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1.
Food Chem ; 399: 134010, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36058099

RESUMO

A method using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed for the determination of toxoflavin and fervenulin in 6 types of food.The limits of detection (LODs, S/N ≥ 3) of toxoflavin and fervenulin reached 12 µg/kg and 24 µg/kg, respectively.The recoveries ranged from 70.1 % to 108.7 %.Intra-day RSDs (n = 5) and inter-day RSDs (n = 3) ranged from 0.9 % to 9.5 %.The method was successfully applied to analyse 36 samples, and one Tremella fuciformis Berk. sample was found with 7.5 mg/kg toxoflavin and 3.2 mg/kg fervenulin. Toxoflavin and fervenulin were acidic compounds and easily degraded in 0.1 % ammonia solution (v/v),degradation products were identified by ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS).


Assuntos
Toxinas Bacterianas , Espectrometria de Massas em Tandem , Cromatografia Líquida de Alta Pressão/métodos , Pirimidinonas , Triazinas
2.
PLoS Pathog ; 18(9): e1010713, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36107831

RESUMO

Enteric microbial pathogens, including Escherichia coli, Shigella and Cryptosporidium species, take a particularly heavy toll in low-income countries and are highly associated with infant mortality. We describe here a means to display anti-infective agents on the surface of a probiotic bacterium. Because of their stability and versatility, VHHs, the variable domains of camelid heavy-chain-only antibodies, have potential as components of novel agents to treat or prevent enteric infectious disease. We isolated and characterized VHHs targeting several enteropathogenic E. coli (EPEC) virulence factors: flagellin (Fla), which is required for bacterial motility and promotes colonization; both intimin and the translocated intimin receptor (Tir), which together play key roles in attachment to enterocytes; and E. coli secreted protein A (EspA), an essential component of the type III secretion system (T3SS) that is required for virulence. Several VHHs that recognize Fla, intimin, or Tir blocked function in vitro. The probiotic strain E. coli Nissle 1917 (EcN) produces on the bacterial surface curli fibers, which are the major proteinaceous component of E. coli biofilms. A subset of Fla-, intimin-, or Tir-binding VHHs, as well as VHHs that recognize either a T3SS of another important bacterial pathogen (Shigella flexneri), a soluble bacterial toxin (Shiga toxin or Clostridioides difficile toxin TcdA), or a major surface antigen of an important eukaryotic pathogen (Cryptosporidium parvum) were fused to CsgA, the major curli fiber subunit. Scanning electron micrographs indicated CsgA-VHH fusions were assembled into curli fibers on the EcN surface, and Congo Red binding indicated that these recombinant curli fibers were produced at high levels. Ectopic production of these VHHs conferred on EcN the cognate binding activity and, in the case of anti-Shiga toxin, was neutralizing. Taken together, these results demonstrate the potential of the curli-based pathogen sequestration strategy described herein and contribute to the development of novel VHH-based gut therapeutics.


Assuntos
Toxinas Bacterianas , Criptosporidiose , Cryptosporidium , Escherichia coli Enteropatogênica , Probióticos , Anticorpos de Domínio Único , Antígenos de Superfície , Vermelho Congo , Estranos , Flagelina , Humanos , Nitrilas , Sistemas de Secreção Tipo III , Fatores de Virulência/genética
3.
Commun Biol ; 5(1): 963, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-36109664

RESUMO

Of the 10 paralogs of MazEF Toxin-Antitoxin system in Mycobacterium tuberculosis, MazEF6 plays an important role in multidrug tolerance, virulence, stress adaptation and Non Replicative Persistant (NRP) state establishment. The solution structures of the DNA binding domain of MazE6 and of its complex with the cognate operator DNA show that transcriptional regulation occurs by binding of MazE6 to an 18 bp operator sequence bearing the TANNNT motif (-10 region). Kinetics and thermodynamics of association, as determined by NMR and ITC, indicate that the nMazE6-DNA complex is of high affinity. Residues in N-terminal region of MazE6 that are key for its homodimerization, DNA binding specificity, and the base pairs in the operator DNA essential for the protein-DNA interaction, have been identified. It provides a basis for design of chemotherapeutic agents that will act via disruption of TA autoregulation, leading to cell death.


Assuntos
Toxinas Bacterianas , Sistemas Toxina-Antitoxina , Toxinas Bacterianas/metabolismo , DNA/metabolismo , Homeostase , Ligação Proteica , Sistemas Toxina-Antitoxina/genética
4.
BMC Microbiol ; 22(1): 219, 2022 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-36115948

RESUMO

BACKGROUND: The prevalence of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin (PVL) gene is higher in Africa (≈50%) compared to Europe (< 5%). The study aimed to measure anti-PVL-antibodies in Africans and Germans in a multi-center study and to test whether detected antibodies can neutralize the cytotoxic effect of PVL on polymorphonuclear leukocytes (PMNs). METHODS: Sera from asymptomatic Africans (n = 22, Nigeria, Gabon) and Caucasians (n = 22, Germany) were used to quantify antibody titers against PVL and α-hemolysin (in arbitrary units [AU]) by ELISA. PMNs from one African and German donor were exposed to 5 nM recombinant PVL to measure the neutralizing effect of serial dilutions of pooled sera from African and Caucasian participants, or donor sera at 0.625 and 2.5% (v/v). RESULTS: Anti-PVL-antibodies were significantly higher in Africans than in Germans (1.9 vs. 0.7 AU, p < 0.0001). The pooled sera from the study participants neutralized the cytotoxic effect of PVL on African and German PMNs in a dose dependent manner. Also, neutralization of PVL on PMNs from the African and German donors had a stronger effect with African sera (half-maximal inhibitory concentration (IC50) = 0.27 and 0.47%, respectively) compared to Caucasian sera (IC50 = 3.51 and 3.59% respectively). CONCLUSION: Africans have higher levels of neutralizing anti-PVL-antibodies. It remains unclear if or at what level these antibodies protect against PVL-related diseases.


Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Toxinas Bacterianas , Exotoxinas , Proteínas Hemolisinas , Humanos , Leucocidinas , Nigéria , Infecções Estafilocócicas/epidemiologia
5.
Commun Biol ; 5(1): 910, 2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36065015

RESUMO

Phenol-soluble modulin α (PSMα) is identified as potent virulence factors in Staphylococcus aureus (S. aureus) infections. Very little is known about the role of PSMß which belongs to the same toxin family. Here we compared the role of PSMs in S. aureus-induced septic arthritis in a murine model using three isogenic S. aureus strains differing in the expression of PSMs (Newman, Δpsmα, and Δpsmß). The effects of PSMs on neutrophil NADPH-oxidase activity were determined in vitro. We show that the PSMα activates neutrophils via the formyl peptide receptor (FPR) 2 and reduces their NADPH-oxidase activity in response to the phorbol ester PMA. Despite being a poor neutrophil activator, PSMß has the ability to reduce the neutrophil activating effect of PSMα and to partly reverse the effect of PSMα on the neutrophil response to PMA. Mice infected with S. aureus lacking PSMα had better weight development and lower bacterial burden in the kidneys compared to mice infected with the parental strain, whereas mice infected with bacteria lacking PSMß strain developed more severe septic arthritis accompanied with higher IL-6 and KC. We conclude that PSMα and PSMß play distinct roles in septic arthritis: PSMα aggravates systemic infection, whereas PSMß protects arthritis development.


Assuntos
Artrite Infecciosa , Infecções Estafilocócicas , Animais , Toxinas Bacterianas , Camundongos , NADP/metabolismo , Oxirredutases/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia
6.
Front Immunol ; 13: 956340, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36072579

RESUMO

Clostridioides difficile (C. difficile) produces toxins A (TcdA) and B (TcdB), both associated with intestinal damage and diarrhea. Pannexin-1 (Panx1) channels allows the passage of messenger molecules, such as adenosine triphosphate (ATP), which in turn activate the P2X7 receptors (P2X7R) that regulate inflammation and cell death in inflammatory bowel diseases. The aim of this study was to verify the effect of C. difficile infection (CDI) in the expression of Panx1 and P2X7R in intestinal tissues of mice, as well as their role in cell death and IL-6 expression induced by TcdA and TcdB in enteric glial cells (EGCs). Male C57BL/6 mice (8 weeks of age) were infected with C. difficile VPI10463, and the control group received only vehicle per gavage. After three days post-infection (p.i.), cecum and colon samples were collected to evaluate the expression of Panx1 by immunohistochemistry. In vitro, EGCs (PK060399egfr) were challenged with TcdA or TcdB, in the presence or absence of the Panx1 inhibitor (10Panx trifluoroacetate) or P2X7R antagonist (A438079), and Panx1 and P2X7R expression, caspase-3/7 activity and phosphatidylserine binding to annexin-V, as well as IL-6 expression were assessed. CDI increased the levels of Panx1 in cecum and colon of mice compared to the control group. Panx1 inhibitor decreased caspase-3/7 activity and phosphatidylserine-annexin-V binding, but not IL-6 gene expression in TcdA and TcdB-challenged EGCs. P2X7 receptor antagonist accentually reduced caspase-3/7 activity, phosphatidylserine-annexin-V binding, and IL-6 gene expression in TcdA and TcdB-challenged EGCs. In conclusion, Panx1 is increased during CDI and plays an important role in the effects of C. difficile toxins in EGCs, participating in cell death induced by both toxins by promoting caspase-3/7 activation via P2X7R, which is also involved in IL-6 expression induced by both toxins.


Assuntos
Toxinas Bacterianas , Clostridioides difficile , Conexinas , Proteínas do Tecido Nervoso , Receptores Purinérgicos P2X7 , Animais , Anexinas , Toxinas Bacterianas/metabolismo , Caspase 3/metabolismo , Morte Celular , Conexinas/genética , Conexinas/metabolismo , Mediadores da Inflamação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/metabolismo , Fosfatidilserinas , Receptores Purinérgicos P2X7/genética
7.
J Cell Biol ; 221(10)2022 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-36074064

RESUMO

Mitochondria are dynamic organelles that play essential roles in cell growth and survival. Processes of fission and fusion are critical for the distribution, segregation, and maintenance of mitochondria and their genomes (mtDNA). While recent work has revealed the significance of mitochondrial organization for mtDNA maintenance, the impact of mtDNA perturbations on mitochondrial dynamics remains less understood. Here, we develop a tool to induce mitochondria-specific DNA damage using a mitochondrial-targeted base modifying bacterial toxin, DarT. Following damage, we observe dynamic reorganization of mitochondrial networks, likely driven by mitochondrial dysfunction. Changes in the organization are associated with the loss of mtDNA, independent of mitophagy. Unexpectedly, perturbation to exonuclease function of mtDNA replicative polymerase, Mip1, results in rapid loss of mtDNA. Our data suggest that, under damage, partitioning of defective mtDNA and organelle are de-coupled, with emphasis on mitochondrial segregation independent of its DNA. Together, our work underscores the importance of genome maintenance on mitochondrial function, which can act as a modulator of organelle organization and segregation.


Assuntos
DNA Mitocondrial , Mitocôndrias , Toxinas Bacterianas , Dano ao DNA , DNA Polimerase I , DNA Mitocondrial/genética , Exonucleases , Mitocôndrias/genética , Dinâmica Mitocondrial/genética , Mitofagia/genética
8.
Int J Mol Sci ; 23(17)2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36077344

RESUMO

C. novyi type A produces the alpha-toxin (TcnA) that belongs to the large clostridial glucosylating toxins (LCGTs) and is able to modify small GTPases by N-acetylglucosamination on conserved threonine residues. In contrast, other LCGTs including Clostridioides difficile toxin A and toxin B (TcdA; TcdB) modify small GTPases by mono-o-glucosylation. Both modifications inactivate the GTPases and cause strong effects on GTPase-dependent signal transduction pathways and the consequent reorganization of the actin cytoskeleton leading to cell rounding and finally cell death. However, the effect of TcnA on target cells is largely unexplored. Therefore, we performed a comprehensive screening approach of TcnA treated HEp-2 cells and analyzed their proteome and their phosphoproteome using LC-MS-based methods. With this data-dependent acquisition (DDA) approach, 5086 proteins and 9427 phosphosites could be identified and quantified. Of these, 35 proteins were found to be significantly altered after toxin treatment, and 1832 phosphosites were responsive to TcnA treatment. By analyzing the TcnA-induced proteomic effects of HEp-2 cells, 23 common signaling pathways were identified to be altered, including Actin Cytoskeleton Signaling, Epithelial Adherens Junction Signaling, and Signaling by Rho Family GTPases. All these pathways are also regulated after application of TcdA or TcdB of C. difficile. After TcnA treatment the regulation on phosphorylation level was much stronger compared to the proteome level, in terms of both strength of regulation and the number of regulated phosphosites. Interestingly, various signaling pathways such as Signaling by Rho Family GTPases or Integrin Signaling were activated on proteome level while being inhibited on phosphorylation level or vice versa as observed for the Role of BRCA1 in DNA Damage Response. ZIP kinase, as well as Calmodulin-dependent protein kinases IV & II, were observed as activated while Aurora-A kinase and CDK kinases tended to be inhibited in cells treated with TcnA based on their substrate regulation pattern.


Assuntos
Toxinas Bacterianas , Clostridioides difficile , Proteínas Monoméricas de Ligação ao GTP , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Enterotoxinas/química , Glicosilação , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Fosfolipases Tipo C/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
9.
Gut Microbes ; 14(1): 2117504, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36045589

RESUMO

Clostridioides difficile is the most common cause of infectious antibiotic-associated diarrhea, with disease mediated by two major toxins TcdA and TcdB. In severe cases, systemic disease complications may arise, resulting in fatal disease. Systemic disease in animal models has been described, with thymic damage an observable consequence of severe disease in mice. Using a mouse model of C. difficile infection, we examined this disease phenotype, focussing on the thymus and serum markers of systemic disease. The efficacy of bezlotoxumab, a monoclonal TcdB therapeutic, to prevent toxin mediated systemic disease complications was also examined. C. difficile infection causes toxin-dependent thymic damage and CD4+CD8+ thymocyte depletion in mice. These systemic complications coincide with changes in biochemical markers of liver and kidney function, including increased serum urea and creatinine, and hypoglycemia. Administration of bezlotoxumab during C. difficile infection prevents systemic disease and thymic atrophy, without blocking gut damage, suggesting the leakage of gut contents into circulation may influence systemic disease. As the thymus has such a crucial role in T cell production and immune system development, these findings may have important implications in relapse of C. difficile disease and impaired immunity during C. difficile infection. The prevention of thymic atrophy and reduced systemic response following bezlotoxumab treatment, without altering colonic damage, highlights the importance of systemic disease in C. difficile infection, and provides new insights into the mechanism of action for this therapeutic.Abbreviations: Acute kidney injury (AKI); Alanine Transaminase (ALT); Aspartate Aminotransferase (AST); C. difficile infection (CDI); chronic kidney disease (CKD); combined repetitive oligo-peptides (CROPS); cardiovascular disease (CVD); Double positive (DP); hematoxylin and eosin (H&E); immunohistochemical (IHC); multiple organ dysfunction syndrome (MODS); phosphate buffered saline (PBS); standard error of the mean (SEM); surface layer proteins (SLP); Single positive (SP); wild-type (WT).


Assuntos
Toxinas Bacterianas , Clostridioides difficile , Infecções por Clostridium , Microbioma Gastrointestinal , Animais , Anticorpos Monoclonais , Atrofia , Proteínas de Bactérias/genética , Toxinas Bacterianas/metabolismo , Anticorpos Amplamente Neutralizantes , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/prevenção & controle , Enterotoxinas/metabolismo
10.
Commun Biol ; 5(1): 906, 2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36064583

RESUMO

Clostridium novyi alpha-toxin (Tcnα) is a potent exotoxin that induces severe symptoms including gas gangrene, myositis, necrotic hepatitis, and sepsis. Tcnα binds to sulfated glycosaminoglycans (sGAG) for cell-surface attachment and utilizes low-density lipoprotein receptor (LDLR) for rapid entry. However, it was also shown that Tcnα may use alternative entry receptors other than LDLR. Here, we define that LRP1 and Megalin can also facilitate the cellular entry of Tcnα by employing reconstitutive LDLR family proteins. LDLR, LRP1, and Megalin recognize Tcnα via their ligand-binding domains (also known as LDL receptor type A repeats). Notably, LDLR and LRP1 have contrasting expression levels in many different cells, thus the dominant entry receptor for Tcnα could be cell-type dependent. These findings together increase our knowledge of the Tcnα actions and further help to understand the pathogenesis of C. novyi infection-associated diseases.


Assuntos
Toxinas Bacterianas , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Toxinas Bacterianas/metabolismo , Transporte Biológico , Proteínas de Transporte/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo
11.
PLoS One ; 17(9): e0273474, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36074767

RESUMO

Epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) is continually changing. Frequency of genotypes typical for community-associated MRSA (CA-MRSA) is increasing in hospitals, as well as resistance to antimicrobial agents. Moreover, different clones predominate in different geographic regions, and temporal shifts occur in the predominant clonal type. The aim of this study was to estimate the prevalence of MRSA, CA-MRSA and PVL-positive MRSA isolates from patients hospitalised in the Military Medical Academy (MMA) and from outpatients, and to perform genotyping of PVL-positive MRSA isolates. MRSA isolates were obtained by standard microbiological techniques. PVL-positive MRSA were detected by single PCR. Determination of SCCmec types in MRSA isolates was done using multiplex PCR and genotyping of PVL-positive MRSA by PFGE, MLST and spa typing. The prevalence of MRSA among S. aureus isolates from different clinical specimens was 43.4%. In outpatients the prevalence of MRSA was 3.2%. SCCmec types specific for CA-MRSA were found in 26% of MRSA isolates from hospitalised patients. In groups, hospitalised patients and outpatients, the prevalence of PVL-positive MRSA isolates was 4%, and all of them harboured SCCmec type V genetic element. PFGE revealed minor differences between four groups of PVL-positive MRSA isolates, but all of them belonged to ST152, and all except one were of the t355 spa type. High prevalence of MRSA and CA-MRSA in MMA, especially the presence of PVL-positive CA-MRSA, represent a serious health threat for patients. Genotype t355/ST152/SCCmec V is the dominant MRSA clone among PVL-positive CA-MRSA.


Assuntos
Toxinas Bacterianas , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Antibacterianos , Toxinas Bacterianas/genética , Genótipo , Humanos , Leucocidinas/genética , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Sérvia/epidemiologia , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Centros de Atenção Terciária
12.
Front Cell Infect Microbiol ; 12: 941939, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35967844

RESUMO

Lymphostatin is a virulence factor of enteropathogenic E. coli (EPEC) and non-O157 serogroup enterohaemorrhagic E. coli. Previous studies using whole-cell lysates of EPEC showed that lymphostatin inhibits the mitogen-activated proliferation of bulk human peripheral blood mononuclear cells (PBMCs) and the production of cytokines IL-2, IL-4, IL-5, and IFN-γ. Here, we used highly purified lymphostatin and PBMC-derived T cells to show that lymphostatin inhibits anti-CD3/anti-CD28-activated proliferation of human CD4+ and CD8+ T cells and blocks the synthesis of IL-2, IL-4, IL-10 and IFN-γ without affecting cell viability and in a manner dependent on an N-terminal DTD glycosyltransferase motif. Such inhibition was not observed with T cells activated by phorbol 12-myristate 13-acetate and ionomycin, implying that lymphostatin targets T cell receptor signaling. Analysis of the expression of CD69 indicated that lymphostatin suppresses T cell activation at an early stage and no impacts on apoptosis or necrosis were observed. Flow cytometric analysis of the DNA content of lymphostatin-treated CD4+ and CD8+ T cells showed a concentration- and DTD-dependent accumulation of the cells in the G0/G1 phase of the cell cycle, and corresponding reduction of the percentage of cells in S phase. Consistent with this, we found a marked reduction in the abundance of cyclins D3, E and A and loss of phosphorylated Rb over time in activated T cells from 8 donors treated with lymphostatin. Moreover, the cyclin-dependent kinase (cdk) inhibitor p27kip1, which inhibits progression of the cell cycle at G1 by acting on cyclin E-cdk2 or cyclin D-cdk4 complexes, was found to be accumulated in lymphostatin-treated T cells. Analysis of the abundance of phosphorylated kinases involved in signal transduction found that 30 of 39 were reduced in abundance following lymphostatin treatment of T cells from 5 donors, albeit not significantly so. Our data provide novel insights into the mode of action of lymphostatin on human T lymphocytes.


Assuntos
Toxinas Bacterianas , Infecções por Escherichia coli , Proteínas de Escherichia coli , Escherichia coli , Linfócitos T , Apoptose , Toxinas Bacterianas/imunologia , Linfócitos T CD8-Positivos/imunologia , Pontos de Checagem do Ciclo Celular/imunologia , Divisão Celular , Proliferação de Células/fisiologia , Citocinas/biossíntese , Citocinas/imunologia , Escherichia coli Enteropatogênica/imunologia , Escherichia coli Enteropatogênica/patogenicidade , Escherichia coli/imunologia , Escherichia coli/patogenicidade , Infecções por Escherichia coli/imunologia , Proteínas de Escherichia coli/imunologia , Humanos , Interleucina-2 , Interleucina-4 , Leucócitos Mononucleares/imunologia , Necrose , Linfócitos T/imunologia , Fatores de Virulência/imunologia
13.
ACS Chem Biol ; 17(9): 2507-2518, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36038138

RESUMO

Toxins TcdA and TcdB from Clostridioides difficile glucosylate human colon Rho GTPases. TcdA and TcdB glucosylation of RhoGTPases results in cytoskeletal changes, causing cell rounding and loss of intestinal integrity. Clostridial toxins TcdA and TcdB are proposed to catalyze glucosylation of Rho GTPases with retention of stereochemistry from UDP-glucose. We used kinetic isotope effects to analyze the mechanisms and transition-state structures of the glucohydrolase and glucosyltransferase activities of TcdB. TcdB catalyzes Rho GTPase glucosylation with retention of stereochemistry, while hydrolysis of UDP-glucose by TcdB causes inversion of stereochemistry. Kinetic analysis revealed TcdB glucosylation via the formation of a ternary complex with no intermediate, supporting an SNi mechanism with nucleophilic attack and leaving group departure occurring on the same face of the glucose ring. Kinetic isotope effects combined with quantum mechanical calculations revealed that the transition states of both glucohydrolase and glucosyltransferase activities of TcdB are highly dissociative. Specifically, the TcdB glucosyltransferase reaction proceeds via an SNi mechanism with the formation of a distinct oxocarbenium phosphate ion pair transition state where the glycosidic bond to the UDP leaving group breaks prior to attack of the threonine nucleophile from Rho GTPase.


Assuntos
Toxinas Bacterianas , Clostridioides difficile , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Glucose , Glucosiltransferases/metabolismo , Humanos , Cinética , Fosfatos , Toxina Tetânica , Treonina , Uridina Difosfato Glucose , Proteínas rho de Ligação ao GTP
14.
Appl Environ Microbiol ; 88(17): e0095922, 2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-35972240

RESUMO

There are no licensed vaccines against enterotoxigenic Escherichia coli (ETEC), a leading cause of children's diarrhea and travelers' diarrhea. Recently, protein-based vaccine candidate MecVax was demonstrated to induce functional antibodies against both ETEC toxins (heat-stable toxin [STa] and heat-labile toxin [LT]) and seven ETEC adhesins (CFA/I and CS1 to CS6) and to protect against ETEC clinical diarrhea or intestinal colonization preclinically. Those studies used intraperitoneal, intramuscular, and intradermal routes, and a dose range for MecVax protein antigens, toxoid fusion 3xSTaN12S-mnLTR192G/L211A, and adhesin CFA/I/II/IV MEFA has not been investigated. Here, we further characterized MecVax broad immunogenicity, utilizing a subcutaneous route, and examined vaccine dose-dependent antibody response effects and also antibody functional activities against ETEC enterotoxicity and bacterial adherence. Data showed that mice immunized subcutaneously with MecVax developed robust IgG responses to seven ETEC adhesins (CFA/I, as well as CS1 to CS6) and two toxins (STa and LT). At a subcutaneous dose of 25, 20, or 10 µg or at an intramuscular dose of 12, 6, or 3 µg, MecVax induced similar levels IgG responses to the targeted toxins and adhesins, and these antibodies exhibited equivalent functional activities against ETEC toxin enterotoxicity and bacterial adherence. Once the intramuscular dose was decreased to 1 µg, vaccine-induced antibodies were significantly reduced and no longer neutralized STa enterotoxicity. The results indicated that MecVax administered subcutaneously is broadly immunogenic and, at an intramuscular dose of 3 µg, can induce functional antitoxin and anti-adhesin antibodies in mice, providing instructive information for future vaccine dose studies in humans and accelerating MecVax vaccine development. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) is a leading cause of children's diarrhea and the most common cause of travelers' diarrhea. ETEC infections are responsible for >200 million diarrhea clinical cases and near 100,000 deaths annually. Currently, there are no licensed vaccines for ETEC diarrhea. The protein-based vaccine candidate MecVax unprecedentedly targets two ETEC toxins (STa and LT, produced by all ETEC strains) and seven ETEC adhesins (CFA/I, as well as CS1 to CS6, associated with >60% of ETEC clinical diarrhea cases) and has been demonstrated to be broadly immunogenic and cross protective; as such, it represents a potentially effective multivalent vaccine against ETEC-associated children's and travelers' diarrhea. This study further confirmed MecVax broad immunogenicity and evaluated the vaccine antigen dose effect on the induction of antigen-specific antibody responses in mice and on antibody functional activities against ETEC toxin enterotoxicity and bacterial adherence, yielding useful information for future human volunteer studies and the development of MecVax as an effective ETEC vaccine.


Assuntos
Toxinas Bacterianas , Escherichia coli Enterotoxigênica , Infecções por Escherichia coli , Proteínas de Escherichia coli , Adesinas Bacterianas/metabolismo , Animais , Anticorpos Antibacterianos , Toxinas Bacterianas/metabolismo , Criança , Diarreia/microbiologia , Modelos Animais de Doenças , Enterotoxinas , Infecções por Escherichia coli/microbiologia , Humanos , Imunoglobulina G/metabolismo , Camundongos , Viagem , Vacinas Combinadas
15.
Trends Microbiol ; 30(10): 920-921, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35989163

RESUMO

Toxin-antitoxin systems can defend bacteria against phages by shutting down infected cells, but the links between their molecular mechanisms and biological functions have remained underexplored. LeRoux et al. now show how the DNA-targeting ADP-ribosylation activity of DarTG impairs phage replication but is overcome by dedicated viral inhibitors and evolved tolerance.


Assuntos
Toxinas Bacterianas , Bacteriófagos , Difosfato de Adenosina , Antivirais
16.
World J Microbiol Biotechnol ; 38(11): 200, 2022 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-35995893

RESUMO

Staphylococcus aureus (S. aureus), a Gram-positive bacteria, is an incurable cause of hospital and community-acquired infections. Inhibition bacterial virulence is a viable strategy against S. aureus infections based on the multiple virulence factors secreted by S. aureus. Alpha-hemolysin (Hla) plays a crucial role in bacteria virulence without affecting bacterial viability. Here, we identified that 7,8-Dihydroxyflavone (7,8-DHF), a natural compound, was able to decrease the expression of and did not affect the in vitro growth of S. aureus USA300 at a concentration of 32 µg/mL. It was verified by western blot and RT-qPCR that the natural compound could inhibit the transcription and translation of Hla. Further mechanism studies revealed that 7,8-DHF has a negative effect on transcriptional regulator agrA and RNAIII, preventing the upregulation of virulence gene. Cytotoxicity assays showed that 7,8-DHF did not produce significant cytotoxicity to A549 cells. Animal experiments showed that the combination of 7,8-DHF and vancomycin had a more significant therapeutic effect on S. aureus infection, reflecting the synergistic effect of 7,8-DHF with antibiotics. In conclusion, 7,8-DHF was able to target Hla to protect host cells from hemolysis while limiting the development of bacterial resistance.


Assuntos
Toxinas Bacterianas , Flavonas , Infecções Estafilocócicas , Staphylococcus aureus , Células A549 , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Toxinas Bacterianas/metabolismo , Flavonas/farmacologia , Proteínas Hemolisinas/genética , Humanos , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
17.
J Am Chem Soc ; 144(32): 14627-14637, 2022 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-35916199

RESUMO

Cylindrospermopsin, a major cyanotoxin, is produced by freshwater cyanobacteria. Its biosynthesis starts from arginine and glycine and involves five polyketide synthases and several tailoring enzymes. We report the identification of 7-deoxy-desulfo-argino-cylindrospermopsin in several cylindrospermopsin-producing cyanobacteria using mass spectrometry experiments. We have purified this new metabolite and established its structure by 1D and 2D NMR spectroscopy using scalar-based 1H-1H, 1H-13C, and 1H-15N as well as 2D 1H-1H ROESY correlation experiments. Using labeled arginines in isotopic incorporation experiments, we have shown that arginine is fully incorporated into 7-deoxy-desulfo-argino-cylindrospermopsin and that the uracil ring of cylindrospermopsin originates from the guanidino moiety of arginine, thus solving a long-standing puzzling question. CyrG and CyrH from the cylindrospermopsin-producing Oscillatoria sp. PCC 6506 were overproduced in Escherichia coli and purified to homogeneity. We showed that CyrG is a zinc-dependent hydrolase, homologous to adenosine deaminases, that transforms 7-deoxy-desulfo-argino-cylindrospermopsin into 7-deoxy-desulfo-cylindrospermopsin and ornithine, with the following kinetic parameters: KM = 0.21 ± 0.05 µM and kcat = 0.19 ± 0.02 min-1. CyrG contained 0.55 mol of zinc per mol of monomer but could be activated by FeII or CoII. CyrH contained almost no metal and showed no such activity even in the presence of excess metal. Using structure-based alignments and secondary structure predictions, we propose that the fifth and last polyketide synthase CyrF in cylindrospermopsin biosynthesis contains an unprecedented C-terminal domain homologous to N-acetyltransferases. We suggest that this domain catalyzes the condensation of the CyrF product with arginine to give 7-deoxy-desulfo-argino-cylindrospermopsin. This would be an unprecedented termination step for a polyketide synthase.


Assuntos
Toxinas Bacterianas , Cianobactérias , Arginina/metabolismo , Toxinas Bacterianas/química , Cianobactérias/metabolismo , Toxinas de Cianobactérias , Policetídeo Sintases/metabolismo , Uracila/química , Zinco/metabolismo
18.
Molecules ; 27(16)2022 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-36014383

RESUMO

Clostridium perfringens (C. perfringens) is an important foodborne pathogen that can cause diseases such as gas gangrene and necrotizing enteritis in a variety of economic animals, seriously affecting public health and the economic benefits and healthy development of the livestock and poultry breeding industry. Perfringolysin O (PFO) is an important virulence factor of C. perfringens and plays critical roles in necrotic enteritis and gas gangrene, rendering it an ideal target for developing new drugs against infections caused by this pathogen. In this study, based on biological activity inhibition assays, oligomerization tests and computational biology assays, we found that the foodborne natural component piceatannol reduced pore-forming activity with an inhibitory ratio of 83.84% in the concentration of 16 µg/mL (IC50 = 7.83 µg/mL) by binding with PFO directly and changing some of its secondary structures, including 3-Helix, A-helix, bend, and in turn, ultimately affecting oligomer formation. Furthermore, we confirmed that piceatannol protected human intestinal epithelial cells from the damage induced by PFO with LDH release reduced by 38.44% at 16 µg/mL, based on a cytotoxicity test. By performing an animal experiment, we found the C. perfringens clones showed an approximate 10-fold reduction in infected mice. These results suggest that piceatannol may be a candidate for anti-C. perfringens drug development.


Assuntos
Enterite , Gangrena Gasosa , Doenças das Aves Domésticas , Animais , Toxinas Bacterianas , Clostridium perfringens , Proteínas Hemolisinas , Humanos , Camundongos , Estilbenos , Virulência
19.
Clin Lab ; 68(8)2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35975492

RESUMO

BACKGROUND: In addition to antibiotic resistance, the entry of Helicobacter pylori into the persistence phase leads to recurrent and chronic infections, as well as the development of antibiotic resistance in persister cells. METHODS: In this study, after genetic confirmation of H. pylori in 20 biopsy specimens, the prevalence of the type II TA systems mazEF, relEB, yafQ/dinJ was investigated. Also, the most common system observed in the study in terms of structure, evolution, and molecular interaction was evaluated by bioinformatics tools. RESULTS: The results of the PCR test on 20 biopsy samples were positive for ureA and glmM genes. Moreover, yafQ/ dinJ was the only module positive in half of the samples (10 samples) in the PCR technique. The toxin residues and their interactions with the cognate antitoxin residues are revealed by docking analysis results. Furthermore, the multiple sequence alignment (MSA) of the YafQ toxin showed that this toxin has a low polymorphism among H. pylori species. The evolutionary study showed that the yafQ toxin had the highest sequence similarity among the bacteria Helicobacter cetorm (60% similarity) and Muricauda olearia (57.35 % similarity). CONCLUSIONS: Collectively, the data of the present study indicate that the YafQ/DinJ is the dominant type II TA system and has the highest frequency among the studied systems in H. pylori, and further studies are required to elucidate its exact role in this bacterium.


Assuntos
Antitoxinas , Proteínas de Bactérias , Toxinas Bacterianas , Helicobacter pylori , Sistemas Toxina-Antitoxina , Antitoxinas/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Helicobacter pylori/genética , Humanos , Sistemas Toxina-Antitoxina/genética
20.
Int Immunopharmacol ; 110: 109076, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35978517

RESUMO

Immunotoxins are regarded as a type of targeted therapy for killing cells by highly potent bacterial, fungal or plant toxins. Shiga like toxins (SLTs) are a group of bacterial AB5 protein toxins that inhibit host cell protein synthesis through the removal of a single adenine residue from the 28S rRNA and lead to apoptosis. Here, we described the design and usage of a Stx-based immunotoxin that can induce the selective cytotoxicity and apoptosis in Fn-14-positive cells related to the colon and lung cancer. In the present study, the Stx2a-PE15-P4A8 fusion protein was expressed efficiently in E. coli (DE3) system when driven from inclusion bodies by 8 M urea. The Stx2a-PE15-P4A8 fusion protein was expressed efficiently in E. coli (DE3) system and then purified. The purified fusion protein could specifically target Fn-14 receptor existed on colon and lung cancer cell lines and suppress these cells in a dose-dependent manner. In addition, the protein was able to nearly 50 % of apoptotic cell death and maintains about 54 % of its stability after 24 h of incubation in mouse serum at 37 °C. Compared to PE38-P4A8 construct in our previous study, these results showed that the Stx2a-PE15-P4A8 construct can be an efficient therapeutic candidate for cancer immunotherapy.


Assuntos
Toxinas Bacterianas , Neoplasias Colorretais , Imunotoxinas , Neoplasias Pulmonares , Animais , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Neoplasias Colorretais/tratamento farmacológico , Escherichia coli/genética , Escherichia coli/metabolismo , Imunotoxinas/genética , Neoplasias Pulmonares/tratamento farmacológico , Camundongos
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