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1.
Shokuhin Eiseigaku Zasshi ; 61(4): 138-142, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-33012768

RESUMO

Staphylococcus aureus food poisoning is caused by the intoxication of staphylococcal enterotoxin (SE) produced in foods. Staphylococcal food poisoning is mostly due to staphylococcal enterotoxin type A (SEA) among SEs. There have been many studies on the growth and SEA production of S. aureus in various foods, but few studies in bread. Thus, the SEA production by S. aureus in dough during fermentation and the SEA inactivation in dough during baking were studied in the normal production processes of bread in this study. No growth of S. aureus or SEA production in dough, whose total weight was about 470 g, was observed during the fermentation at 25 and 35℃ for four hr, suggesting that the risk of SEA production in dough during fermentation under these conditions would be negligible. Any SEA injected at 6.0 and 0.56 ng/g in dough could not be detected after 20 and 10 min of baking at 200℃, respectively. These results showed that the baking process, which was completed in 25 min, was enough to inactivate SEA at those doses of SEA in the dough. The results on the production and inactivation of SEA in dough during the production processes in this study would be useful information on microbiological food safety of bread making.


Assuntos
Pão , Enterotoxinas , Viabilidade Microbiana , Staphylococcus aureus , Pão/microbiologia , Enterotoxinas/metabolismo , Temperatura Alta , Humanos , Intoxicação Alimentar Estafilocócica/microbiologia , Intoxicação Alimentar Estafilocócica/prevenção & controle , Staphylococcus aureus/metabolismo
2.
PLoS One ; 15(9): e0238153, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32998160

RESUMO

Bacillus cereus is a foodborne pathogen causing emesis and diarrhea in those affected. It is assumed that the non-hemolytic enterotoxin (Nhe) plays a key role in B. cereus induced diarrhea. The ability to trace Nhe activity is important for food safety. While assays such as PCR and ELISA exist to detect Nhe, those methods cannot differentiate between active and inactive forms of Nhe. The existing rabbit ileal loop bioassay used to detect Nhe activity is ethically disfavored because it uses live experimental animals. Here we present a custom built low-cost CCD based luminometer and applied it in conjunction with a cell-based assay using Vero cells transduced to express the luciferase enzyme. The activity of Nhe was measured as its ability to inhibit synthesis of luciferase as quantified by reduction of light emission by the luciferase reaction. Emitted light intensity was observed to be inversely proportional to Nhe concentration over a range of 7 ng/ml to 125 ng/ml, with a limit of detection of 7 ng/ml Nhe.


Assuntos
Enterotoxinas/metabolismo , Medições Luminescentes , Animais , Biocatálise , Chlorocebus aethiops , Células HEK293 , Humanos , Luciferases/genética , Luciferases/metabolismo , Células Vero
3.
Int J Food Microbiol ; 334: 108816, 2020 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-32835996

RESUMO

Bee-pollen is a functional food sold for human and animal consumption but also is a favorable microhabitat for many spore-forming bacteria. Among them, Bacillus cereus can produce several toxins and other virulence factors, causing an emetic or diarrheal syndrome after ingestion. The study involved 36 bee-pollen samples obtained from different sampling points throughout the production process (collecting, freezing, drying, and cleaning) in Argentina. Fifty isolates of B. cereus yielded 24 different fingerprint patterns with BOX and ERIC primers. Only three fingerprint patterns were maintained throughout the production process. In contrast, others were lost or incorporated during the different steps, suggesting that cross-contamination occurred as shown by differences in fingerprint patterns after freezing, drying, and cleaning steps compared to the initial collection step. Genes encoding for cereulide (ces), cytotoxin K (cytK), sphingomyelinase (sph), the components of hemolysin BL (hblA, hblB, hblC, hblD) and non-hemolytic complex (nheAB) were studied. All the isolates displayed one or more enterotoxin genes. The most frequent virulence genes detected belong to the HBL complex, being the most abundant hblA (98%), followed by hblD (64%), hblB (54%), and hblC (32%), respectively. Ten strains (20%), present at all sampling points, carried all the subunits of the HBL complex. The non-hemolytic enterotoxic complex (nheAB) was found in 48 strains (96%), while seven strains (14%) present at all sampling points showed the amplification product for sphingomyelinase (sph). One cereulide-producer was isolated at the cleaning step; this strain contained all the components for the hemolytic enterotoxin complex HBL, the NHE complex, and cytotoxin K related to the foodborne diarrhoeal syndrome. In total, 11 different virulence patterns were observed, and also a correlation between rep-fingerprint and virulence patterns. The results suggest that bee-pollen can be contaminated at any point in the production process with potential enterotoxic B. cereus strains, emphasizing the importance of hygienic processing.


Assuntos
Bacillus cereus/patogenicidade , Abelhas , Enterotoxinas/genética , Microbiologia de Alimentos , Pólen , Animais , Argentina , Bacillus cereus/genética , Bacillus cereus/isolamento & purificação , Enterotoxinas/metabolismo , Manipulação de Alimentos , Pólen/microbiologia , Pólen/toxicidade , Virulência/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
4.
J Int Med Res ; 48(8): 300060520933810, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32776805

RESUMO

The sudden outbreak of severe acute respiratory syndrome coronavirus 2 pneumonia posed a significant challenge to medical professionals because treatment of critically ill patients requires the efforts of a multidisciplinary team. To highlight this principle, we examined acute kidney injury (AKI) in IgA-dominant infection-associated glomerulonephritis (GN) and menstrual toxic shock syndrome (mTSS). Both GN and mTSS are rare diseases caused by staphylococcal infection, and renal function is frequently impaired. The resulting AKIs are disparate pathological entities driven by distinct immune mechanisms. We begin by describing the case of a diabetic man with pyopneumothorax following methicillin-resistant Staphylococcus aureus (MRSA). He had endocapillary proliferative GN with in situ IgA-dominant immune-complex formation in the mesangium accompanied by complement C3 deposition in the glomerular capillary wall. By contrast, acute tubular necrosis was observed in a case of mTSS; the patient's immune response was stimulated differently by MRSA enterotoxin and exotoxin resulting in aberrant IgA deposition, complement activation, and insufficient antibody production. As a multidisciplinary communication covering the fields of nephrology, immunology, and pathology, this report may help clinicians to understand these distinct renal lesions and make optimal therapeutic decisions expeditiously.


Assuntos
Lesão Renal Aguda/patologia , Glomerulonefrite por IGA/patologia , Imunoglobulina A/imunologia , Distúrbios Menstruais/patologia , Choque Séptico/patologia , Infecções Estafilocócicas/patologia , Lesão Renal Aguda/microbiologia , Adolescente , Betacoronavirus , Ativação do Complemento/imunologia , Infecções por Coronavirus/patologia , Enterotoxinas/metabolismo , Feminino , Glomerulonefrite por IGA/microbiologia , Humanos , Rim/patologia , Masculino , Distúrbios Menstruais/microbiologia , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/patologia , Pneumotórax/microbiologia , Pneumotórax/patologia , Choque Séptico/microbiologia
5.
Lett Appl Microbiol ; 71(4): 394-399, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32557652

RESUMO

The aims of the present study were to examine the occurrence of Staphylococcus spp. in the tonsils of slaughtered pigs in a regional slaughterhouse in Greece, the antibiotic resistance of the Staphylococcus spp. isolates, and the enteroxigenicity of the S. aureus isolates. Staphylococcus spp. were isolated in 70 (48·61%) out of the total 144 tonsil samples. The predominant species was S. aureus in coagulase-positive staphylococci (CoPS), while the predominant species were Staphylococcus epidermidis and Staphylococcus saprophyticus in the coagulase-negative staphylococci (CoNS). Staphylococcus spp. isolates presented high antibiotic resistance frequencies to tetracycline (97·1%) or clindamycin (80·0%) and low antibiotic resistance frequencies to fusidic acid (14·3%). No methicillin-resistant S. aureus (MRSA) strains were identified, and all Staphylococcus spp. isolates were susceptible to vancomycin. Among the 26 S. aureus isolates, 21 (80·76%) possessed staphylococcal enterotoxin genes with seven different enterotoxin gene profiles. The predominant enterotoxin profile was seg, sei and sej with seven S. aureus isolates. The occurrence of multidrug resistant Staphylococcus spp. in pig tonsils indicate public health risk to pork consumers and handlers in developing antimicrobial resistance.


Assuntos
Farmacorresistência Bacteriana , Enterotoxinas/metabolismo , Tonsila Palatina/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus/isolamento & purificação , Doenças dos Suínos/microbiologia , Matadouros/estatística & dados numéricos , Animais , Antibacterianos/farmacologia , Clindamicina/farmacologia , Grécia/epidemiologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus/classificação , Staphylococcus/efeitos dos fármacos , Staphylococcus/genética , Suínos , Doenças dos Suínos/epidemiologia
6.
Am J Physiol Gastrointest Liver Physiol ; 318(5): G870-G888, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32223302

RESUMO

Clostridioides difficile is an important nosocomial pathogen that produces toxins to cause life-threatening diarrhea and colitis. Toxins bind to epithelial receptors and promote the collapse of the actin cytoskeleton. C. difficile toxin activity is commonly studied in cancer-derived and immortalized cell lines. However, the biological relevance of these models is limited. Moreover, no model is available for examining C. difficile-induced enteritis, an understudied health problem. We hypothesized that human intestinal enteroids (HIEs) express toxin receptors and provide a new model to dissect C. difficile cytotoxicity in the small intestine. We generated biopsy-derived jejunal HIE and Vero cells, which stably express LifeAct-Ruby, a fluorescent label of F-actin, to monitor actin cytoskeleton rearrangement by live-cell microscopy. Imaging analysis revealed that toxins from pathogenic C. difficile strains elicited cell rounding in a strain-dependent manner, and HIEs were tenfold more sensitive to toxin A (TcdA) than toxin B (TcdB). By quantitative PCR, we paradoxically found that HIEs expressed greater quantities of toxin receptor mRNA and yet exhibited decreased sensitivity to toxins when compared with traditionally used cell lines. We reasoned that these differences may be explained by components, such as mucins, that are present in HIEs cultures, that are absent in immortalized cell lines. Addition of human-derived mucin 2 (MUC2) to Vero cells delayed cell rounding, indicating that mucus serves as a barrier to toxin-receptor binding. This work highlights that investigation of C. difficile infection in that HIEs can provide important insights into the intricate interactions between toxins and the human intestinal epithelium.NEW & NOTEWORTHY In this article, we developed a novel model of Clostridioides difficile-induced enteritis using jejunal-derived human intestinal enteroids (HIEs) transduced with fluorescently tagged F-actin. Using live-imaging, we identified that jejunal HIEs express high levels of TcdA and CDT receptors, are more sensitive to TcdA than TcdB, and secrete mucus, which delays toxin-epithelial interactions. This work also optimizes optically clear C. difficile-conditioned media suitable for live-cell imaging.


Assuntos
/patogenicidade , Infecções por Clostridium/microbiologia , Enterite/microbiologia , Jejuno/microbiologia , ADP Ribose Transferases/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/microbiologia , Citoesqueleto de Actina/ultraestrutura , Animais , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Forma Celular , Chlorocebus aethiops , Infecções por Clostridium/metabolismo , Infecções por Clostridium/patologia , Enterite/metabolismo , Enterite/patologia , Enterotoxinas/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Jejuno/metabolismo , Jejuno/ultraestrutura , Mucina-2/metabolismo , Organoides , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Fatores de Tempo , Células Vero , Virulência
7.
J Dairy Sci ; 103(5): 4100-4108, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32197850

RESUMO

Staphylococcus aureus is one of the main causative agents of food poisoning. This bacterium is an important component of cheese microbiota and plays an important role in foodborne diseases. Another important component of the microbiota is the lactic acid bacterium, which actively participates in processes that define the physicochemical, sensorial, and microbiological features of cheese. Of the various microbiological interactions in cheese, the interaction between lactic acid bacteria and Staph. aureus is most relevant. To this end, we evaluated the viability of Staph. aureus strains and the expression of their enterotoxins in cheeses produced experimentally, using Weissella paramesenteroides GIR16L4 or Lactobacillus rhamnosus D1 or both as starter cultures. Over 7 d, we observed that the presence of lactic acid bacteria did not impair Staph. aureus growth. However, via qPCR we observed a change in the gene expression of staphylococcal enterotoxins, suggesting that molecular communication exists between Staph. aureus strains and lactic acid bacteria in cheese.


Assuntos
Toxinas Bacterianas/metabolismo , Queijo/microbiologia , Enterotoxinas/metabolismo , Lactobacillus rhamnosus/crescimento & desenvolvimento , Staphylococcus aureus/crescimento & desenvolvimento , Superantígenos/metabolismo , Weissella/crescimento & desenvolvimento , Animais , Toxinas Bacterianas/genética , Queijo/análise , Enterotoxinas/genética , Microbiologia de Alimentos , Lactobacillales/metabolismo , Lactobacillus rhamnosus/metabolismo , Leite , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Superantígenos/genética , Transcriptoma , Weissella/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(11): 6139-6144, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123082

RESUMO

Clostridioides difficile is a Gram-positive, pathogenic bacterium and a prominent cause of hospital-acquired diarrhea in the United States. The symptoms of C. difficile infection are caused by the activity of three large toxins known as toxin A (TcdA), toxin B (TcdB), and the C. difficile transferase toxin (CDT). Reported here is a 3.8-Å cryo-electron microscopy (cryo-EM) structure of CDT, a bipartite toxin comprised of the proteins CDTa and CDTb. We observe a single molecule of CDTa bound to a CDTb heptamer. The formation of the CDT complex relies on the interaction of an N-terminal adaptor and pseudoenzyme domain of CDTa with six subunits of the CDTb heptamer. CDTb is observed in a preinsertion state, a conformation observed in the transition of prepore to ß-barrel pore, although we also observe a single bound CDTa in the prepore and ß-barrel conformations of CDTb. The binding interaction appears to prime CDTa for translocation as the adaptor subdomain enters the lumen of the preinsertion state channel. These structural observations advance the understanding of how a single protein, CDTb, can mediate the delivery of a large enzyme, CDTa, into the cytosol of mammalian cells.


Assuntos
Toxinas Bacterianas/metabolismo , Enterotoxinas/metabolismo , Transferases/ultraestrutura , Microscopia Crioeletrônica , Conformação Proteica em Folha beta , Multimerização Proteica , Transferases/metabolismo
9.
J Dairy Sci ; 103(4): 3076-3081, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32037175

RESUMO

Our objective was to assess the enterotoxigenic potential of coagulase-negative staphylococci (CNS) isolated from bovine milk in Poland. We analyzed CNS isolates collected from 133 bovine milk batches from dairy farms in the Western Pomerania district during 2 milking seasons. A total of 163 isolates were screened by multiplex/duplex PCR for the presence of 18 of 25 enterotoxin genes identified so far in Staphylococcus aureus. The CNS strains presumed to be potentially enterotoxigenic were identified at the species level based on MALDI-TOF mass spectrometry analysis. Based on the presence of an amplicon matching D, G, or O enterotoxin genes, we initially identified 32 of the 163 CNS isolates tested as potentially enterotoxigenic. However, only 8 of these strains were confirmed as such. All 8 of these CNS strains, identified as Staphylococcus haemolyticus, harbored the seg genes, a prerequisite for enterotoxin G production, but so far not connected with staphylococcal foodborne poisoning cases. None of the CNS bovine milk isolates tested was a potential producer of classical A to E staphylococcal enterotoxins. Results of our surveys revealed a low prevalence of enterotoxigenic CNS among the milk isolates from dairy farms in the Western Pomerania district, Poland, suggesting that they pose only a mild health risk in milk. In our opinion, confirmed formation of nonspecific amplicons leading to false-positive results excludes multiplex/duplex PCR as the sole method for assessing the enterotoxigenic potential of CNS.


Assuntos
Enterotoxinas/metabolismo , Leite/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus/genética , Animais , Bovinos , Enterotoxinas/genética , Enterotoxinas/isolamento & purificação , Feminino , Polônia/epidemiologia , Reação em Cadeia da Polimerase/veterinária , Infecções Estafilocócicas/microbiologia , Staphylococcus/isolamento & purificação , Staphylococcus/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação
10.
J Med Microbiol ; 69(2): 290-297, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32004137

RESUMO

Introduction. Staphylococcus aureus is a recognised cause of foodborne intoxication and antibiotic-associated diarrhoea (AAD), which are both mediated by staphylococcal enterotoxins. However, unlike foodborne intoxication, AAD appears to require infection of the host. While S. aureus intoxication is widely studied, little is known about S. aureus pathogenesis in the context of gastrointestinal infection.Aim. To develop a mouse model of S. aureus gastrointestinal infection.Methodology. An established AAD mouse model was adapted for S. aureus infection, and damage observed via histopathological analysis and immunostaining of intestinal tissues.Results. Various strains colonised the mouse model, and analysis showed that although clinical signs of disease were not seen, S. aureus infection induced damage in the small intestine, disrupting host structures essential for epithelial integrity. Studies using a staphylococcal enterotoxin B mutant showed that this toxin may contribute to damage during gastrointestinal infection.Conclusion. This work presents a new mouse model of S. aureus gastrointestinal infection, while also providing insight into the pathogenesis of S. aureus in the gut.


Assuntos
Intestino Delgado/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia , Animais , Modelos Animais de Doenças , Enterotoxinas/genética , Enterotoxinas/metabolismo , Enterotoxinas/toxicidade , Fezes/microbiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Staphylococcus aureus/genética
11.
Curr Pharm Biotechnol ; 21(10): 973-979, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32101119

RESUMO

BACKGROUND: Heat-Labile enterotoxin B subunit (LTB) produced by Escherichia coli, a non-toxic protein subunit with potential biological properties, is a powerful mucosal and parenteral adjuvant which can induce a strong immune response against co-administered antigens. OBJECTIVE: In the present study, LTB protein, encoded by the optimized ltb (also known synthetic ltb, s-ltb) gene in centella plant (Centella asiatica) for use as an antigen, has been discussed. METHODS: The s-ltb gene was cloned into a plant expression vector, pMYO51, adjacent to the CaMV 35S promoter and was then introduced into centella plant by biolistic transformation. PCR amplification was conducted to determine the presence of s-ltb gene in the transgenic centella plant. The expression of s-ltb gene was analyzed by immunoblotting and quantified by ELISA. In vitro activity of LTB protein was determined by GM1-ELISA. RESULTS: PCR amplification has found seven transgenic centella individuals. However, only five of them produced LTB protein. ELISA analysis showed that the highest amount of LTB protein detected in transgenic centella leaves was about 0.8% of the total soluble protein. GM1-ELISA assay indicated that plant LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers. CONCLUSION: The s-ltb gene that was successfully transformed into centella plants by the biolistic method has produced a relatively high amount of plant LTB protein in the pentameric quaternary structure that has GM1-ganglioside binding affinity, a receptor on the intestinal epithelial membrane.


Assuntos
Toxinas Bacterianas/genética , Biolística/métodos , Centella/genética , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Plantas Geneticamente Modificadas/genética , Animais , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/metabolismo , Centella/metabolismo , Enterotoxinas/química , Enterotoxinas/imunologia , Enterotoxinas/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/imunologia , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Temperatura Alta , Plantas Geneticamente Modificadas/metabolismo , Triterpenos
12.
Sci Rep ; 10(1): 2887, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32076011

RESUMO

Bacillus cereus is increasingly recognized as an opportunistic pathogen causing local and systemic infections. The causative strains typically produce three pore-forming enterotoxins. This study focusses on the tripartite non-hemolytic enterotoxin (Nhe). Until today, studies have tried to elucidate the structure, complex formation and cell binding mechanisms of the tripartite Nhe toxin. Here, we demonstrate the synthesis of the functional tripartite Nhe toxin using eukaryotic cell-free systems. Single subunits, combinations of two Nhe subunits as well as the complete tripartite toxin were tested. Functional activity was determined by hemolytic activity on sheep blood agar plates, planar lipid bilayer measurements as well as cell viability assessment using the MTT assay. Our results demonstrate that cell-free protein synthesis based on translationally active eukaryotic lysates is a platform technology for the fast and efficient synthesis of functionally active, multicomponent toxins.


Assuntos
Bacillus cereus/metabolismo , Enterotoxinas/metabolismo , Mamíferos/metabolismo , Animais , Células CHO , Células CACO-2 , Morte Celular , Sistema Livre de Células , Cricetulus , Humanos , Bicamadas Lipídicas/metabolismo , Biossíntese de Proteínas , Ovinos
13.
Proc Natl Acad Sci U S A ; 117(2): 1049-1058, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31896582

RESUMO

Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For AsymCDTb, a Ca2+ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile.


Assuntos
ADP Ribose Transferases/química , ADP Ribose Transferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Enterotoxinas/química , Enterotoxinas/metabolismo , ADP Ribose Transferases/genética , Animais , Proteínas de Bactérias/genética , Sítios de Ligação , Fenômenos Biofísicos , Chlorocebus aethiops , Microscopia Crioeletrônica , Cristalografia por Raios X , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Domínios Proteicos , Células Vero
14.
FASEB J ; 34(2): 2198-2212, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31907988

RESUMO

Clostridioides difficile (formerly Clostridium difficile; C difficile), the leading cause of nosocomial antibiotic-associated colitis and diarrhea in the industrialized world, triggers colonic disease through the release two toxins, toxin A (TcdA) and toxin B (TcdB), glucosyltransferases that modulate monomeric G-protein function and alter cytoskeletal function. The initial degree of the host immune response to C difficile and its pathogenic toxins is a common indicator of disease severity and infection recurrence. Thus, targeting the intestinal inflammatory response during infection could significantly decrease disease morbidity and mortality. In the current study, we sought to interrogate the influence of the pregnane X receptor (PXR), a modulator of xenobiotic and detoxification responses, which can sense and respond to microbial metabolites and modulates inflammatory activity, during exposure to TcdA and TcdB. Following intrarectal exposure to TcdA/B, PXR-deficient mice (Nr1i2-/- ) exhibited reduced survival, an effect that was associated with increased levels of innate immune cell influx. This exacerbated response was associated with a twofold increase in the expression of Tlr4. Furthermore, while broad-spectrum antibiotic treatment (to deplete the intestinal microbiota) did not alter the responses in Nr1i2-/- mice, blocking TLR4 signaling significantly reduced TcdA/B-induced disease severity and immune responses in these mice. Lastly, to assess the therapeutic potential of targeting the PXR, we activated the PXR with pregnenolone 16α-carbonitrile (PCN) in wild-type mice, which greatly reduced the severity of TcdA/B-induced damage and intestinal inflammation. Taken together, these data suggest that the PXR plays a role in the host's response to TcdA/B and may provide a novel target to dampen the inflammatory tissue damage in C difficile infections.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Enterocolite Pseudomembranosa/metabolismo , Enterotoxinas/metabolismo , Receptor de Pregnano X/metabolismo , Transdução de Sinais , Animais , /patogenicidade , Enterocolite Pseudomembranosa/genética , Enterocolite Pseudomembranosa/patologia , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Knockout , Receptor de Pregnano X/genética , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
16.
mBio ; 10(6)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848291

RESUMO

Clostridium perfringens type F strains cause gastrointestinal disease when they produce a pore-forming toxin named C. perfringens enterotoxin (CPE). In human enterocyte-like Caco-2 cells, low CPE concentrations cause caspase-3-dependent apoptosis, while high CPE concentrations cause necrosis. Since necrosis or apoptosis sometimes involves receptor-interacting serine/threonine-protein kinase-1 or 3 (RIP1 or RIP3), this study examined whether those kinases are important for CPE-induced apoptosis or necrosis. Highly specific RIP1 or RIP3 inhibitors reduced both CPE-induced apoptosis and necrosis in Caco-2 cells. Those findings suggested that the form of necrosis induced by treating Caco-2 cells with high CPE concentrations involves necroptosis, which was confirmed when high, but not low, CPE concentrations were shown to induce oligomerization of mixed-lineage kinase domain-like pseudokinase (MLKL), a key late step in necroptosis. Furthermore, an MLKL oligomerization inhibitor reduced cell death caused by high, but not low, CPE concentrations. Supporting RIP1 and RIP3 involvement in CPE-induced necroptosis, inhibitors of those kinases also reduced MLKL oligomerization during treatment with high CPE concentrations. Calpain inhibitors similarly blocked MLKL oligomerization induced by high CPE concentrations, implicating calpain activation as a key intermediate in initiating CPE-induced necroptosis. In two other CPE-sensitive cell lines, i.e., Vero cells and human enterocyte-like T84 cells, low CPE concentrations also caused primarily apoptosis/late apoptosis, while high CPE concentrations mainly induced necroptosis. Collectively, these results establish that high, but not low, CPE concentrations cause necroptosis and suggest that RIP1, RIP3, MLKL, or calpain inhibitors can be explored as potential therapeutics against CPE effects in vivo IMPORTANCE C. perfringens type F strains are a common cause of food poisoning and antibiotic-associated diarrhea. Type F strain virulence requires production of C. perfringens enterotoxin (CPE). In Caco-2 cells, high CPE concentrations cause necrosis while low enterotoxin concentrations induce apoptosis. The current study determined that receptor-interacting serine/threonine-protein kinases 1 and 3 are involved in both CPE-induced apoptosis and necrosis in Caco-2 cells, while mixed-lineage kinase domain-like pseudokinase (MLKL) oligomerization is involved in CPE-induced necrosis, thereby indicating that this form of CPE-induced cell death involves necroptosis. High CPE concentrations also caused necroptosis in T84 and Vero cells. Calpain activation was identified as a key intermediate for CPE-induced necroptosis. These results suggest inhibitors of RIP1, RIP3, MLKL oligomerization, or calpain are useful therapeutics against CPE-mediated diseases.


Assuntos
Infecções por Clostridium/metabolismo , Infecções por Clostridium/microbiologia , Clostridium perfringens/fisiologia , Enterotoxinas/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Apoptose , Células CACO-2 , Chlorocebus aethiops , Interações Hospedeiro-Patógeno , Humanos , Modelos Biológicos , Proteínas Quinases/química , Multimerização Proteica , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Células Vero
17.
Artigo em Inglês | MEDLINE | ID: mdl-31681620

RESUMO

Pet and EspC are toxins secreted by enteroaggregative (EAEC) and enteropathogenic (EPEC) diarrheagenic Escherichia coli pathotypes, respectively. Both toxins are members of the Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) family. Pet and EspC are important virulence factors that produce cytotoxic and enterotoxic effects on enterocytes. Here, we evaluated the effect of curcumin, a polyphenolic compound obtained from the rhizomes of Curcuma longa L. (Zingiberaceae) on the secretion and cytotoxic effects of Pet and EspC proteins. We found that curcumin prevents Pet and EspC secretion without affecting bacterial growth or the expression of pet and espC. Our results show that curcumin affects the release of these SPATEs from the translocation domain, thereby affecting the pathogenesis of EAEC and EPEC. Curcumin-treated EAEC and EPEC did not induce significant cell damage like the ability to disrupt the actin cytoskeleton, without affecting their characteristic adherence patterns on epithelial cells. A molecular model of docking predicted that curcumin interacts with the determinant residues Asp1018-Asp1019 and Asp1029-Asp1030 of the translocation domain required for the release of Pet and EspC, respectively. Consequently, curcumin blocks Pet and EspC cytotoxicity on epithelial cells by preventing their release from the outer membrane.


Assuntos
Membrana Externa Bacteriana/metabolismo , Toxinas Bacterianas/metabolismo , Curcumina/farmacologia , Escherichia coli Enteropatogênica/efeitos dos fármacos , Escherichia coli Enteropatogênica/fisiologia , Enterotoxinas/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Serina Endopeptidases/metabolismo , Toxinas Bacterianas/química , Sítios de Ligação , Curcumina/química , Citoesqueleto/metabolismo , Enterotoxinas/química , Proteínas de Escherichia coli/química , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Proteólise , Serina Endopeptidases/química , Relação Estrutura-Atividade
18.
Artigo em Inglês | MEDLINE | ID: mdl-31681632

RESUMO

Clostridium difficile is a Gram-positive, spore-forming bacterium, and major cause of nosocomial diarrhea. Related studies have identified numerous factors that influence virulence traits such as the production of the two primary toxins, toxin A (TcdA) and toxin B (TcdB), as well as sporulation, motility, and biofilm formation. However, multiple putative transcriptional regulators are reportedly encoded in the genome, and additional factors are likely involved in virulence regulation. Although the leucine-responsive regulatory protein (Lrp) has been studied extensively in Gram-negative bacteria, little is known about its function in Gram-positive bacteria, although homologs have been identified in the genome. This study revealed that disruption of the lone lrp homolog in C. difficile decelerated growth under nutrient-limiting conditions, increased TcdA and TcdB production. Lrp was also found to negatively regulate sporulation while positively regulate swimming motility in strain R20291, but not in strain 630. The C. difficile Lrp appeared to function through transcriptional repression or activation. In addition, the lrp mutant was relatively virulent in a mouse model of infection. The results of this study collectively demonstrated that Lrp has broad regulatory function in C. difficile toxin expression, sporulation, motility, and pathogenesis.


Assuntos
Toxinas Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Proteína Reguladora de Resposta a Leucina/metabolismo , Esporos Bacterianos , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Biofilmes , Linhagem Celular , Chlorocebus aethiops , Infecções por Clostridium/microbiologia , Modelos Animais de Doenças , Enterotoxinas/genética , Enterotoxinas/metabolismo , Humanos , Proteína Reguladora de Resposta a Leucina/química , Proteína Reguladora de Resposta a Leucina/genética , Masculino , Camundongos , Mutação , Transcrição Genética , Células Vero
19.
BMC Infect Dis ; 19(1): 961, 2019 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-31711425

RESUMO

BACKGROUND: Clostridioides difficile is considered the main pathogen responsible for hospital-acquired infections. This prospective study determined the prevalence, molecular epidemiological characteristics, and risk factors for C. difficile infection (CDI) and C. difficile colonization (CDC) among patients in the intensive care unit (ICU) of a large-scale tertiary hospital in China, with the aim of providing strategies for efficient CDI and CDC prevention and control. METHODS: Stool samples were collected and anaerobically cultured for C. difficile detection. The identified isolates were examined for toxin genes and subjected to multilocus sequence typing. Patients were classified into CDI, CDC, and control groups, and their medical records were analyzed to determine the risk factors for CDI and CDC. RESULTS: Of the 800 patients included in the study, 33 (4.12%) and 25 (3.12%) were identified to have CDI and CDC, respectively. Associations with CDI were found for fever (OR = 13.993), metabolic disorder (OR = 7.972), and treatment with fluoroquinolone (OR = 42.696) or combined antibiotics (OR = 2.856). CDC patients were characterized by prolonged hospital stay (OR = 1.137), increased number of comorbidities (OR = 36.509), respiratory diseases (OR = 0.043), and treatment with vancomycin (OR = 18.168). Notably, treatment with metronidazole was found to be a protective factor in both groups (CDI: OR = 0.042; CDC: OR = 0.013). Eighteen sequence types (STs) were identified. In the CDI group, the isolated strains were predominantly toxin A and toxin B positive (A + B+) and the epidemic clone was genotype ST2. In the CDC group, the dominant strains were A + B+ and the epidemic clone was ST81. CONCLUSIONS: The prevalences of CDC and CDI in our ICU were relatively high, suggesting the importance of routine screening for acquisition of C. difficile. Future prevention and treatment strategies for CDC and CDI should consider hospital stay, enteral nutrition, underlying comorbidities, and use of combined antibiotics. Moreover, metronidazole may be a protective factor for both CDI and CDC, and could be used empirically.


Assuntos
/isolamento & purificação , Infecções por Clostridium/epidemiologia , Unidades de Terapia Intensiva , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , China/epidemiologia , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/prevenção & controle , Comorbidade , Enterotoxinas/metabolismo , Feminino , Genótipo , Humanos , Tempo de Internação , Masculino , Programas de Rastreamento , Metronidazol/uso terapêutico , Pessoa de Meia-Idade , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Prevalência , Estudos Prospectivos , Fatores de Risco
20.
Int J Mol Sci ; 20(23)2019 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-31775346

RESUMO

Staphylococcus aureus strains produce a unique family of immunostimulatory exotoxins termed as bacterial superantigens (SAgs), which cross-link major histocompatibility complex class II (MHC II) molecule and T-cell receptor (TCR) to stimulate large numbers of T cells at extremely low concentrations. SAgs are associated with food poisoning and toxic shock syndrome. To date, 26 genetically distinct staphylococcal SAgs have been reported. This study reports the first X-ray structure of newly characterized staphylococcal enterotoxin N (SEN). SEN possesses the classical two domain architecture that includes an N-terminal oligonucleotide-binding fold and a C-terminal ß-grasp domain. Amino acid and structure alignments revealed that several critical amino acids that are proposed to be responsible for MHC II and TCR molecule engagements are variable in SEN, suggesting that SEN may adopt a different binding mode to its cellular receptors. This work helps better understand the mechanisms of action of SAgs.


Assuntos
Enterotoxinas/química , Enterotoxinas/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Conformação Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Staphylococcus aureus/metabolismo , Superantígenos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Antígenos de Histocompatibilidade Classe II/química , Humanos , Modelos Moleculares , Receptores de Antígenos de Linfócitos T/química , Homologia de Sequência , Superantígenos/química
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