RESUMO
Pathogenic Bacillus and clostridial (i.e., Clostridium and Clostridioides) bacteria express a diverse repertoire of effector proteins to promote disease. This includes production of binary toxins, which enter host epithelial cells and seriously damage the intestinal tracts of insects, animals, and humans. In particular, binary toxins form an AB-type complex composed of a catalytic subunit that is toxic (A) and an oligomeric cell-binding and delivery subunit (B), where upon delivery of A into the cytoplasm of the host cell it catalytically ADP-ribosylates actin and rapidly induces host cell death. In this review, binary toxins expressed by Bacillus thuringiensis, Clostridioides difficile, and Clostridium perfringens will be discussed, with particular focus placed upon the structural elucidations of their respective B subunits and how these findings help to deconvolute how toxic enzyme delivery into target host cells is achieved by these deadly bacteria.
Assuntos
Toxinas Bacterianas , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Humanos , Animais , Clostridium perfringens/metabolismo , Bacillus thuringiensis/metabolismo , Clostridioides difficile/metabolismoRESUMO
Clostridium perfringens type A causes gas gangrene, which involves muscle infection. Both alpha toxin (PLC), encoded by the plc gene, and perfringolysin O (PFO), encoded by the pfoA gene, are important when type A strains cause gas gangrene in a mouse model. This study used the differentiated C2C12 muscle cell line to test the hypothesis that one or both of those toxins contributes to gas gangrene pathogenesis by releasing growth nutrients from muscle cells. RT-qPCR analyses showed that the presence of differentiated C2C12 cells induces C. perfringens type A strain ATCC3624 to upregulate plc and pfoA expression, as well as increase expression of several regulatory genes, including virS/R, agrB/D, and eutV/W. The VirS/R two component regulatory system (TCRS) and its coupled Agr-like quorum sensing system, along with the EutV/W TCRS (which regulates expression of genes involved in ethanolamine [EA] utilization), were shown to mediate the C2C12 cell-induced increase in plc and pfoA expression. EA was demonstrated to increase toxin gene expression. ATCC3624 growth increased in the presence of differentiated C2C12 muscle cells and this effect was shown to involve both PFO and PLC. Those membrane-active toxins were each cytotoxic for differentiated C2C12 cells, suggesting they support ATCC3624 growth by releasing nutrients from differentiated C2C12 cells. These findings support a model where, during gas gangrene, increased production of PFO and PLC in the presence of muscle cells causes more damage to those host cells, which release nutrients like EA that are then used to support C. perfringens growth in muscle.
Assuntos
Toxinas Bacterianas , Clostridium perfringens , Gangrena Gasosa , Fosfolipases Tipo C , Clostridium perfringens/genética , Clostridium perfringens/crescimento & desenvolvimento , Clostridium perfringens/metabolismo , Clostridium perfringens/fisiologia , Camundongos , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Linhagem Celular , Gangrena Gasosa/microbiologia , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo , Diferenciação Celular , Células Musculares/microbiologia , Células Musculares/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Percepção de QuorumRESUMO
Clostridium perfringens causes multiple diseases in humans and animals. Its pathogenic effect is supported by a broad and heterogeneous arsenal of toxins and other virulence factors associated with a specific host tropism. Molecular approaches have indicated that most C. perfringens toxins produce membrane pores, leading to osmotic cell disruption and apoptosis. However, identifying mechanisms involved in cell tropism and selective toxicity effects should be studied more. The differential presence and polymorphisms of toxin-encoding genes and genes encoding other virulence factors suggest that molecular mechanisms might exist associated with host preference, receptor binding, and impact on the host; however, this information has not been reviewed in detail. Therefore, this review aims to clarify the current state of knowledge on the structural features and mechanisms of action of the major toxins and virulence factors of C. perfringens and discuss the impact of genetic diversity of toxinotypes in tropism for several hosts.
Assuntos
Toxinas Bacterianas , Infecções por Clostridium , Clostridium perfringens , Fatores de Virulência , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Humanos , Animais , Clostridium perfringens/genética , Clostridium perfringens/patogenicidade , Clostridium perfringens/metabolismo , Infecções por Clostridium/microbiologiaRESUMO
Clostridium perfringens ε-toxin has long been associated with a severe enterotoxaemia of livestock animals, and more recently, was proposed to play a role in the etiology of multiple sclerosis in humans. The remarkable potency of the toxin has intrigued researchers for many decades, who suggested that this indicated an enzymatic mode of action. Recently, there have been major breakthroughs by finding that it is a pore-forming toxin which shows exquisite specificity for cells bearing the myelin and lymphocyte protein (MAL) receptor. This review details the molecular structures of the toxin, the evidence which identifies MAL as the receptor and the possible roles of other cell membrane components in toxin binding. The information on structure and mode of action has allowed the functions of individual amino acids to be investigated and has led to the creation of mutants with reduced toxicity that could serve as vaccines. In spite of this progress, there are still a number of key questions around the mode of action of the toxin which need to be further investigated.
Assuntos
Toxinas Bacterianas , Clostridium perfringens , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Clostridium perfringens/metabolismoRESUMO
Bile acids (BAs) are steroid detergents in bile that contribute to the absorption of fats and fat-soluble vitamins while shaping the gut microbiome because of their antimicrobial properties1-4. Here we identify the enzyme responsible for a mechanism of BA metabolism by the gut microbiota involving amino acid conjugation to the acyl-site of BAs, thus producing a diverse suite of microbially conjugated bile acids (MCBAs). We show that this transformation is mediated by acyltransferase activity of bile salt hydrolase (bile salt hydrolase/transferase, BSH/T). Clostridium perfringens BSH/T rapidly performed acyl transfer when provided various amino acids and taurocholate, glycocholate or cholate, with an optimum at pH 5.3. Amino acid conjugation by C. perfringens BSH/T was diverse, including all proteinaceous amino acids except proline and aspartate. MCBA production was widespread among gut bacteria, with strain-specific amino acid use. Species with similar BSH/T amino acid sequences had similar conjugation profiles and several bsh/t alleles correlated with increased conjugation diversity. Tertiary structure mapping of BSH/T followed by mutagenesis experiments showed that active site structure affects amino acid selectivity. These MCBA products had antimicrobial properties, where greater amino acid hydrophobicity showed greater antimicrobial activity. Inhibitory concentrations of MCBAs reached those measured natively in the mammalian gut. MCBAs fed to mice entered enterohepatic circulation, in which liver and gallbladder concentrations varied depending on the conjugated amino acid. Quantifying MCBAs in human faecal samples showed that they reach concentrations equal to or greater than secondary and primary BAs and were reduced after bariatric surgery, thus supporting MCBAs as a significant component of the BA pool that can be altered by changes in gastrointestinal physiology. In conclusion, the inherent acyltransferase activity of BSH/T greatly diversifies BA chemistry, creating a set of previously underappreciated metabolites with the potential to affect the microbiome and human health.
Assuntos
Aciltransferases , Amidoidrolases , Ácidos e Sais Biliares , Clostridium perfringens , Microbioma Gastrointestinal , Animais , Humanos , Camundongos , Aciltransferases/química , Aciltransferases/metabolismo , Alelos , Amidoidrolases/química , Amidoidrolases/metabolismo , Aminoácidos/metabolismo , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Cirurgia Bariátrica , Ácidos e Sais Biliares/química , Ácidos e Sais Biliares/metabolismo , Domínio Catalítico , Clostridium perfringens/enzimologia , Clostridium perfringens/metabolismo , Fezes/química , Vesícula Biliar/metabolismo , Microbioma Gastrointestinal/fisiologia , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Fígado/metabolismo , Ácido Taurocólico/metabolismoRESUMO
Enterotoxaemia (ET) is a severe disease that affects domestic ruminants, including sheep and goats, and is caused by Clostridium perfringens type B and D strains. The disease is characterized by the production of Epsilon toxin (ETX), which has a significant impact on the farming industry due to its high lethality. The binding of ETX to the host cell receptor is crucial, but still poorly understood. Therefore, the structural features of goat Myelin and lymphocytic (MAL) protein were investigated and defined in this study. We induced the mutations in aromatic amino acid residues of ETX and substituted them with aliphatic residues at domains I and II. Subsequently, protein-protein interactions (PPI) were performed between ETX (wild)-MAL and ETX (mutated)-MAL protein predicting the domain sites of ETX structure. Further, molecular dynamics (MD) simulation studies were performed for both complexes to investigate the dynamic behavior of the proteins. The binding efficiency between 'ETX (wild)-MAL protein' and 'ETX (mutated)-MAL protein complex' interactions were compared and showed that the former had stronger interactions and binding efficiency due to the higher stability of the complex. The MD analysis showed destabilization and higher fluctuations in the PPI of the mutated heterodimeric ETX-MAL complex which is otherwise essential for its functional conformation. Such kind of interactions with mutated functional domains of ligands provided much-needed clarity in understanding the pre-pore complex formation of epsilon toxin with the MAL protein receptor of goats. The findings from this study would provide an impetus for designing a novel vaccine for Enterotoxaemia in goats.Communicated by Ramaswamy H. Sarma.
Assuntos
Toxinas Bacterianas , Clostridium perfringens , Bainha de Mielina , Animais , Aminoácidos/metabolismo , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Enterotoxemia , Cabras , Linfócitos , Mutação , Proteínas da Mielina/genética , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismoRESUMO
Clostridium perfringens (C. perfringens) is a bacterium that causes serious problems in humans and animals such as food poisoning, gas gangrene and infections. C. perfringens has three sialidases (NanH, NanI, NanJ) and inhibition of NanI constitutes an approach in the treatment of C. perfringens since NanI provides the carbohydrate source necessary for the growth of bacteria. In our study, the inhibition effect of some drugs belonging to different drug groups on NanI activity was investigated. Among these drugs, orlistat (0.21±0.05â µM) was determined to have a lower IC50 value than the positive control quercetin (15.58±1.59â µM). It was determined inâ vitro by spectrofluorometric method. Additionally, NanI molecular docking studies with orlistatand quercetin were performed using iGemdock, DockThor and SwissDock. Orlistat (-93.93, -8.649 and -10.03â kcal/mol, respectively) was found to have a higher binding affinity than quercetin (-92.68, -7.491 and -8.70â kcal/mol, respectively), and the results were in line with inâ vitro studies. The results may suggest that orlistat is a molecule with drug potential for C. perfringens because it inhibits the drug target NanI, and that the inhibition efficiency can be increased by studies with orlistat derivatives.
Assuntos
Clostridium perfringens , Neuraminidase , Humanos , Animais , Clostridium perfringens/metabolismo , Orlistate/farmacologia , Orlistate/metabolismo , Simulação de Acoplamento Molecular , Quercetina/farmacologiaRESUMO
Clostridium perfringens iota-toxin is composed of two separate proteins: a binding protein (Ib) that recognizes a host cell receptor and promotes the cellular uptake of a catalytic protein and (Ia) possessing ADP-ribosyltransferase activity that induces actin cytoskeleton disorganization. Ib exhibits the overall structure of bacterial pore-forming toxins (PFTs). Lipolysis-stimulated lipoprotein receptor (LSR) is defined as a host cell receptor for Ib. The binding of Ib to LSR causes an oligomer formation of Ib in lipid rafts of plasma membranes, mediating the entry of Ia into the cytoplasm. Ia induces actin cytoskeleton disruption via the ADP-ribosylation of G-actin and causes cell rounding and death. The binding protein alone disrupts the cell membrane and induces cytotoxicity in sensitive cells. Host cells permeabilized by the pore formation of Ib are repaired by a Ca2+-dependent plasma repair pathway. This review shows that the cellular uptake of iota-toxin utilizes a pathway of plasma membrane repair and that Ib alone induces cytotoxicity.
Assuntos
Actinas , Clostridium perfringens , Animais , Chlorocebus aethiops , Clostridium perfringens/metabolismo , Transporte Biológico , Actinas/metabolismo , Células Vero , ADP Ribose Transferases/químicaRESUMO
Clostridium perfringens enterotoxin (CpE) is a ß-pore forming toxin that disrupts gastrointestinal homeostasis in mammals by binding membrane protein receptors called claudins. Although structures of CpE fragments bound to claudins have been determined, the mechanisms that trigger CpE activation and oligomerization that lead to the formation of cytotoxic ß-pores remain undetermined. Proteolysis of CpE in the gut by trypsin has been shown to play a role in this and subsequent cytotoxicity processes. Here, we report solution structures of full-length and trypsinized CpE using small-angle X-ray scattering (SAXS) and crystal structures of trypsinized CpE and its C-terminal claudin-binding domain (cCpE) using X-ray crystallography. Mass spectrometry and SAXS uncover that removal of the CpE N-terminus by trypsin alters the CpE structure to expose areas that are normally unexposed. Crystal structures of trypsinized CpE and cCpE reveal unique dimer interfaces that could serve as oligomerization sites. Moreover, comparisons of these structures to existing ones predict the functional implications of oligomerization in the contexts of cell receptor binding and ß-pore formation. This study sheds light on trypsin's role in altering CpE structure to activate its function via inducing oligomerization on its path toward cytotoxic ß-pore formation. Its findings can incite new approaches to inhibit CpE-based cytotoxicity with oligomer-disrupting therapeutics.
Assuntos
Clostridium perfringens , Enterotoxinas , Animais , Tripsina , Espalhamento a Baixo Ângulo , Clostridium perfringens/metabolismo , Difração de Raios X , Enterotoxinas/metabolismo , Claudinas/metabolismo , Mamíferos/metabolismoRESUMO
Concurrent with an increase in the human population on the earth, more than ever, the creation of energy and maintenance of health is necessary, and nowadays, various sources of energy supply are being developed. The general global view in this regard is to provide protein and energy from available and cheap sources. Iran is no exception to this general rule, only in the field of ensuring the health of livestock resources every year, about 10 tons of peptone is needed for producing clostridial vaccines. Vermicomposting worms (Esienia fetida) with high protein percentages and rapid reproductions are a suitable source for peptone production. Based on this, the vaccine strain of Clostridium perfringens type D cultivated in two different media contain peptone produced from worms and meat peptone. The growth rate, epsilon toxin (ETX), and alpha toxin (CPA) of Cl. perfringens have been compared in two media. The results showed that the growth rate of bacteria in the worm peptone medium in 48 h was 22% higher than that of the meat peptone. Additionally, the activity of alpha toxin (phospholipase C) was in worm peptone 15% higher than meat peptone during 80 min of measurement. Regarding epsilon toxin lethality, all three mice of the N-worm peptone group died, while all three mice of the meat peptone group survived even 72 h after injection. The average survival time of mice in the N-worm peptone group was 1700 min. Therefore, we suggest the worms' protein is more suitable than industrial meat in peptone production for vicinal propose. To eliminate the need for hydrolyzed protein in the production of vaccines in the future, we suggest an increase in the fields of employment and the development of fertilizer and worm farms in Iran.
Assuntos
Clostridium perfringens , Peptonas , Humanos , Animais , Camundongos , Clostridium perfringens/metabolismo , Peptonas/metabolismo , Carne/microbiologia , Irã (Geográfico)RESUMO
As Clostridium perfringens (C. perfringens) epsilon toxin (ETX) ranks as the third most potent clostridial toxin after botulinum and tetanus toxins, vaccination is necessary for creatures that can be affected by it to be safe from the effects of this toxin. Nowadays, nanostructures are good choices for carriers for biological environments. We aimed to synthesize biomimetic biodegradable nanodevices to enhance the efficiency of the ETX vaccine. For this purpose, poly(lactic-co-glycolic acid) (PLGA) copolymer loaded with purified epsilon protoxin (proETX) to create nanoparticles called nanotoxins (NTs) and then coated by RBC membrane-derived vesicles (RVs) to form epsilon nanotoxoids (RV-NTs). The resulting RV-NTs shaped smooth spherical surfaces with double-layer core/shell structure with an average particle size of 105.9 ± 35.1 nm and encapsulation efficiency of 97.5% ± 0.13%. Compared with NTs, the RV-NTs were more stable for 15 consecutive days. In addition, although both structures showed a long-term cumulative release, the release rates from RV-NTs were slower than NTs during 144 hours. According to the results of cell viability, ETX loading in PLGA and entrapment in the RBC membrane decreased the toxicity of the toxin. The presence of PLGA enhances the uptake of proETX, and the synthesized structures showed no significant lesion after injection. These results demonstrate that NTs and RV-NTs could serve as an effective vaccine platform to deliver ETX for future in vivo assays.
Assuntos
Clostridium perfringens , Nanopartículas , Clostridium perfringens/química , Clostridium perfringens/metabolismo , Biomimética , Membrana Celular/metabolismo , Nanopartículas/toxicidadeRESUMO
OBJECTIVE: Clostridium perfringens causes food poisoning and gas gangrene, a serious wound-associated infection. C. perfringens cells adhere to collagen via fibronectin (Fn). We investigated whether the peptidoglycan hydrolase of C. perfringens, i.e., autolysin (Acp), is implicated in Fn binding to C. perfringens cells. METHODS: This study used recombinant Acp fragments, human Fn and knockout mutants (C. perfringens 13 acp::erm and HN13 ΔfbpC ΔfbpD). Ligand blotting, Western blotting analysis, and complementation tests were performed. The Fn-binding activity of each mutant was evaluated by ELISA. RESULTS: From an Fn-binding assay using recombinant Acp fragments, Fn was found to bind to the catalytic domain of Acp. In mutant cells lacking Acp, Fn binding was significantly decreased, but was restored by the complementation of the acp gene. There are three known kinds of Fn-binding proteins in C. perfringens: FbpC, FbpD, and glyceraldehyde-3-phosphate dehydrogenase. We found no difference in Fn-binding activity between the mutant cells lacking both FbpC and FbpD (SAK3 cells) and the wild-type cells, indicating that these Fn-binding proteins are not involved in Fn binding to C. perfringens cells. CONCLUSIONS: We found that the Acp is an Fn-binding protein that acts as an Fn receptor on the surface of C. perfringens cells.
Assuntos
Clostridium perfringens , Gangrena Gasosa , Humanos , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Integrina alfa5beta1/metabolismo , Ligação Proteica , Proteínas de Transporte/metabolismoRESUMO
Clostridium perfringens (types A and C) can cause several diseases by secreting alpha (CPA) and beta (CPB) exotoxins in the gastrointestinal tract. Although vaccination is the main measure of immunization against C. perfringens, available vaccines have limitations in terms of productivity and safety. Thus, recombinant vaccines are an important, more effective, practical, and safer strategy in the immunization of animals. In this study, we evaluated the immunization of sheep with recombinant Escherichia coli bacterins expressing CPA and CPB complete proteins (co-administered), the immunogenic nontoxic domains rCPA-C247-370 and rCPB-C143-311 co-administered or fused as a bivalent chimera (rCPBcAc). For this, in silico analysis was performed to design rCPBcAc, considering the stability of the mRNA (-278.80 kcal/mol), the degree of antigenicity (0.7557), the epitopes of the B cell ligand, and different physicochemical characteristics. All proteins were expressed in vitro. In vivo, animals vaccinated with the co-administered antigens rCPA + rCPB and rCPA-C+ rCPB-C (200 µg each) had mean CPA and CPB neutralizing antitoxin titers of 4, 10, 4.8, and 14.4 IU/mL, respectively, while those vaccinated with 200 µg of rCPBcAc chimera (approximately 100 µg of each antigen) had titers of <4 and 12 IU/mL of CPA and CPB antitoxins, respectively, 56 days after the administration of the first dose. In addition, the chimera was considered to be immunogenic for inducing antitoxin titers using the half dose. In this study, we presented a new recombinant antigen potentially applicable for vaccines against the CPA and CPB toxins for preventing diseases caused by Clostridium perfringens.
Assuntos
Antitoxinas , Toxinas Bacterianas , Infecções por Clostridium , Animais , Ovinos , Clostridium perfringens/metabolismo , Infecções por Clostridium/prevenção & controle , Infecções por Clostridium/veterinária , Vacinas Bacterianas , Imunização , Vacinação , Vacinas Sintéticas , Anticorpos AntibacterianosRESUMO
Clostridium perfringens epsilon toxin (ETX) is the third most lethal bacterial toxin and has been suggested to be an environmental trigger of multiple sclerosis, an immune-mediated disease of the human central nervous system. However, ETX cytotoxicity on primary human cells has not been investigated. In this article, we demonstrate that ETX preferentially binds to and kills human lymphocytes expressing increased levels of the myelin and lymphocyte protein MAL. Using flow cytometry, ETX binding was determined to be time and dose dependent and was highest for CD4+ cells, followed by CD8+ and then CD19+ cells. Similar results were seen with ETX-induced cytotoxicity. To determine if ETX preference for CD4+ cells was related to MAL expression, MAL gene expression was determined by RT-qPCR. CD4+ cells had the highest amount of Mal gene expression followed by CD8+ and CD19+ cells. These data indicate that primary human cells are susceptible to ETX and support the hypothesis that MAL is a main receptor for ETX. Interestingly, ETX bindings to human lymphocytes suggest that ETX may influence immune response in multiple sclerosis.
Assuntos
Toxinas Bacterianas , Esclerose Múltipla , Humanos , Clostridium perfringens/metabolismo , Linfócitos , Sistema Nervoso Central , Toxinas Bacterianas/metabolismoRESUMO
Clostridium perfringens beta-1 toxin (CPB1) is responsible for necrotizing enteritis and enterotoxemia. However, whether the release of host inflammatory factors caused by CPB1 is related to pyroptosis, an inflammatory form of programmed cell death, has not been reported. A construct expressing recombinant Clostridium perfringens beta-1 toxin (rCPB1) was created, and the cytotoxic activity of the purified rCPB1 toxin was assessed via CCK-8 assay. The rCPB1-induced macrophage pyroptosis by assessing changes to the expression of pyroptosis-related signal molecules and the pyroptosis pathway of macrophages using quantitative real-time PCR, immunoblotting, ELISA, immunofluorescence, and electron microscopic assays. The results showed that the intact rCPB1 protein was purified from an E. coli expression system, which exhibited moderate cytotoxicity on mouse mononuclear macrophage leukemia cells (RAW264.7), normal colon mucosal epithelial cells (NCM460), and human umbilical vein endothelial cells (HUVEC). rCPB1 could induce pyroptosis in macrophages and HUVEC cells, in part through the Caspase-1-dependent pathway. The rCPB1-induced pyroptosis of RAW264.7 cells could be blocked by inflammasome inhibitor MCC950. These results demonstrated that rCPB1 treatment of macrophages promoted the assembly of NLRP3 inflammasomes and activated Caspase 1; the activated Caspase 1 caused gasdermin D to form plasma membrane pores, leading to the release of inflammatory factors IL-18 and IL-1ß, resulting in macrophage pyroptosis. NLRP3 may be a potential therapeutic target for Clostridium perfringes disease. This study provided a novel insight into the pathogenesis of CPB1.
Assuntos
Proteína 3 que Contém Domínio de Pirina da Família NLR , Piroptose , Humanos , Animais , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose/fisiologia , Clostridium perfringens/metabolismo , Caspase 1/metabolismo , Escherichia coli/metabolismo , Inflamassomos/metabolismo , Macrófagos/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Interleucina-1beta/metabolismoRESUMO
OBJECTIVES: Clostridium perfringens epsilon-toxin is considered to be a crucial agent in enterotoxemia in domestic animals. Epsilon-toxin enters host cells via endocytosis and results in the formation of late endosome/lysosome-derived vacuoles. In the present study, we found that acid sphingomyelinase promotes the internalization of epsilon-toxin in MDCK cells. METHODS: We measured the extracellular release of acid sphingomyelinase (ASMase) by epsilon-toxin. We examined the role of ASMase in epsilon-toxin-induced cytotoxicity using selective inhibitors and knockdown of ASMase. Production of ceramide after toxin treatment was determined by immunofluorescence technique. RESULTS: Blocking agents of ASMase and exocytosis of lysosomes inhibited this epsilon-toxin-induced vacuole formation. Lysosomal ASMase was liberated to extracellular space during treatment of the cells with epsilon-toxin in the presence of Ca2+. RNAi-mediated attenuation of ASMase blocked epsilon-toxin-induced vacuolation. Moreover, incubation of MDCK cells with epsilon-toxin led to production of ceramide. The ceramide colocalized with lipid raft-binding cholera toxin subunit B (CTB) in the cell membrane, indicating that conversion of lipid raft associated sphingomyelin to ceramide by ASMase facilitates lesion of MDCK cells and internalization of epsilon-toxin. CONCLUSIONS: Based on the present results, ASMase is required for efficient internalization of epsilon-toxin.
Assuntos
Toxinas Bacterianas , Esfingomielina Fosfodiesterase , Animais , Cães , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Células Madin Darby de Rim Canino , Ceramidas/metabolismo , Clostridium perfringens/metabolismoRESUMO
The Clostridium perfringens (C. perfringen) beta2 (CPB2) toxin produced by C. perfringens type C (CpC) can cause necrotizing enteritis in piglets. Immune system activation in response to inflammation and pathogen infection is aided by long non-coding RNAs (lncRNAs). In our previous work, we revealed the differential expression of the novel lncRNA LNC_001186 in CpC-infected ileum versus healthy piglets. This implied that LNC_001186 may be a regulatory factor essential for CpC infection in piglets. Herein, we analyzed the coding ability, chromosomal location and subcellular localization of LNC_001186 and explored its regulatory role in CPB2 toxin-induced apoptosis of porcine small intestinal epithelial (IPEC-J2) cells. RT-qPCR results indicated that LNC_001186 expression was highly enriched in the intestines of healthy piglets and significantly increased in CpC-infected piglets' ileum tissue and CPB2 toxin-treated IPEC-J2 cells. The total sequence length of LNC_001186 was 1323 bp through RACE assay. CPC and CPAT, two online databases, both confirmed that LNC_001186 had a low coding ability. It was present on pig chromosome 3. Cytoplasmic and nuclear RNA isolation and RNA-FISH assays showed that LNC_001186 was present in the nucleus and cytoplasm of IPEC-J2 cells. Furthermore, six target genes of LNC_001186 were predicted using cis and trans approaches. Meanwhile, we constructed ceRNA regulatory networks with LNC_001186 as the center. Finally, LNC_001186 overexpression inhibited IPEC-J2 cells' apoptosis caused by CPB2 toxin and promoted cell viability. In summary, we determined the role of LNC_001186 in IPEC-J2 cells' apoptosis caused by CPB2 toxin, which assisted us in exploring the molecular mechanism of LNC_001186 in CpC-induced diarrhea in piglets.
Assuntos
Toxinas Bacterianas , RNA Longo não Codificante , Animais , Suínos/genética , RNA Longo não Codificante/genética , Toxinas Bacterianas/genética , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Apoptose/genética , IntestinosRESUMO
Piglet diarrhea caused by Clostridium perfringens (C. perfringens) type C (CpC) seriously endangers the development of the pig production industry. C. perfringens beta2 (CPB2) toxin is a virulent toxin produced by CpC. Long non-coding RNAs (lncRNAs) are key regulators in the immune inflammatory response to bacterial infection. Nevertheless, the functional mechanism of lncRNAs in bacterial piglet diarrhea is unclear. Herein, a novel lncRNA lnc001776 expression was confirmed to be substantially elevated in the ileum tissue of CpC-infected diarrhea piglets and in CPB2 toxin-treated porcine small intestinal epithelial cells (IPEC-J2). lnc001776 knockdown restrained CPB2 toxin-induced apoptosis, inflammatory injury, barrier dysfunction and activation of JNK/NF-kB pathway in IPEC-J2 cells. Additionally, ssc-let-7i-5p was identified as sponge for lnc001776. Overexpression of ssc-let-7i-5p repressed CPB2-induced injury in IPEC-J2 cells. Interleukin 6 (IL-6), a target gene of ssc-let-7i-5p, was enhanced in CPB2 toxin-treated IPEC-J2 cells. Rescue experiments demonstrated that a ssc-let-7i-5p mimic reversed the effect of lnc001776 overexpression on CPB2 toxin-induced IPEC-J2 cell injury and JNK/NF-kB pathway, whereas IL-6 overexpression partially restored the impact of lnc001776. Overall, lnc001776 overexpression exacerbated CPB2 toxin-induced IPEC-J2 cell damage by sponging ssc-let-7i-5p to regulate IL-6 to activate JNK/NF-kB pathway, indicating that lnc001776 could be a key target for piglet resistance to CpC-induced diarrhea.
Assuntos
Toxinas Bacterianas , RNA Longo não Codificante , Animais , Suínos , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Interleucina-6/metabolismo , NF-kappa B/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Toxinas Bacterianas/toxicidade , Toxinas Bacterianas/metabolismo , Células Epiteliais/metabolismo , Diarreia/microbiologiaRESUMO
Inflammasome signaling is a central pillar of innate immunity triggering inflammation and cell death in response to microbes and danger signals. Here, we show that two virulence factors from the human bacterial pathogen Clostridium perfringens are nonredundant activators of the NLRP3 inflammasome in mice and humans. C. perfringens lecithinase (also known as phospolipase C) and C. perfringens perfringolysin O induce distinct mechanisms of activation. Lecithinase enters LAMP1+ vesicular structures and induces lysosomal membrane destabilization. Furthermore, lecithinase induces the release of the inflammasome-dependent cytokines IL-1ß and IL-18, and the induction of cell death independently of the pore-forming proteins gasdermin D, MLKL and the cell death effector protein ninjurin-1 or NINJ1. We also show that lecithinase triggers inflammation via the NLRP3 inflammasome in vivo and that pharmacological blockade of NLRP3 using MCC950 partially prevents lecithinase-induced lethality. Together, these findings reveal that lecithinase activates an alternative pathway to induce inflammation during C. perfringens infection and that this mode of action can be similarly exploited for sensing by a single inflammasome.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Humanos , Camundongos , Animais , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Clostridium perfringens/metabolismo , Fatores de Virulência , Inflamação , Interleucina-1beta/metabolismo , Fatores de Crescimento Neural , Moléculas de Adesão Celular NeuronaisRESUMO
Hemolysis is the process of rupturing erythrocytes (red blood cells) by forming nanopores on their membranes using hemolysins, which then impede membrane permeability. However, the self-assembly process before the state of transmembrane pores and underlying mechanisms of conformational change are not fully understood. In this work, theoretical and experimental evidence of the pre-pore morphology of Clostridium perfringens epsilon toxin (ETX), a typical hemolysin, is provided using in situ atomic force microscopy (AFM) complemented by molecular dynamics (MD) simulations to detect the conformational distribution of different states in Mica. The AFM suggests that the ETX pore is formed in two stages: ETX monomers first attach to the membrane and form a pre-pore in no special conditions required, which then undergo a conformational change to form a transmembrane pore at temperatures above the critical point in the presence of receptors. The authors' MD simulations reveal that initial nucleation occurs when specific amino acids adsorb to negatively charged mica cavities. This work fills the knowledge gap in understanding the early stage of hemolysis and the oligomerization of hemolysins. Moreover, the newly identified pre-pore of ETX holds promise as a candidate for nanopore applications.