Pathogen Clostridium Perfringens Research Articles

Responses of feeding prebiotics on nutrient digestibility, faecal microbiota composition and short-chain fatty acid concentrations in dogs: a meta-analysis

The effects of prebiotics on digestibility, short-chain fatty acid (SCFA) concentrations and bacterial populations in the faeces and immunity in dogs were evaluated by meta-analyses. Overall, data from 15 published studies containing 65 different treatment means of 418 observations from different breeds of dogs were included in the data set. Feeding of prebiotics to dogs did not affect the nutrient intake (P > 0.10), nor did prebiotics change (P > 0.10) the digestibility of dry matter (DM) and fat. However, crude protein (CP) digestibility tended to decrease quadratically (P = 0.06) with increasing dosages of prebiotics, although the degree of prediction was low (R2 = 0.33). The concentration of total SCFA (P = 0.08; R2 = 0.90) tended to increase linearly, whereas concentration of acetate (R2 = 0.25), propionate (R2 = 0.88) and butyrate (R2 = 0.85) increased quadratically with increasing dosage of prebiotics in the faeces of dogs. The numbers of beneficial bifidobacteria (P < 0.01; R2 = 0.62) increased quadratically, but lactobacilli (P < 0.01; R2 = 0.66) increased linearly with increasing supplementation of prebiotics. The changes in healthy bacterial numbers were affected by the interaction of initial bacterial numbers and dose of prebiotics; bacterial numbers increased relatively more when initial bacterial numbers were low. Dietary composition did not influence the response of prebiotics on lactobacilli and bifidobacterial numbers in this study. The numbers of pathogenic Clostridium perfringens and Escherichia coli were not affected by prebiotics. Prebiotics did not affect the serum immunoglobulin (Ig) concentrations such as IgG, IgA and IgM in dogs. Although prebiotics may tend to have an adverse effect on CP digestibility, prebiotics at doses up to 1.40% food intake (DM basis) might increase the beneficial bacterial populations and SCFA concentrations in the faeces of dogs. Thus, the feeding of prebiotics has a great prospective to improve the intestinal health of dogs.

Lytic enzyme discovery through multigenomic sequence analysis in Clostridium perfringens

With their ability to lyse Gram-positive bacteria, phage lytic enzymes (or lysins) have received a great deal of attention as novel anti-infective agents. The number of known genes encoding these peptidoglycan hydrolases has increased markedly in recent years, due in large part to advances in DNA sequencing technology. As the genomes of more and more bacterial species/strains are sequenced, lysin-encoding open reading frames (ORFs) can be readily identified in lysogenized prophage regions. In the current study, we sought to assess lysin diversity for the medically relevant pathogen Clostridium perfringens. The sequenced genomes of nine C. perfringens strains were computationally mined for prophage lysins and lysin-like ORFs, revealing several dozen proteins of various enzymatic classes. Of these lysins, a muramidase from strain ATCC 13124 (termed PlyCM) was chosen for recombinant analysis based on its dissimilarity to previously characterized C. perfringens lysins. Following expression and purification, various biochemical properties of PlyCM were determined in vitro, including pH/salt-dependence and temperature stability. The enzyme exhibited activity at low μg/ml concentrations, a typical value for phage lysins. It was active against 23 of 24 strains of C. perfringens tested, with virtually no activity against other clostridial or non-clostridial species. Overall, PlyCM shows potential for development as an enzybiotic agent, demonstrating how expanding genomic databases can serve as rich pools for biotechnologically relevant proteins.

EtfA catalyses the formation of dipicolinic acid inClostridium perfringens

Dipicolinic acid (DPA) is a major component of bacterial endospores, comprising 5-15% of the spore dry weight, and is important for spore stability and resistance properties. The biosynthetic precursor to DPA, dihydro-dipicolinic acid (DHDPA), is produced by DHDPA synthase within the lysine biosynthesis pathway. In Bacillus subtilis, and most other bacilli and clostridia, DHDPA is oxidized to DPA by the products of the spoVF operon. Analysis of the genomes of the clostridia in Cluster I, including the pathogens Clostridium perfringens, Clostridium botulinum and Clostridium tetani, has shown that no spoVF orthologues exist in these organisms. DPA synthase was purified from extracts of sporulating C. perfringens cells. Peptide sequencing identified an electron transfer flavoprotein, EtfA, in this purified protein fraction. A C. perfringens strain with etfA inactivated is blocked in late stage sporulation and produces < or = 11% of wild-type DPA levels. C. perfringens EtfA was expressed in and purified from Escherichia coli, and this protein catalysed DPA formation in vitro. The sequential production of DHDPA and DPA in C. perfringens appears to be catalysed by DHDPA synthase followed by EtfA. Genome sequence data and the taxonomy of spore-forming species suggest that this may be the ancestral mechanism for DPA synthesis.

Evaluation of PCR and DNA Sequencing for Direct Detection of Clostridium perfringens in the Intestinal Tract of Broilers

The aim of this investigation was to determine the presence of the opportunistic pathogen Clostridium perfringens by PCR and DNA sequencing, without previous cultivation. This methodology was then used to investigate how C. perfringens was affected by different preventive measures, such as ionophores and feed additives, for necrotic enteritis in broilers chickens. DNA was extracted from the intestinal content or intestinal tissue by DNA extraction kits. Detection limits for 16S rRNA, alpha-toxin, and cpb2 PCR gene targets were approximately 1 x 10(3), 5 x 10(4), and 1 x 10(6) cells per g of intestinal content or tissue, respectively, as determined with samples spiked with C. perfringens. The method was evaluated with samples from single conventional broilers or from pools of six birds of experimentally reared broilers. Conventional chickens, raised with salinomycin in their feed, showed reduced numbers of C. perfringens-positive samples (P < 0.05) for all three PCR tests. With respect to cpb2, a tendency to detect more samples as positive for C. perfringens was observed with increasing age. The addition of sodium butyrate and lactic acid in the feed for experimental birds had a minor effect (P < 0.10) on positive samples, as detected with the 16S rRNA PCR. For experimental birds fed whole wheat, only three out of six pools of six birds allowed detection of C. perfringens by the 16S rRNA PCR, compared to five for the untreated controls or the Avilamycin- or prebiotic-treated birds. All 16S rRNA partial gene sequences obtained were identical and were 99.5% similar to the rrnB gene of the type strain of C. perfringens. Two types of the partial cpb2 gene sequence were detected with a similarity of 93%. One type was translated into protein, whereas a stop codon was found in the other type. Both types were located in the "atypical" phylogenetic group of the cpb2 gene sequences. The PCR test, based on extraction of DNA from intestinal content, provided rapid screening of poultry for C. perfringens without the need to have access to facilities in order to immediately cultivate and identify bacteria at the location of sampling. Further work is suggested to determine the relationship between the degree of necrotic enteritis, the actual level of C. perfringens in the animal, and the detection achieved by PCR.

Influence of high gas production during thermophilic anaerobic digestion in pilot-scale and lab-scale reactors on survival of the thermotolerant pathogens Clostridium perfringens and Campylobacter jejuni in piggery wastewater

Safe reuse of animal wastes to capture energy and nutrients, through anaerobic digestion processes, is becoming an increasingly desirable solution to environmental pollution. Pathogen decay is the most important safety consideration and is in general, improved at elevated temperatures and longer hydraulic residence times. During routine sampling to assess pathogen decay in thermophilic digestion, an inversely proportional relationship between levels of Clostridium perfringens and gas production was observed. Further samples were collected from pilot-scale, bench-scale thermophilic reactors and batch scale vials to assess whether gas production (predominantly methane) could be a useful indicator of decay of the thermotolerant pathogens C. perfringens and Campylobacter jejuni. Pathogen levels did appear to be lower where gas production and levels of methanogens were higher. This was evident at each operating temperature (50, 57, 65 °C) in the pilot-scale thermophilic digesters, although higher temperatures also reduced the numbers of pathogens detected. When methane production was higher, either when feed rate was increased, or pH was lowered from 8.2 (piggery wastewater) to 6.5, lower numbers of pathogens were detected. Although a number of related factors are known to influence the amount and rate of methane production, it may be a useful indicator of the removal of the pathogens C. perfringens and C. jejuni.

The Putative Coupling Protein TcpA Interacts with Other pCW3-Encoded Proteins To Form an Essential Part of the Conjugation Complex

Conjugative plasmids encode antibiotic resistance determinants or toxin genes in the anaerobic pathogen Clostridium perfringens. The paradigm conjugative plasmid in this bacterium is pCW3, a 47-kb tetracycline resistance plasmid that encodes the unique tcp transfer locus. The tcp locus consists of 11 genes, intP and tcpA-tcpJ, at least three of which, tcpA, tcpF, and tcpH, are essential for the conjugative transfer of pCW3. In this study we examined protein-protein interactions involving TcpA, the putative coupling protein. Use of a bacterial two-hybrid system identified interactions between TcpA and TcpC, TcpG, and TcpH. This analysis also demonstrated TcpA, TcpC, and TcpG self-interactions, which were confirmed by chemical cross-linking studies. Examination of a series of deletion and site-directed derivatives of TcpA identified the domains and motifs required for these interactions. Based on these results, we have constructed a model for this unique conjugative transfer apparatus.

Characterization of two different endo- -N-acetylgalactosaminidases from probiotic and pathogenic enterobacteria, Bifidobacterium longum and Clostridium perfringens

Endo-alpha-N-acetylgalactosaminidase (endo-alpha-GalNAc-ase) catalyzes the hydrolysis of the O-glycosidic bond between alpha-GalNAc at the reducing end of mucin-type sugar chains and serine/threonine of proteins to release oligosaccharides. Previously, we identified the gene engBF encoding endo-alpha-GalNAc-ase from Bifidobacterium longum, which specifically released the disaccharide Gal beta 1-3GalNAc (Fujita K, Oura F, Nagamine N, Katayama T, Hiratake J, Sakata K, Kumagai H, Yamamoto K. 2005. Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo-alpha-N-acetylgalactosaminidase from Bifidobacterium longum. J Biol Chem. 280:37415-37422). Here we cloned a similar gene named engCP from Clostridium perfringens, a pathogenic enterobacterium, and characterized the gene product EngCP. Detailed analyses on substrate specificities of EngCP and EngBF using a series of p-nitrophenyl-alpha-glycosides chemically synthesized by the di-tert-butylsilylene-directed method revealed that both enzymes released Hex/HexNAc beta 1-3GalNAc (Hex = Gal or Glc). EngCP could also release the core 2 trisaccharide Gal beta 1-3(GlcNAc beta 1-6)GalNAc, core 8 disaccharide Gal alpha 1-3GalNAc, and monosaccharide GalNAc. Our results suggest that EngCP possesses broader substrate specificity than EngBF. Actions of the two enzymes on native glycoproteins and cell surface glycoproteins were also investigated.

The Solution Structure of the C-terminal Modular Pair from Clostridium perfringens μ-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.

RNA-dependent lipid remodeling by bacterial multiple peptide resistance factors

Multiple peptide resistance (MprF) virulence factors control cellular permeability to cationic antibiotics by aminoacylating inner membrane lipids. It has been shown previously that one class of MprF can use Lys-tRNA(Lys) to modify phosphatidylglycerol (PG), but the mechanism of recognition and possible role of other MprFs are unknown. Here, we used an in vitro reconstituted lipid aminoacylation system to investigate the two phylogenetically distinct MprF paralogs (MprF1 and MprF2) found in the bacterial pathogen Clostridium perfringens. Although both forms of MprF aminoacylate PG, they do so with different amino acids; MprF1 is specific for Ala-tRNA(Ala), and MprF2 utilizes Lys-tRNA(Lys). This provides a mechanism by which the cell can fine tune the charge of the inner membrane by using the neutral amino acid alanine, potentially providing resistance to a broader range of antibiotics than offered by lysine modification alone. Mutation of tRNA(Ala) and tRNA(Lys) had little effect on either MprF activity, indicating that the aminoacyl moiety is the primary determinant for aminoacyl-tRNA recognition. The lack of discrimination of the tRNA is consistent with the role of MprF as a virulence factor, because species-specific differences in tRNA sequence would not present a barrier to horizontal gene transfer. Taken together, our findings reveal how the MprF proteins provide a potent virulence mechanism by which pathogens can readily acquire resistance to chemically diverse antibiotics.

Antibacterial activity of two Phlomis essential oils against food pathogens

Phlomis species from the Lamiaceae family are widely distributed in Turkey. In this study, the essential oils of Phlomis russeliana (Sims.) Bentham and Phlomis grandiflora H.S. Thompson var. grandiflora collected from North and Southern parts of Turkey, were obtained by hydrodistillation of the aerial parts. The essential oils were subsequently analysed by gas chromatography (GC) and gas chromatography–mass spectrometry (GC/MS). The major constituents of P. russeliana essential oil were identified as sesquiterpenes β-caryophyllene (23%), germacrene-D (15%), and caryophyllene oxide (8%). Analysis of P. grandiflora var. grandiflora oil also showed oxygenated sesquiterpenes such as β-eudesmol (42%) and α-eudesmol (16%) as major constituents. Furthermore, essential oils were tested in vitro against common food borne bacteria such as Aeromonas hydrophila, Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Yersinia enterocolitica, and the anaerobic pathogen Clostridium perfringens using the micro-broth dilution assay. When compared with antimicrobial standards weak to moderate (125 to >1000 μg/ml) minimum inhibitory concentrations (MIC) were observed. The results show that Phlomis essential oils might be an alternative to conventional antimicrobials in various foods.

Conservation and Evolutionary Dynamics of the agr Cell-to-Cell Communication System across Firmicutes

We present evidence that the agr cell-to-cell communication system is present across firmicutes, including the human pathogen Clostridium perfringens. Although we find that the agr system is evolutionarily conserved and that the general functions which it regulates are similar in different species, the individual regulated genes are not the same. This suggests that the regulatory network controlled by agr is dynamic and evolves rapidly.

TcpA, an FtsK/SpoIIIE Homolog, Is Essential for Transfer of the Conjugative Plasmid pCW3 inClostridium perfringens

The conjugative tetracycline resistance plasmid pCW3 is the paradigm conjugative plasmid in the anaerobic gram-positive pathogen Clostridium perfringens. Two closely related FtsK/SpoIIIE homologs, TcpA and TcpB, are encoded on pCW3, which is significant since FtsK domains are found in coupling proteins of gram-negative conjugation systems. To develop an understanding of the mechanism of conjugative transfer in C. perfringens, we determined the role of these proteins in the conjugation process. Mutation and complementation analysis was used to show that the tcpA gene was essential for the conjugative transfer of pCW3 and that the tcpB gene was not required for transfer. Furthermore, complementation of a pCW3DeltatcpA mutant with divergent tcpA homologs provided experimental evidence that all of the known conjugative plasmids from C. perfringens use a similar transfer mechanism. Functional genetic analysis of the TcpA protein established the essential role in conjugative transfer of its Walker A and Walker B ATP-binding motifs and its FtsK-like RAAG motif. It is postulated that TcpA is the essential DNA translocase or coupling protein encoded by pCW3 and as such represents a key component of the unique conjugation process in C. perfringens.

Environmental Survey for Four Pathogenic Bacteria and Closely Related Species Using Phylogenetic and Functional Genes*

Bacterial species with high DNA sequence similarity to pathogens could affect the specificity of assays designed to detect biological threat agents in environmental samples. The natural presence of four pathogenic bacteria, Bacillus anthracis, Clostridium perfringens, Francisella tularensis, and Yersinia pestis and their closely related species, was determined for a large collection of soil and aerosol samples. Polymerase chain reaction (PCR) and gene sequencing were used using group-specific 16S rRNA primers to identify pathogens and related species, and pathogen-specific virulence genes. Close relatives of B. anthracis (B. cereus group species) were detected in 37% of the soils and 25% of the aerosol samples. The B. anthracis protective antigen (pag) gene or a close homolog was detected in 16 of these samples. For the other three pathogen groups, the frequency of detection was much lower, and none of the samples were positive with both the phylogenetic and virulence gene primer sets.

Comparison of chicken gut colonisation by the pathogens Campylobacter jejuni and Clostridium perfringens by real-time quantitative PCR

We compared the colonisation of the chicken gut by the two important pathogens Campylobacter jejuni (frequent food-borne pathogen) and alpha-toxin gene containing Clostridium perfringens (causative agent of necrotic enteritis in chickens) using a new high-throughput automated DNA purification method for microbial biodiversity analyses. The method gave high reproducibility (standard deviation of 1.1 C(T)-values for a universal 16S rDNA real-time PCR), and inhibition was observed in only 0.9% of the individual DNA purifications (n = 753). We analysed 253 randomly collected chicken caecal samples (sampled in 2001 and 2003) from Norwegian chicken flocks by real-time quantitative PCR. Our results showed positive correlation (P = 0.009) in chicken caecal colonisation between C. jejuni and Cl. perfringens. We also found that there was a significant underrepresentation (P = 0.008) of chickens containing high levels of Cl. perfringens and low levels of C. jejuni. This indicates a possible interaction between these bacteria. Potential interaction between pathogens and other bacteria in the gut will certainly be important research fields in the future. As demonstrated here, the development of new tools for high-throughput analyses will be of key importance for these studies.

Helical crystallization on nickel–lipid nanotubes: Perfringolysin O as a model protein

To facilitate purification and subsequent structural studies of recombinant proteins the most widely used genetically encoded tag is the histidine tag (His-tag) which specifically binds to N-nitrilotriacetic-acid-chelated nickel ions. Lipids derivatized with a nickel-chelating head group can be mixed with galactosylceramide glycolipids to prepare lipid nanotubes that bind His-tagged proteins. In this study, we use His-tagged perfringolysin O (PFO), a soluble toxin secreted by the bacterial pathogen Clostridium perfringens, as a model protein to test the utility of nickel–lipid nanotubes as a tool for structural studies of His-tagged proteins. PFO is a member of the cholesterol dependent cytolysin family (CDC) of oligomerizing, pore-forming toxins found in a variety of Gram-positive bacterial pathogens. CDC pores have been difficult to study by X-ray crystallography because they are membrane associated and vary in size. We demonstrate that both a wild-type and a mutant form of PFO form helical arrays on nickel–lipid containing nanotubes. Cryo-electron microscopy and image analysis of the helical arrays were used to reconstruct a 3D density map of wild-type PFO. This study suggests that the use of nickel–lipid nanotubes may offer a general approach for structural studies of recombinant proteins and may provide insights into the molecular interactions of proteins that have a natural affinity for a membrane surface.

Attempts to Isolate Naturally Occurring Campylobacter, Salmonella, and Clostridium perfringens from the Ductus Deferens, Testes, and Ceca of Commercial Broiler Breeder Roosters

Recent studies have shown a significant level of Campylobacter in the semen of midlife and late-life broiler breeder roosters. The present study was conducted to determine if several foodborne pathogens (Campylobacter, Salmonella, and Clostridium perfringens) could be isolated from the ductus deferens, testes, and ceca of 45- to 65-wk-old commercial broiler breeder roosters. Five roosters from each of 3 separate commercial housed breeder farms were transported to the laboratory. An aseptic necropsy was performed to remove the ductus deferens, testes, and ceca without surface contamination from blood and other tissues. All samples were analyzed for each of the 3 previously mentioned bacteria, total aerobic bacteria, and Enterobacteriaceae. None of the 3 foodborne pathogens were isolated from the testes of the 15 commercial roosters. Clostridium perfringens was isolated from 1 of the 15 ductus deferens, whereas no Campylobacter or Salmonella were isolated from this tissue. Campylobacter was cultured from the ceca of all 15 roosters, Clostridium perfringens from 14/15, and Salmonella from 2/15. One-quarter of all commercial broiler breeder's semen samples were found to be contaminated with Campylobacter in a previous study; however, this organism as well as Salmonella and Clostridium perfringens were not found in the reproductive tissues located in the abdominal region. Escherichia coli and Enterococcus faecium were occasionally cultured from the ductus deferens. These data suggest that the contamination of semen by these foodborne pathogens is via fecal or cecal contamination as the semen passes through the cloaca and not from bacterial colonization of the testes and ductus deferens.

Prepore to pore transition of a cholesterol-dependent cytolysin visualized by electron microscopy

Perfringolysin O (PFO), a soluble toxin secreted by the pathogenic Clostridium perfringens, forms large homo-oligomeric pore complexes comprising up to 50 PFO molecules in cholesterol-containing membranes. In this study, electron microscopy (EM) and single-particle image analysis were used to reconstruct two-dimensional (2D) projection maps from images of oligomeric PFO prepore and pore complexes formed on cholesterol-rich lipid layers. The projection maps are characterized by an outer and an inner ring of density peaks. The outer rings of the prepore and pore complexes are very similar; however, the protein densities that make up the inner ring of the pore complex are more intense and discretely resolved than they are for the prepore complex. The change in inner-ring protein density is consistent with a mechanism in which the monomers within the prepore complex make a transition from a partially disordered state to a more ordered transmembrane β-barrel in the pore complex. Finally, the orientation of the monomers within the oligomeric complexes was determined by visualization of streptavidin (SA) molecules bound to biotinylated cysteine-substituted residues predicted to face either the inner or outer surface of the oligomeric pore complex. This study provides an unprecedented view of the conversion of the PFO prepore to pore complex.

In vitro and in vivo antibacterial activity of shrimp chitosan against some intestinal bacteria

The effects of shrimp chitosan with deacetylation degrees (DD) of 50%, 70% and 95% (DD50, DD70, DD95) on the growth of the intestinal bacteria were investigated in vitro in the laboratory media, and in vivo by an oral feeding test using hamsters as the animal model. The antibacterial activities of these chitosan products against one strain of pathogenic Clostridium perfringens and 13 strains of probiotics, including seven strains of Lactobacillus, and six strains of Bifidobacterium were evaluated. In vitro, the antibacterial activities of DD95 and DD70 were much higher than that of DD50. The strains of probiotics were more resistant to chitosan than the pathogen of C. perfringens. The minimal lethal concentration for DD95 against C. perfringens was 250 p.p.m., whereas the survival percentages for most probiotics tested were above 90% for DD95 at 500 p.p.m. The animals were fed on either a control diet, or diets containing powdered chitosan instead of 5% cellulose in the control diet for 4 weeks. The cecal bacterial counts of total aerobes, total anaerobes, lactobacilli, bifidobacteria and clostridia were similar for the control and experimental groups. The reasons for the differences in the antibacterial activity in vitro and in vivo are discussed.

The SKHR motif is required for biological function of the VirR response regulator from Clostridium perfringens.

The response regulator VirR and its cognate sensor histidine kinase, VirS, are responsible for toxin gene regulation in the human pathogen Clostridium perfringens. The C-terminal domain of VirR (VirRc) contains the functional FxRxHrS motif, which is involved in DNA binding and is conserved in many regulatory proteins. VirRc was cloned, purified, and shown by in vivo and in vitro studies to comprise an independent DNA binding domain. Random and site-directed mutagenesis was used to identify further amino acids that were required for the functional integrity of the protein. Random mutagenesis identified a unique residue, Met-172, that was required for biological function. Site-directed mutagenesis of the SKHR motif (amino acids 216 to 219) revealed that these residues were also required for biological activity. Analysis of the mutated proteins indicated that they were unable to bind to the DNA target with the same efficiency as the wild-type protein.

Structural insights into the membrane-anchoring mechanism of a cholesterol-dependent cytolysin.

Perfringolysin O (PFO), a cytolytic toxin secreted by pathogenic Clostridium perfringens, forms large pores in cholesterol-containing membranes. Domain 4 (D4) of the protein interacts first with the membrane and is responsible for cholesterol recognition. By using several independent fluorescence techniques, we have determined the topography of D4 in the membrane-inserted oligomeric form of the toxin. Only the short hydrophobic loops at the tip of the D4 beta-sandwich are exposed to the bilayer interior, whereas the remainder of D4 projects from the membrane surface and is surrounded by water, making little or no contact with adjacent protein monomers in the oligomer. Thus, a limited interaction of D4 with the bilayer core seems to be sufficient to accomplish cholesterol recognition and initial binding of PFO to the membrane. Furthermore, D4 serves as the fulcrum around which extensive structural changes occur during the formation and insertion of the large transmembrane beta-barrel into the bilayer.