Ruminococcin C, a new anti-Clostridium perfringens bacteriocin produced in the gut by the commensal bacterium Ruminococcus gnavus E1

When colonizing the digestive tract of mono-associated rats, Ruminococcus gnavus E1 - a bacterium isolated from human faeces - produced a trypsin-dependent anti-Clostridium perfringens substance collectively named Ruminococcin C (RumC). RumC was isolated from the caecal contents of E1-monocontaminated rats and found to consist of two antimicrobial fractions: a single peptide (RumCsp) of 4235 Da, and a mixture of two other peptides (RumCdp) with distinct molecular masses of 4324 Da and 4456 Da. Both RumCsp and RumCdp were as effective as metronidazole in combating C. perfringens and their activity spectra against different pathogens were established. Even if devoid of synergistic activity, the combination of RumCsp and RumCdp was observed to be much more resistant to acidic pH and high temperature than each fraction tested individually. N-terminal sequence analysis showed that the primary structures of these three peptides shared a high degree of homology, but were clearly distinct from previously reported amino acid sequences. Amino acid composition of the three RumC peptides did not highlight the presence of any Lanthionine residue. However, Edman degradation could not run beyond the 11th amino acid residue. Five genes encoding putative pre-RumC-like peptides were identified in the genome of strain E1, confirming that RumC was a bacteriocin. This is the first time that a bacteriocin produced in vivo by a human commensal bacterium was purified and characterized.     

Crystal structure of the Clostridium limosum C3 exoenzyme

C3-like toxins ADP-ribosylate and inactivate Rho GTPases. Seven C3-like ADP-ribosyltransferases produced by Clostridium botulinum, Clostridium limosum, Bacillus cereus and Staphylococcus aureus were identified and two representatives - C3bot from C. botulinum and C3stau2 from S. aureus - were crystallized. Here we present the 1.8 Å structure of C. limosum C3 transferase C3lim and compare it to the structures of other family members. In contrast to the structure of apo-C3bot, the canonical ADP-ribosylating turn turn motif is observed in a primed conformation, ready for NAD binding. This suggests an impact on the binding mode of NAD and on the transferase reaction. The crystal structure explains why auto-ADP-ribosylation of C3lim at Arg41 interferes with the ADP-ribosyltransferase activity of the toxin.     

The Level of Expression of α-toxin by Different Strains ofClostridium perfringensis Dependent on Differences in Promoter Structure and Genetic Background

The control of expression of the α-toxin gene (cpaorplc) ofClostridium perfringenshas been studied in three strains shown to have high (NCTC8237), intermediate (strain 13) and low (NCTC8533) phospholipase C activity in the culture supernatant. The phospholipase C activity was shown to be related tocpamRNA levels. Primer extension studies were performed to locate thecpapromoter regions in strains NCTC8237 and 13. Differences in promoter sequences could account for the differences in α-toxin production between strains 13 and NCTC8237. In contrast, the differences in α-toxin production between strains NCTC8237 and NCTC8533 were unlikely to be due to promoter differences because the upstream promoter-containing sequences were identical in these strains. The recombinant plasmid carrying the NCTC8237cpagene was introduced into strains 13 and NCTC8533. The level of production of the α-toxin was 16-fold higher in strain 13, indicating the presence of strain-dependant regulatory systems.     

A quantitative electrochemiluminescence assay for Clostridium perfringens alpha toxin

Described is a rapid direct sandwich format electrochemiluminescence assay for identifying and assaying Clostridium perfringens alpha toxin. Biotinylated antibodies to C. perfringens alpha toxin bound to streptavidin paramagnetic beads specifically immunoadsorbed soluble sample alpha toxin which subsequently selectively immunoadsorbed ruthenium (Ru)-labeled detection antibodies. The ruthenium chelate of detection antibodies chemically reacted in the presence of tripropylamine and upon electronic stimulation emitted photons (electrochemiluminescence) that were detected by the photodiode of the detector. Elevated toxin concentrations increased toxin immunoadsorption and the specific immunoadsorption of Ru-labeled antibodies to alpha toxin, which resulted in increased dose-dependent electrochemiluminescent signals. The standardized assay was rapid (single 2.5-h coincubation of all reagents), required no wash steps, and had a sensitivity of about 1 ng/ml of toxin. The assay had excellent accuracy and precision and was validated in buffer, serum, and urine with no apparent matrix effects.     

Prevalence and characterization of Clostridium perfringens from spices in Argentina

Spices can present high microbial counts and Clostridium perfringens, Bacillus cereus, Salmonella and Shigella, among others have been isolated from spices. C. perfringens is an important pathogen agent causing, among other diseases, enteritis in humans caused by C. perfringens enterotoxin (CPE) which causes human food poisoning and enterotoxemia in domestic animals. The aims of the present work were (i) to establish the hygienic sanitary quality of some spices in San Luis, Argentina; (ii) to determine the presence of C. perfringens in these spices by means of the most probable number (MPN) and count on plate methods; (iii) to characterize the enterotoxigenic strains of C. perfringens by PCR and immunological methods such as reverse passive latex agglutination (RPLA) and (iv) to type by PCR C. perfringens strains isolated. A total of 115 samples of spices, 67 of which were purchased in local retail stores and 48 domestically collected were analysed. Total aerobe counts on tryptone glucose yeast extract agar medium of the 115 samples were between <10 and 10(6) CFU/g. The colifecal counts using Mac Conkey broth of the 115 samples were <4-10(3)CFU/g, with 28 samples (24.34%) exceeding the limit established by the Spanish Alimentary Code (10 CFU/g) while 2 samples (1.73%) had a sulfite reducing anaerobe load above standard limits. A total of 14 C. perfringens strains (12.17%) were isolated and characterized from 115 samples by the standard biochemical tests. Four of which (28.60%) turned out to be enterotoxigenic by PCR and RPLA. In order to type C. perfringens strains based on their main toxins, the 14 strains were analysed by PCR. All strains belonged to type A. All RPLA positive strains were cpe(+) by PCR. The percentage of enterotoxigenic strains was more elevated that those reported in other studies for this type of sample. These results indicate that sanitary conditions in different production stages of species must be improved to reduce health hazards. The high percentage (24.34%) of samples with colifecal values above standard limits is an indication of deficient sanitary conditions. These results suggest the need to provide legislation on the sanitary and hygienic quality of spices in our country.     

Crystal structure of the C-terminal three-helix bundle subdomain of C. elegans Hsp70

Hsp70 chaperones are composed of two domains; the 40 kDa N-terminal nucleotide-binding domain (NDB) and the 30 kDa C-terminal substrate-binding domain (SBD). Structures of the SBD from Escherichia coli homologues DnaK and HscA show it can be further divided into an 18 kDa beta-sandwich subdomain, which forms the hydrophobic binding pocket, and a 10 kDa C-terminal three-helix bundle that forms a lid over the binding pocket. Across prokaryotes and eukaryotes, the NBD and beta-sandwich subdomain are well conserved in both sequence and structure. The C-terminal subdomain is, however, more evolutionary variable and the only eukaryotic structure from rat Hsc70 revealed a diverged helix-loop-helix fold. We have solved the crystal structure of the C-terminal 10 kDa subdomain from Caenorhabditis elegans Hsp70 which forms a helical-bundle similar to the prokaryotic homologues. This provides the first confirmation of the structural conservation of this subdomain in eukaryotes. Comparison with the rat structure reveals a domain-swap dimerisation mechanism; however, the C. elegans subdomain exists exclusively as a monomer in solution in agreement with the hypothesis that regions out with the C-terminal subdomain are necessary for Hsp70 self-association.     

Identification and modeling of a drug target for Clostridium perfringens SM101

In the present study, comparative genome analysis between Clostridium perfringens and the human genome was carried out to identify genes that are essential for the pathogen's survival, and non-homologous to the genes of human host, that can be used as potential drug targets. The study resulted in the identification of 426 such genes. The number of these potential drug targets thus identified is significantly lower than the genome's protein coding capacity (2558 protein coding genes). The 426 genes of C. perfringens were further analyzed for overall similarities with the essential genes of 14 different bacterial species present in Database of Essential Genes (DEG). Our results show that there are only 5 essential genes of C. perfringens that exhibit similarity with 12 species of the 14 different bacterial species present in DEG database. Of these, 1 gene was similar in 12 species and 4 genes were similar in 11 species. Thus, the study opens a new avenue for the development of potential drugs against the highly pathogenic bacterium. Further, by selecting these essential genes of C. perfringens, which are common and essential for other pathogenic microbial species, a broad spectrum anti-microbial drug can be developed. As a case study, we have built a homology model of one of the potential drug targets, ABC transporter-ATP binding protein, which can be employed for in silico docking studies by suitable inhibitors.     

Molecular typing and antimicrobial susceptibility of Clostridium perfringens from broiler chickens

Clostridium perfringens (Cp) causes necrotic enteritis disease in commercial poultry. Antimicrobials are used to control and treat this disease and sometimes clinical outbreaks do not respond well to certain treatments. This study was designed to isolate Cp from clinical cases, type these isolates by multiplex PCR, and determine their antimicrobial susceptibility by micro-dilution method. A total of 67 Cp isolates were obtained from 155 broiler chicken flocks. All isolates were classified as type A and non-enterotoxin producers. Lincomycin, erythromycins, and tilmicosin showed very high minimal inhibitory concentration (MIC) 50 of ≥256 μg/ml. However, tylosin, amoxicillin, ampicillin, penicillin, florfenicol, danofloxacin, enrofloxacin, chlortetracycline, doxycycline, and oxytetracycline had variable MIC?? of 64, 0.5, 1, 1, 8, 4, 8, 4, 8, 0.5 μg/ml, respectively. It is recommended that Cp infections in Jordan be treated with either penicillins or tetracyclines especially amoxicillin and oxytetracycline.     

Prevention of the cytopathic effect induced by Clostridium difficile Toxin B by active Rac1

Clostridium difficile Toxin B (TcdB) glucosylates low molecular weight GTP-binding proteins of the Rho subfamily and thereby causes actin re-organization (cell rounding). This "cytopathic effect" has been generally attributed to RhoA inactivation. Here we show that cells expressing non-glucosylatable Rac1-Q61L are protected from the cytopathic effect of TcdB. In contrast, cells expressing RhoA-Q63L or mock-transfected cells are fully susceptible for the cytopathic effect of TcdB. These findings are extended to the Rac1/RhoG mimic IpgB1 and the RhoA mimic IpgB2 from Shigella. Ectopic expression of IpgB1, but not IpgB2, counteracts the cytopathic effect of TcdB. These data strongly suggest that Rac1 rather than RhoA glucosylation is critical for the cytopathic effect of TcdB.     

Clostridium perfringens alpha toxin is produced in the intestines of broiler chicks inoculated with an alpha toxin mutant

Poultry necrotic enteritis (NE) is caused by specific strains of Clostridium perfringens, most of which are type A. The role of alpha toxin (CPA) in NE has been called into question by the finding that an engineered cpa mutant retains full virulence in vivo[9]. This is in contrast to the finding that immunization with CPA toxoids protects against NE. We confirmed the earlier findings, in that 14-day-old Cornish × Rock broiler chicks challenged with a cpa mutant developed lesions compatible with NE in >90% of birds inoculated with the mutant. However, CPA was detected in amounts ranging from 10 to >100 ng per g of gut contents and mucosa in birds inoculated with the cpa mutant, the wildtype strain from which the mutant was constructed, and our positive control strain. There was a direct relationship between lesion severity and amount of CPA detected (R = 0.89-0.99). These findings suggest that the role of CPA in pathogenesis of NE requires further investigation.     

Acid phosphatase test proves superior to standard phenotypic identification procedure for Clostridium perfringens strains isolated from water

Clostridium perfringens is used as an indicator for persistent faecal pollution as well as to monitor the efficacy of water treatment processes. For these purposes, differentiation between C. perfringens and other Clostridia is essential and is routinely carried out by phenotypic standard tests as proposed in the ISO/CD 6461-2:2002 (ISO_LGMN: lactose fermentation, gelatine liquidation, motility and nitrate reduction). Because the ISO_LGMN procedure is time consuming and labour intensive, the acid phosphatase test was investigated as a possible and much more rapid alternative method for confirmation. The aim of our study was to evaluate and compare confirmation results obtained by these two phenotypic methods using genotypically identified strains, what to our knowledge has not been accomplished before. For this purpose, a species specific PCR method was selected based on the results received for type strains and genotypically characterised environmental strains. For the comparative investigation type strains as well as presumptive C. perfringens isolates from water and faeces samples were used. The acid phosphatase test revealed higher percentage (92%) of correctly identified environmental strains (n = 127) than the ISO_LGMN procedure (83%) and proved to be a sensitive and reliable confirmation method.     

ADP-ribosylation of actin from the green algaChara corallina byClostridium botulinum C2 toxin andClostridium perfringens iota toxin

Summary The ability of two bacterial toxins to modify a plant actin by covalent ADP-ribosylation was tested in the green algaChara corallina. Using [³²P]NAD, bothClostridium botulinum C2 toxin andClostridium perfringens iota toxin labelled a protein of M_r 42 kDa which comigrated with actin and was immunoprecipitated by a monoclonal anti-actin antibody. ADP-ribosylation ofChara actin was more efficient with iota toxin than with C2 toxin. The actin bundles in perfusedChara cells were not affected by toxin-containing media competent for ADP-ribosylation. The data indicate that monomeric plant actin is substrate for ADP-ribosylation by the bacterial toxins.Abbreviations ADP adenosine-diphosphate - EGTA ethyleneglycol-bis-(-aminoethyl)N,N,N,N-tetraacetic acid - NAD nicotinamide dinucleotide - pCA -log [Ca²⁺] - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Structural analysis of the PP2C phosphatase tPphA from Thermosynechococcus elongatus: a flexible flap subdomain controls access to the catalytic site

The homologue of the phosphoprotein PII phosphatase PphA from Thermosynechococcus elongatus, termed tPphA, was identified and its structure was resolved in two different space groups, C222₁ and P4₁2₁2, at a resolution of 1.28 and 3.05 Å, respectively. tPphA belongs to a large and widely distributed subfamily of Mg²⁺/Mn²⁺-dependent phosphatases of the PPM superfamily characterized by the lack of catalytic and regulatory domains. The core structure of tPphA shows a high degree of similarity to the two PPM structures identified so far. In contrast to human PP2C, but similar to Mycobacterium tuberculosis phosphatase PstP, the catalytic centre exhibits a third metal ion in addition to the dinuclear metal centre universally conserved in all PPM members. The fact that the third metal is only liganded by amino acids, which are universally conserved in all PPM members, implies that the third metal could be general for all members of this family. As a specific feature of tPphA, a flexible subdomain, previously recognized as a flap domain, could be revealed. Comparison of different structural isomers of tPphA as well as site-specific mutagenesis implied that the flap domain is involved in substrate binding and catalytic activity. The structural arrangement of the flap domain was accompanied by a large side-chain movement of an Arg residue (Arg169) at the basis of the flap. Mutation of this residue strongly impaired protein stability as well as catalytic activity, emphasizing the importance of this amino acid for the regional polysterism of the flap subdomain and confirming the assumption that flap domain flexibility is involved in catalysis.     

Identification of glutamate ABC-Transporter component in Clostridium perfringens as a putative drug target

Clostridium perfringens is an anaerobic pathogen known to cause vast number of diseases in mammals and birds. Various toxins and hydrolysing enzymes released by the organism are responsible for the necrosis of soft tissues. Due to serious safety issues associated with current vaccines against C. perfringens, there is a need for new drug or vaccine targets. C. perfringens is extremely dependent on its host for nutrition which can be targeted for vaccine development or drug design. Therefore, it is of interest to identify the unique transport systems used by C. perfringens involved in uptake of essential amino acids that are synthesized by the host, so that therapeutic agents can be designed to target the specific transport systems. Use of bioinformatics tools resulted in the identification of a protein component of the glutamate transport system that is not present in the host. Analysis of the conservation profile of the protein domain indicated it to be a glutamate binding protein which also stimulates the ATPase activity of ATP Binding Cassettes (ABC) transporters. Homology modelling of the protein showed two distinct lobes, which is a characteristic of substrate binding proteins. This suggests that the carboxylates of glutamate might be stabilized by electrostatic interactions with basic residues as is observed with other binding proteins. Hence, the homology model of this potential drug target can be employed for in silico docking studies by suitable inhibitors.     

Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier.

Claudins, comprising a multigene family, constitute tight junction (TJ) strands. Clostridium perfringens enterotoxin (CPE), a single approximately 35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half. We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4. C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2. In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface. In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation. At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased. In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner. These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.     

The solution structure of the C-terminal modular pair from Clostridium perfringens mu-toxin reveals a noncellulosomal dockerin module

The genome of the opportunistic pathogen Clostridium perfringens encodes a large number of secreted glycoside hydrolases. Their predicted activities indicate that they are involved in the breakdown of complex carbohydrates and other glycans found in the mucosal layer of the human gastrointestinal tract, within the extracellular matrix, and on the surface of host cells. One such group of these enzymes is the family 84 glycoside hydrolases, which has predicted hyaluronidase activity and comprises five members [C. perfringens glycoside hydrolase family 84 (CpGH84) A-E]. The first identified member, CpGH84A, corresponds to the μ-toxin whose modular architecture includes an N-terminal catalytic domain, four family 32 carbohydrate-binding modules, three FIVAR modules of unknown function, and a C-terminal putative calcium-binding module. Here, we report the solution NMR structure of the C-terminal modular pair from the μ-toxin. The three-helix bundle FIVAR module displays structural homology to a heparin-binding module within the N-terminal of the a C protein from group B Streptoccocus. The C-terminal module has a typical calcium-binding dockerin fold comprising two anti-parallel helices that form a planar face with EF-hand calcium-binding loops at opposite ends of the module. The size of the helical face of the μ-toxin dockerin module is approximately equal to the planar region recently identified on the surface of a cohesin-like X82 module of CpGH84C. Size-exclusion chromatography and heteronuclear NMR-based chemical shift mapping studies indicate that the helical face of the dockerin module recognizes the CpGH84C X82 module. These studies represent the structural characterization of a noncellulolytic dockerin module and its interaction with a cohesin-like X82 module. Dockerin/X82-mediated enzyme complexes may have important implications in the pathogenic properties of C. perfringens.     

Crystal structure of a non-discriminating glutamyl-tRNA synthetase

Error-free protein biosynthesis is dependent on the reliable charging of each tRNA with its cognate amino acid. Many bacteria, however, lack a glutaminyl-tRNA synthetase. In these organisms, tRNA(Gln) is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase (ND-GluRS). This enzyme thus charges both tRNA(Glu) and tRNA(Gln) with glutamate. Discriminating GluRS (D-GluRS), found in some bacteria and all eukaryotes, exclusively generates Glu-tRNA(Glu). Here we present the first crystal structure of a non-discriminating GluRS from Thermosynechococcus elongatus (ND-GluRS(Tel)) in complex with glutamate at a resolution of 2.45 A. Structurally, the enzyme shares the overall architecture of the discriminating GluRS from Thermus thermophilus (D-GluRS(Tth)). We confirm experimentally that GluRS(Tel) is non-discriminating and present kinetic parameters for synthesis of Glu-tRNA(Glu) and of Glu-tRNA(Gln). Anticodons of tRNA(Glu) (34C/UUC36) and tRNA(Gln) (34C/UUG36) differ only in base 36. The pyrimidine base of C36 is specifically recognized in D-GluRS(Tth) by the residue Arg358. In ND-GluRS(Tel) this arginine residue is replaced by glycine (Gly366) presumably allowing both cytosine and the bulkier purine base G36 of tRNA(Gln) to be tolerated. Most other ND-GluRS share this structural feature, leading to relaxed substrate specificity.     

Expression and purification of functional Clostridium perfringens alpha and epsilon toxins in Escherichia coli

The alpha and epsilon toxins are 2 of the 4 major lethal toxins of the pathogen Clostridium perfringens. In this study, the expression of the epsilon toxin (etx) gene of C. perfringens was optimized by replacing rare codons with high-frequency codons, and the optimized gene was synthesized using overlapping PCR. Then, the etx gene or the alpha-toxin gene (cpa) was individually inserted into the pTIG-Trx expression vector with a hexahistidine tag and a thioredoxin (Trx) to facilitate their purification and induce the expression of soluble proteins. The recombinant alpha toxin (rCPA) and epsilon toxin (rETX) were highly expressed as soluble forms in the recipient Escherichia coli BL21 strain, respectively. The rCPA and rETX were purified using Ni(2+)-chelating chromatography and size-exclusion chromatography. And the entire purification process recovered about 40% of each target protein from the starting materials. The purified target toxins formed single band at about 42kDa (rCPA) or 31kDa (rETX) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their functional activity was confirmed by bioactivity assays. We have shown that the production of large amounts of soluble and functional proteins by using the pTIG-Trx vector in E. coli is a good alternative for the production of native alpha and epsilon toxins and could also be useful for the production of other toxic proteins with soluble forms.     

Recombinant expression of in silico identified Bcell epitope of epsilon toxin of Clostridium perfringens in translational fusion with a carrier protein

Epsilon toxin secreted by Clostridium perfringens types B and D has been directly implicated as the causative agent of fatal enterotoxemia in domestic animals. The aim of the present study is to use in silico approach for identification of B-cell epitope(s) of epsilon toxin, and its expression in fusion with a carrier protein to analyze its potential as vaccine candidate(s). Using different computational analyses and bioinformatics tools, a number of antigenic determinant regions of epsilon toxin were identified. One of the B cell epitopes of epsilon toxin comprising the region (amino acids 40-62) was identified as a promising antigenic determinant. This Etx epitope (Etx_40-62) was cloned and expressed as a translational fusion with B-subunit of heat labile enterotoxin (LTB) of E. coli in a secretory expression system. Similar to the native LTB, the recombinant fusion protein retained the ability to pentamerize and bind to GM₁ ganglioside receptor of LTB. The rLTB.Etx_40-62 could be detected both with anti-Etx and anti-LTB antisera. The rLTB.Etx_40-62 fusion protein thus can be evaluated as a potential vaccine candidate against C. perfringens.

Abbreviations

aa - amino acid(s), Etx - epsilon toxin of Clostridium perfringens, LTB - B-subunit of heat labile enterotoxin of E. coli.