Cysteine-Scanning Mutagenesis Supports the Importance of Clostridium perfringens Enterotoxin Amino Acids 80 to 106 for Membrane Insertion and Pore Formation

ABSTRACTClostridium perfringensenterotoxin (CPE) causes the gastrointestinal symptoms of the second most common bacterial food-borne illness. Previous studies suggested that a region named TM1, which has amphipathic characteristics and spans from amino acids 81 to 106 of the native CPE protein, forms a β-hairpin involved in β-barrel pore formation. To further explore the potential role of TM1 in pore formation, the single Cys naturally present in CPE at residue 186 was first altered to alanine by mutagenesis; the resultant rCPE variant, named C186A, was shown to retain cytotoxic properties. Cys-scanning mutagenesis was then performed in which individual Cys mutations were introduced into each TM1 residue of the C186A variant. When those Cys variants were characterized, three variants were identified that exhibit reduced cytotoxicity despite possessing binding and oligomerization abilities similar to those of the C186A variant from which they were derived. Pronase challenge experiments suggested that the reduced cytotoxicity of those two Cys variants, i.e., the F91C and F95C variants, which model to the tip of the β-hairpin, was attributable to a lessened ability of these variants to insert into membranes after oligomerization. In contrast, another Cys variant, i.e., the G103C variant, with impaired cytotoxicity apparently inserted into membranes after oligomerization but could not form a pore with a fully functional channel. Collectively, these results support the TM1 region forming a β-hairpin as an important step in CPE insertion and pore formation. Furthermore, this work identifies the first amino acid residues specifically involved in those two steps in CPE action.

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Human Claudin-8 and -14 Are Receptors Capable of Conveying the Cytotoxic Effects of Clostridium perfringens Enterotoxin

mBio ◽

10.1128/mbio.00594-12 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 31

Author(s):

Archana Shrestha ◽

Bruce A. McClane

Keyword(s):

Drug Delivery ◽

Clostridium Perfringens ◽

Gastrointestinal Disease ◽

Gastrointestinal Symptoms ◽

Site Directed Mutagenesis ◽

Cytotoxic Effects ◽

Content Type ◽

Food Borne ◽

Food Borne Illness ◽

Normal Human

ABSTRACTClostridium perfringensenterotoxin (CPE) contributes to several important human gastrointestinal (GI) diseases. This toxin and its derivatives are also being explored for translational applications, i.e., cancer therapy or drug delivery. Some, but not all, members of the 24-member claudin (Cldn) family of mammalian tight junction proteins can serve as CPE receptors. Among the human Cldns (hCldns), hCldn-3 and -4 are known to convey CPE sensitivity when expressed by fibroblast transfectants. However, other Cldns are also reportedly expressed in the intestines, where they might contribute to natural CPE-mediated GI disease, and in other organs, where they might react with CPE-based therapeutics. Therefore, the current study assessed whether two additional hCldns beside hCldn-3 and -4 are also functional CPE receptors. Using Cldn-expressing transfectants, hCldn-8 and -14 were shown to convey CPE-mediated cytotoxicity at pathophysiologically relevant concentrations of this toxin, although ~2-to-10-fold less efficiently than hCldn-4. Site-directed mutagenesis then demonstrated that the N146residue in hCldn-14 and the S151residue in hCldn-8 are largely responsible for modulating the weaker CPE binding properties of hCldn-8 and -14 versus hCldn-4, which broadens understanding of Cldn:CPE binding interactions. Since Cldn-8 and -14 are reportedly expressed in mammalian intestines, the current results support the possibility that these two hCldns contribute to natural CPE-mediated gastrointestinal disease and could be CPE-based therapeutic targets for cancers overexpressing those claudins. However, these results also suggest caution during therapeutic use of CPE, which might trigger toxic side effects in normal human tissues producing hCldn-8 or -14, as well as in those producing hCldn-3 or -4.IMPORTANCEClostridium perfringensenterotoxin (CPE) is responsible for the gastrointestinal symptoms of the second-most-common bacterial food-borne illness and is also being explored for use as a cancer therapeutic or for increasing drug delivery. Until now, the only known human CPE receptors were claudin-3 and -4. This work shows that human claudin-8 and -14 can also bind CPE and convey cytotoxicity, although slightly less efficiently than claudin-3 and -4. The claudin-8 and -14 residues responsible for this weaker CPE binding were identified, shedding new light on CPE:claudin interactions.

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

mSphere ◽

10.1128/msphere.00352-17 ◽

2017 ◽

Vol 2 (4) ◽

Cited By ~ 6

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.

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Aspergillus fumigatus Intrinsic Fluconazole Resistance Is Due to the Naturally Occurring T301I Substitution in Cyp51Ap

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00905-16 ◽

2016 ◽

Vol 60 (9) ◽

pp. 5420-5426 ◽

Cited By ~ 26

Author(s):

Florencia Leonardelli ◽

Daiana Macedo ◽

Catiana Dudiuk ◽

Matias S. Cabeza ◽

Soledad Gamarra ◽

...

Keyword(s):

Candida Albicans ◽

Aspergillus Fumigatus ◽

Parental Strain ◽

Amino Acid Residues ◽

Intrinsic Resistance ◽

Single Nucleotide ◽

Fluconazole Resistance ◽

Content Type ◽

Natural Polymorphism

ABSTRACTAspergillus fumigatusintrinsic fluconazole resistance has been demonstrated to be linked to theCYP51Agene, although the precise molecular mechanism has not been elucidated yet. Comparisons betweenA. fumigatusCyp51Ap andCandida albicansErg11p sequences showed differences in amino acid residues already associated with fluconazole resistance inC. albicans. The aim of this study was to analyze the role of the natural polymorphism I301 inAspergillus fumigatusCyp51Ap in the intrinsic fluconazole resistance phenotype of this pathogen. The I301 residue inA. fumigatusCyp51Ap was replaced with a threonine (analogue to T315 atCandida albicansfluconazole-susceptible Erg11p) by changing one single nucleotide in theCYP51Agene. Also, aCYP51Aknockout strain was obtained using the same parental strain. Both mutants' antifungal susceptibilities were tested. The I301T mutant exhibited a lower level of resistance to fluconazole (MIC, 20 μg/ml) than the parental strain (MIC, 640 μg/ml), while no changes in MIC were observed for other azole- and non-azole-based drugs. These data strongly implicate theA. fumigatusCyp51Ap I301 residue in the intrinsic resistance to fluconazole.

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Observations on the Role of TcdE Isoforms in Clostridium difficile Toxin Secretion

Journal of Bacteriology ◽

10.1128/jb.00224-15 ◽

2015 ◽

Vol 197 (15) ◽

pp. 2600-2609 ◽

Cited By ~ 20

Author(s):

Revathi Govind ◽

Leah Fitzwater ◽

Rebekah Nichols

Keyword(s):

Amino Acids ◽

Clostridium Difficile ◽

Cell Viability ◽

Virulence Factors ◽

Start Codon ◽

Nosocomial Pathogen ◽

Content Type ◽

Antibiotic Associated Diarrhea ◽

Toxin Secretion

ABSTRACTClostridium difficileis a major nosocomial pathogen and the principal causative agent of antibiotic-associated diarrhea. The toxigenicC. difficilestrains that cause disease secrete virulence factors, toxin A and toxin B, that cause colonic injury and inflammation.C. difficiletoxins have no export signature and are secreted by an unusual mechanism that involves TcdE, a holin-like protein. We isolated a TcdE mutant of the epidemic R20291 strain with impaired toxin secretion, which was restored by complementation with functional TcdE. In the TcdE open reading frame (ORF), we identified three possible translation start sites; each translated isoform may play a specific role in TcdE-controlled toxin release. We created plasmid constructs that express only one of the three TcdE isoforms and complemented the TcdE mutant with these isoforms. Western blot analysis of the complemented strains demonstrated that TcdE is translated efficiently from the start codon at the 25th and 27th positions in the predicted ORF, producing proteins with 142 amino acids (TcdE142) and 140 amino acids (TcdE140), respectively. TcdE166was not detected when expressed from its own ribosomal binding site (RBS). The effects of all three TcdE isoforms onC. difficilecell viability and toxin release were determined. Among the three isoforms, overexpression of TcdE166and TcdE142had a profound effect on cell viability compared to the TcdE140isoform. Similarly, TcdE166and TcdE142facilitated toxin release more efficiently than did TcdE140. The importance of these variations among TcdE isoforms and their role in toxin release are discussed.IMPORTANCEC. difficileis a nosocomial pathogen that has become the most prevalent cause of antibiotic-associated diarrhea in North America and in several countries in Europe. Most strains ofC. difficileproduce two high-molecular-weight toxins that are regarded as the primary virulence factors. The mechanism by which these large toxins are secreted from bacterial cells is not yet clear but involves TcdE, a holin-like protein. In this work, we show that TcdE could be translated from three different start codons, resulting in the production of three TcdE isoforms. Furthermore, we investigated the role of these isoforms in toxin release and cell lysis inC. difficile. An understanding of TcdE-dependent toxin secretion may be helpful for the development of strategies for preventing and treatingC. difficileinfections.

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Cysteine Scanning Mutagenesis of α4, a Putative Pore-Lining Helix of the Bacillus thuringiensis Insecticidal Toxin Cry1Aa

Applied and Environmental Microbiology ◽

10.1128/aem.00094-08 ◽

2008 ◽

Vol 74 (9) ◽

pp. 2565-2572 ◽

Cited By ~ 26

Author(s):

Frédéric Girard ◽

Vincent Vachon ◽

Gabrielle Préfontaine ◽

Lucie Marceau ◽

Yanhui Su ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Pore Formation ◽

Membrane Vesicles ◽

Significant Activity ◽

Amino Acid Residues ◽

Insect Larvae ◽

Insecticidal Toxin ◽

Cysteine Scanning ◽

Cysteine Scanning Mutagenesis ◽

Pore Lining

ABSTRACT Helix α4 of Bacillus thuringiensis Cry toxins is thought to line the lumen of the pores they form in the midgut epithelial cells of susceptible insect larvae. To define its functional role in pore formation, most of the α4 amino acid residues were replaced individually by a cysteine in the Cry1Aa toxin. The toxicities and pore-forming abilities of the mutated toxins were examined, respectively, by bioassays using neonate Manduca sexta larvae and by a light-scattering assay using midgut brush border membrane vesicles isolated from M. sexta. A majority of these mutants had considerably reduced toxicities and pore-forming abilities. Most mutations causing substantial or complete loss of activity map on the hydrophilic face of the helix, while most of those having little or only relatively minor effects map on its hydrophobic face. The properties of the pores formed by mutants that retain significant activity appear similar to those of the pores formed by the wild-type toxin, suggesting that mutations resulting in a loss of activity interfere mainly with pore formation.

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

Infection and Immunity ◽

10.1128/iai.00528-10 ◽

2010 ◽

Vol 78 (10) ◽

pp. 4286-4293 ◽

Cited By ~ 51

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.

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Behavior of Different Shiga Toxin-Producing Escherichia coli Serotypes in Various Experimentally Contaminated Raw-Milk Cheeses

Applied and Environmental Microbiology ◽

10.1128/aem.02192-12 ◽

2012 ◽

Vol 79 (1) ◽

pp. 150-158 ◽

Cited By ~ 26

Author(s):

Stéphane D. Miszczycha ◽

Frédérique Perrin ◽

Sarah Ganet ◽

Emmanuel Jamet ◽

Fanny Tenenhaus-Aziza ◽

...

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Human Health Risk ◽

Raw Milk ◽

Public Health Implication ◽

Content Type ◽

Growth And Survival ◽

The Public ◽

Food Borne ◽

Food Borne Illness

ABSTRACTShiga toxin-producingEscherichia coli(STEC) is an important cause of food-borne illness. The public health implication of the presence of STEC in dairy products remains unclear. Knowledge of STEC behavior in cheeses would help to evaluate the human health risk. The aim of our study was to observe the growth and survival of experimentally inoculated STEC strains in raw-milk cheeses manufactured and ripened according to five technological schemes: blue-type cheese, uncooked pressed cheese with long ripening and with short ripening steps, cooked cheese, and lactic cheese. Cheeses were contaminated with different STEC serotypes (O157:H7, O26:H11, O103:H2, and O145:H28) at the milk preparation stage. STEC growth and survival were monitored on selective media during the entire manufacturing process. STEC grew (2 to 3 log10CFU · g−1) in blue-type cheese and the two uncooked pressed cheeses during the first 24 h of cheese making. Then, STEC levels progressively decreased in cheeses that were ripened for more than 6 months. In cooked cheese and in lactic cheese with a long acidic coagulation step (pH < 4.5), STEC did not grow. Their levels decreased after the cooking step in the cooked cheese and after the coagulation step in the lactic cheese, but STEC was still detectable at the end of ripening and storage. A serotype effect was found: in all cheeses studied, serotype O157:H7 grew less strongly and was less persistent than the others serotypes. This study improves knowledge of the behavior of different STEC serotypes in various raw-milk cheeses.

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The Metalloprotease Mpl Supports Listeria monocytogenes Dissemination through Resolution of Membrane Protrusions into Vacuoles

Infection and Immunity ◽

10.1128/iai.00130-16 ◽

2016 ◽

Vol 84 (6) ◽

pp. 1806-1814 ◽

Cited By ~ 7

Author(s):

Diego E. Alvarez ◽

Hervé Agaisse

Keyword(s):

Listeria Monocytogenes ◽

Amino Acid Residues ◽

Content Type ◽

Bacterial Surface ◽

Membrane Protrusions ◽

Dissemination Process ◽

Processing Site ◽

Nucleation Promoting Factor ◽

Mild Defect

Listeria monocytogenesis an intracellular pathogen that disseminates within the intestinal epithelium through acquisition of actin-based motility and formation of plasma membrane protrusions that project into adjacent cells. The resolution of membrane protrusions into vacuoles from which the pathogen escapes results in bacterial spread from cell to cell. This dissemination process relies on themlp-actA-plcBoperon, which encodes ActA, a bacterial nucleation-promoting factor that mediates actin-based motility, and PlcB, a phospholipase that mediates vacuole escape. Here we investigated the role of the metalloprotease Mpl in the dissemination process. In agreement with previous findings showing that Mpl is required for PlcB activation, infection of epithelial cells with the ΔplcBor Δmplstrains resulted in the formation of small infection foci. As expected, the ΔplcBstrain displayed a strong defect in vacuole escape. However, the Δmplstrain showed an unexpected defect in the resolution of protrusions into vacuoles, in addition to the expected but mild defect in vacuole escape. The Δmplstrain displayed increased levels of ActA on the bacterial surface in protrusions. We mapped an Mpl-dependent processing site in ActA between amino acid residues 207 to 238. Similar to the Δmplstrain, the ΔactA207–238strain displayed increased levels of ActA on the bacterial surface in protrusions. Although the ΔactA207–238strain displayed wild-type actin-based motility, it formed small infection foci and failed to resolve protrusions into vacuoles. We propose that, in addition to its role in PlcB processing and vacuole escape, the metalloprotease Mpl is required for ActA processing and protrusion resolution.

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The Agr-Like Quorum Sensing System Is Required for Pathogenesis of Necrotic Enteritis Caused by Clostridium perfringens in Poultry

Infection and Immunity ◽

10.1128/iai.00975-16 ◽

2017 ◽

Vol 85 (6) ◽

Cited By ~ 15

Author(s):

Qiang Yu ◽

Dion Lepp ◽

Iman Mehdizadeh Gohari ◽

Tao Wu ◽

Hongzhuan Zhou ◽

...

Keyword(s):

Quorum Sensing ◽

Clostridium Perfringens ◽

Global Gene Expression ◽

Toxin Production ◽

Phospholipid Metabolism ◽

Necrotic Enteritis ◽

Content Type ◽

Protein Levels ◽

Global Gene Expression Analysis

ABSTRACT Clostridium perfringens encodes at least two different quorum sensing (QS) systems, the Agr-like and LuxS, and recent studies have highlighted their importance in the regulation of toxin production and virulence. The role of QS in the pathogenesis of necrotic enteritis (NE) in poultry and the regulation of NetB, the key toxin involved, has not yet been investigated. We have generated isogenic agrB-null and complemented strains from parent strain CP1 and demonstrated that the virulence of the agrB-null mutant was strongly attenuated in a chicken NE model system and restored by complementation. The production of NetB, a key NE-associated toxin, was dramatically reduced in the agrB mutant at both the transcriptional and protein levels, though not in a luxS mutant. Transwell assays confirmed that the Agr-like QS system controls NetB production through a diffusible signal. Global gene expression analysis of the agrB mutant identified additional genes modulated by Agr-like QS, including operons related to phospholipid metabolism and adherence, which may also play a role in NE pathogenesis. This study provides the first evidence that the Agr-like QS system is critical for NE pathogenesis and identifies a number of Agr-regulated genes, most notably netB, that are potentially involved in mediating its effects. The Agr-like QS system thus may serve as a target for developing novel interventions to prevent NE in chickens.

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

mBio ◽

10.1128/mbio.00019-13 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 36

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.

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