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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.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
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
10.
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
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.
PLoS Negl Trop Dis ; 16(8): e0010207, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35930612

RESUMO

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is one of the top aetiologic agents of diarrhea in children under the age of 5 in low-middle income countries (LMICs). The lack of point of care diagnostic tools for routine ETEC diagnosis results in limited data regarding the actual burden and epidemiology in the endemic areas. We evaluated performance of the novel Rapid LAMP based Diagnostic Test (RLDT) for detection of ETEC in stool as a point of care diagnostic assay in a resource-limited setting. METHODS: We conducted a cross-sectional study of 324 randomly selected stool samples from children under 5 presenting with moderate to severe diarrhea (MSD). The samples were collected between November 2012 to September 2013 at selected health facilities in Zambia. The RLDT was evaluated by targeting three ETEC toxin genes [heat labile toxin (LT) and heat stable toxins (STh and STp)]. Quantitative PCR was used as the "gold standard" to evaluate the diagnostic sensitivity and specificity of RLDT for detection of ETEC. We additionally described the prevalence and seasonality of ETEC. RESULTS: The study included 324 participants, 50.6% of which were female. The overall prevalence of ETEC was 19.8% by qPCR and 19.4% by RLDT. The children between 12 to 59 months had the highest prevalence of 22%. The study determined ETEC toxin distribution was LT 28/321(9%), ST 18/321(6%) and LT/ST 16/321(5%). The sensitivity and specificity of the RLDT compared to qPCR using a Ct 35 as the cut-off, were 90.7% and 97.5% for LT, 85.2% and 99.3% for STh and 100% and 99.7% for STp, respectively. CONCLUSION: The results of this study suggest that RLDT is sufficiently sensitive and specific and easy to implement in the endemic countries. Being rapid and simple, the RLDT also presents as an attractive tool for point-of-care testing at the health facilities and laboratories in the resource-limited settings.


Assuntos
Toxinas Bacterianas , Escherichia coli Enterotoxigênica , Infecções por Escherichia coli , Proteínas de Escherichia coli , Toxinas Bacterianas/genética , Criança , Estudos Transversais , Diarreia/diagnóstico , Diarreia/epidemiologia , Escherichia coli Enterotoxigênica/genética , Enterotoxinas/genética , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/epidemiologia , Proteínas de Escherichia coli/genética , Feminino , Humanos , Masculino , Epidemiologia Molecular , Zâmbia/epidemiologia
13.
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
14.
Res Vet Sci ; 151: 175-183, 2022 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-36041311

RESUMO

Suilysin (Sly) from Streptococcus suis has been shown to elicit strong immune responses and may act as a vaccine adjuvant. In the present study, we tested the adjuvant effect of Sly using an engineered Pasteurella multocida toxin, rPMT-NC, as the antigen. The antigen was also formulated with other conventional adjuvants (aluminum hydroxide, water-in-oil-in-water) for comparison. The efficacy of these vaccine formulations were evaluated in mice. The optimal dosage of purified rSly for enhancing immune responses in mice was first determined to be 40 µg/ml based on significantly (p < 0.05) increased serum antibody titers, expression of cytokines, including interleukin (IL)-4, IL-12, and interferon (IFN)-γ and the survival rate after challenge with P. multocida. Mice immunized with rPMT-NC + rSly had augmented antibody production and cellular immunity compare to those immunized with rPMT-NC plus other adjuvants. In addition, the survival rate of mice immunized with rPMT-NC + rSly was the highest (70% v.s. 30% of mice immunized with rPMT-NC alone) among all groups. In conclusion, rSly has the potential to be used as a biological adjuvant to enhance immune responses and protective efficacy of protein-based vaccines.


Assuntos
Pasteurella multocida , Streptococcus suis , Adjuvantes Imunológicos/farmacologia , Animais , Proteínas de Bactérias , Toxinas Bacterianas , Proteínas Hemolisinas , Camundongos , Água
15.
Biomater Adv ; 136: 212752, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35929287

RESUMO

The ideal vaccine delivery systems can not only deliver antigens in intelligent manners but also act as adjuvants. Recently found that Mn2+ can effectively stimulate anti-tumor immune responses, and Ca2+ can regulate autophagy to promote the cross-presentation of antigens. Thus, we constructed such a manganese-containing multimode vaccine delivery system by using calcium-doped manganese carbonate microspheres (Ca@MnCO3) and perforin-listeria hemolysin (LLO), as termed as Ca@MnCO3/LLO. The two components Ca@MnCO3 and LLO, not only act as vaccine adjuvants by themselves, but also contribute to achieve cellular immunity. Among them, Ca@MnCO3 microspheres as an excellent Mn2+ and Ca2+ reservoir, can continuously release adjuvants Mn2+ and Ca2+ to enhance immune response in dendritic cells, while LLO can contribute to induce lysosomal escape. Particularly, Ca2+ was added firstly to MnCO3 microspheres to improve the stability and load capacity of the microspheres. Along with the degradation of intracellular Ca@MnCO3 microspheres, and the lysosomal membrane-lytic effects of perforin LLO, the Mn2+, Ca2+ and OVA were released to the cytoplasm. These outcomes cooperatively promote antigen cross-presentation, elicit CD8+ T cell proliferation, and finally achieve prominent anti-tumor effects. The results indicate that the manganese-containing vaccine delivery system Ca@MnCO3/LLO provides a promising platform for the construction of tumor vaccines.


Assuntos
Toxinas Bacterianas , Vacinas Anticâncer , Listeria monocytogenes , Adjuvantes Imunológicos , Cálcio , Carbonatos , Proteínas de Choque Térmico , Proteínas Hemolisinas , Imunoterapia , Manganês , Perforina
16.
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
17.
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
18.
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
19.
Appl Microbiol Biotechnol ; 106(17): 5675-5686, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35927333

RESUMO

Bacteria have developed various mechanisms by which they can compete or cooperate with other bacteria. This study showed that in the cocultures of wild-type Sphingomonas melonis TY and its isogenic mutant TYΔndpD grow with nicotine, the former can outcompete the latter. TYΔndpD undergoes growth arrest after four days when cocultured with wild-type TY, whereas the coculture has just entered a stationary phase and the substrate was nearly depleted, and the interaction between the two related strains was revealed by transcriptomic analysis. Analysis of the differential expression genes indicated that wild-type TY inhibited the growth of TYΔndpD mainly through toxin-antitoxin (TA) systems. The four upregulated antitoxin coding genes belong to type II TA systems in which the bactericidal effect of the cognate toxin was mainly through inhibition of translation or DNA replication, whereas wild-type TY with upregulated antitoxin genes can regenerate cognate immunity protein continuously and thus prevent the lethal action of toxin to itself. In addition, colicin-mediated antibacterial activity against closely related species may also be involved in the competition between wild-type TY and TYΔndpD under nutritional stress. Moreover, upregulation of carbon and nitrogen catabolism related-, stress response related-, DNA repair related-, and DNA replication-related genes in wild-type TY showed that it triggered a series of response mechanisms when facing dual stress of competition from isogenic mutant cells and nutritional limitation. Thus, we proposed that S. melonis TY employed the TA systems and colicin to compete with TYΔndpD under nutritional stress, thereby maximally acquiring and exploiting finite resources. KEY POINTS: • Cross-feeding between isogenic mutants and the wild-type strain. • Nutrition stress caused a shift from cooperation to competition. • TYΔndpD undergo growth arrest by exogenous and endogenous toxins.


Assuntos
Antitoxinas , Toxinas Bacterianas , Colicinas , Proteínas de Bactérias , Perfilação da Expressão Gênica , Sphingomonas
20.
Nat Commun ; 13(1): 4846, 2022 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-35978022

RESUMO

V. vulnificus-infected patients suffer from hemolytic anemia and circulatory lesions, often accompanied by venous thrombosis. However, the pathophysiological mechanism of venous thrombosis associated with V. vulnificus infection remains largely unknown. Herein, V. vulnificus infection at the sub-hemolytic level induced shape change of human red blood cells (RBCs) accompanied by phosphatidylserine exposure, and microvesicle generation, leading to the procoagulant activation of RBCs and ultimately, acquisition of prothrombotic activity. Of note, V. vulnificus exposed to RBCs substantially upregulated the rtxA gene encoding multifunctional autoprocessing repeats-in-toxin (MARTX) toxin. Mutant studies showed that V. vulnificus-induced RBC procoagulant activity was due to the pore forming region of the MARTX toxin causing intracellular Ca2+ influx in RBCs. In a rat venous thrombosis model triggered by tissue factor and stasis, the V. vulnificus wild type increased thrombosis while the ΔrtxA mutant failed to increase thrombosis, confirming that V. vulnificus induces thrombosis through the procoagulant activation of RBCs via the mediation of the MARTX toxin.


Assuntos
Toxinas Bacterianas , Trombose , Vibrio vulnificus , Animais , Toxinas Bacterianas/genética , Eritrócitos , Humanos , Fosfatidilserinas , Ratos , Trombose/genética , Vibrio vulnificus/genética
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