that the VirS/VirR system mediates necrotizing enteritis, at least in part, by controlling in vivo CPB production. In addition, vegetative cells of the isogenic virR null mutant were, relative to wild-type vegetative cells, strongly attenuated in their lethality in a mouse enterotoxemia model. Collectively, these results identify the first regulator of in vivo pathogenicity for C. perfringens vegetative cells causing disease originating in the complex intestinal environment. Since VirS/VirR also mediates histotoxic infections, this two-component regulatory system now assumes a global role in regulating a spectrum of infections caused by C. perfringens vegetative cells. IMPORTANCE Clostridium perfringens is an important human and veterinary pathogen. C. perfringens vegetative cells cause both histotoxic infections, e.g., traumatic gas gangrene, and infections originating when this bacterium grows in the intestines. The VirS/VirR two-component regulatory system has been shown to control the pathogenicity of C. perfringens type A strains in a mouse gas gangrene model, but there is no understanding of pathogenicity regulation when C. perfringens vegetative cells cause disease originating in the complex intestinal environment. The current study establishes that VirS/VirR controls vegetative cell pathogenicity when C. perfringens type C isolates cause hemorrhagic necrotic enteritis and lethal enterotoxemia (i.e., toxin absorption from the intestines into the circulation, allowing targeting of internal organs). This effect involves VirS/VirR-mediated regulation of beta toxin production in vivo. Therefore, VirS/VirR is the first identified global in vivo regulator controlling the ability of C. perfringens vegetative cells to cause gas gangrene and, at least some, intestinal infections.

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 Characterization of Clostridium perfringens TpeL Toxin Gene Carriage, Production, Cytotoxic Contributions, and Trypsin Sensitivity

2015 ◽  
Vol 83 (6) ◽  
pp. 2369-2381 ◽  
Author(s):  
Jianming Chen ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Toxin Gene ◽  
Type B ◽  
Content Type ◽  
Positive Type ◽  
Natural Production ◽  
Type D ◽  
Clostridial Species ◽  
Beta Toxin

Large clostridial toxins (LCTs) are produced by at least four pathogenic clostridial species, and several LCTs are proven pivotal virulence factors for both human and veterinary diseases. TpeL is a recently identified LCT produced byClostridium perfringensthat has received relatively limited study. In response, the current study surveyed carriage of thetpeLgene among differentC. perfringensstrains, detecting this toxin gene in some type A, B, and C strains but not in any type D or E strains. This study also determined that all tested strains maximally produce, and extracellularly release, TpeL at the late-log or early-stationary growth stage duringin vitroculture, which is different from the maximal late-stationary-phase production reported previously for other LCTs and for TpeL production byC. perfringensstrain JIR12688. In addition, the present study found that TpeL levels in culture supernatants can be repressed by either glucose or sucrose. It was also shown that, at natural production levels, TpeL is a significant contributor to the cytotoxic activity of supernatants from cultures oftpeL-positive strain CN3685. Lastly, this study identified TpeL, which presumably is produced in the intestines during diseases caused by TpeL-positive type B and C strains, as a toxin whose cytotoxicity decreases after treatment with trypsin; this finding may have pathophysiologic relevance by suggesting that, like beta toxin, TpeL contributes to type B and C infections in hosts with decreased trypsin levels due to disease, diet, or age.

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 Identification and Characterization of Clostridium perfringens Beta Toxin Variants with Differing Trypsin Sensitivity andIn VitroCytotoxicity Activity

2015 ◽  
Vol 83 (4) ◽  
pp. 1477-1486 ◽  
Author(s):  
James R. Theoret ◽  
Francisco A. Uzal ◽  
Bruce A. McClane
Keyword(s):  
Amino Acid ◽  
Host Cell ◽  
Clostridium Perfringens ◽  
Cleavage Site ◽  
Cell Binding ◽  
Primary Host ◽  
Sequence Comparisons ◽  
Content Type ◽  
Significant Difference ◽  
Beta Toxin

By producing toxins,Clostridium perfringenscauses devastating diseases of both humans and animals.C. perfringensbeta toxin (CPB) is the major virulence determinant for type C infections and is also implicated in type B infections, but little is known about the CPB structure-function relationship. Amino acid sequence comparisons of the CPBs made by 8 randomly selected isolates identified two natural variant toxins with four conserved amino acid changes, including a switch of E to K at position 168 (E168K) that introduces a potential trypsin cleavage site into the CPB protein of strain JGS1076. To investigate whether this potential trypsin cleavage site affects sensitivity to trypsin, a primary host defense against this toxin, the two CPB variants were assayed for their trypsin sensitivity. The results demonstrated a significant difference in trypsin sensitivity, which was linked to the E168K switch by using site-directed recombinant CPB (rCPB) mutants. The natural CPB variants also displayed significant differences in their cytotoxicity to human endothelial cells. This cytotoxicity difference was mainly attributable to increased host cell binding rather than the ability to oligomerize or form functional pores. Using rCPB site-directed mutants, differences in cytotoxicity and host cell binding were linked to an A300V amino acid substitution in the strain JGS1076 CPB variant that possessed more cytotoxic activity. Mapping of sequence variations on a CPB structure modeled using related toxins suggests that the E168K substitution is surface localized and so can interact with trypsin and that the A300V substitution is located in a putative binding domain of the CPB toxin.

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