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1.
FASEB J ; 35(9): e21742, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34403506

RESUMO

Withdrawal from contact inhibition is necessary for epithelial cancer precursor cells to initiate cell growth and motility. Nevertheless, little is understood about the mechanism for the sudden initiation of cell growth under static conditions. We focused on cellular junctions as one region where breaking out of contact inhibition occurs. In well-differentiated endometrial cancer cells, Sawano, the ligand administration for tricellular tight junction protein LSR, which transiently decreased the robust junction property, caused an abrupt increase in cell motility and consequent excessive multilayered cell growth despite being under contact inhibition conditions. We observed that macropinocytosis essentially and temporarily occurred as an antecedent event for the above process at intercellular junctions without disruption of the junction apparatus but not at the apical plasma membrane. Collectively, we concluded that the formation of macropinocytosis, which is derived from tight junction-mediated signaling, was triggered for the initiation of cell growth in static precancerous epithelium.


Assuntos
Adesão Celular , Inibição de Contato , Pinocitose , Receptores de Lipoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Toxinas Bacterianas/farmacologia , Sítios de Ligação , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fenótipo , Pinocitose/efeitos dos fármacos , Transporte Proteico , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo
2.
Cell Mol Life Sci ; 78(17-18): 6319-6335, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34308492

RESUMO

The Cytolethal Distending Toxin (CDT) is a bacterial genotoxin produced by pathogenic bacteria causing major foodborne diseases worldwide. CDT activates the DNA Damage Response and modulates the host immune response, but the precise relationship between these outcomes has not been addressed so far. Here, we show that chronic exposure to CDT in HeLa cells or mouse embryonic fibroblasts promotes a strong type I interferon (IFN) response that depends on the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) through the recognition of micronuclei. Indeed, despite active cell cycle checkpoints and in contrast to other DNA damaging agents, cells exposed to CDT reach mitosis where they accumulate massive DNA damage, resulting in chromosome fragmentation and micronucleus formation in daughter cells. These mitotic phenotypes are observed with CDT from various origins and in cancer or normal cell lines. Finally, we show that CDT exposure in immortalized normal colonic epithelial cells is associated to cGAS protein loss and low type I IFN response, implying that CDT immunomodulatory function may vary depending on tissue and cell type. Thus, our results establish a direct link between CDT-induced DNA damage, genetic instability and the cellular immune response that may be relevant in the context of natural infection associated to chronic inflammation or carcinogenesis.


Assuntos
Toxinas Bacterianas/farmacologia , Interferon Tipo I/metabolismo , Nucleotidiltransferases/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HeLa , Humanos , Interferon Tipo I/genética , Camundongos , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/genética
3.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201747

RESUMO

Rett syndrome (RTT) is a rare neurological disorder caused by mutations in the X-linked MECP2 gene and a major cause of intellectual disability in females. No cure exists for RTT. We previously reported that the behavioural phenotype and brain mitochondria dysfunction are widely rescued by a single intracerebroventricular injection of the bacterial toxin CNF1 in a RTT mouse model carrying a truncating mutation of the MeCP2 gene (MeCP2-308 mice). Given the heterogeneity of MECP2 mutations in RTT patients, we tested the CNF1 therapeutic efficacy in a mouse model carrying a null mutation (MeCP2-Bird mice). CNF1 selectively rescued cognitive defects, without improving other RTT-related behavioural alterations, and restored brain mitochondrial respiratory chain complex activity in MeCP2-Bird mice. To shed light on the molecular mechanisms underlying the differential CNF1 effects on the behavioural phenotype, we compared treatment effects on relevant signalling cascades in the brain of the two RTT models. CNF1 provided a significant boost of the mTOR activation in MeCP2-308 hippocampus, which was not observed in the MeCP2-Bird model, possibly explaining the differential effects of CNF1. These results demonstrate that CNF1 efficacy depends on the mutation beared by MeCP2-mutated mice, stressing the need of testing potential therapeutic approaches across RTT models.


Assuntos
Toxinas Bacterianas/farmacologia , Encéfalo/efeitos dos fármacos , Proteínas de Escherichia coli/farmacologia , Proteína 2 de Ligação a Metil-CpG/genética , Mitocôndrias/efeitos dos fármacos , Síndrome de Rett/tratamento farmacológico , Animais , Toxinas Bacterianas/administração & dosagem , Encéfalo/metabolismo , Modelos Animais de Doenças , Proteínas de Escherichia coli/administração & dosagem , Medo/efeitos dos fármacos , Feminino , Infusões Intraventriculares , Mutação com Perda de Função , Masculino , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/etiologia , Camundongos Mutantes , Proteínas dos Microfilamentos/metabolismo , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Síndrome de Rett/etiologia , Serina-Treonina Quinases TOR/metabolismo
4.
Nucleic Acids Res ; 49(14): 8384-8395, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34255843

RESUMO

Bacteria have evolved sophisticated mechanisms to deliver potent toxins into bacterial competitors or into eukaryotic cells in order to destroy rivals and gain access to a specific niche or to hijack essential metabolic or signaling pathways in the host. Delivered effectors carry various activities such as nucleases, phospholipases, peptidoglycan hydrolases, enzymes that deplete the pools of NADH or ATP, compromise the cell division machinery, or the host cell cytoskeleton. Effectors categorized in the family of polymorphic toxins have a modular structure, in which the toxin domain is fused to additional elements acting as cargo to adapt the effector to a specific secretion machinery. Here we show that Photorhabdus laumondii, an entomopathogen species, delivers a polymorphic antibacterial toxin via a type VI secretion system. This toxin inhibits protein synthesis in a NAD+-dependent manner. Using a biotinylated derivative of NAD, we demonstrate that translation is inhibited through ADP-ribosylation of the ribosomal 23S RNA. Mapping of the modification further showed that the adduct locates on helix 44 of the thiostrepton loop located in the GTPase-associated center and decreases the GTPase activity of the EF-G elongation factor.


Assuntos
Toxinas Bacterianas/farmacologia , GTP Fosfo-Hidrolases/genética , RNA Ribossômico 23S/genética , Sistemas de Secreção Tipo VI/efeitos dos fármacos , ADP-Ribosilação/efeitos dos fármacos , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , NAD/genética , Fator G para Elongação de Peptídeos/genética , Photorhabdus/química , Photorhabdus/genética , Biossíntese de Proteínas/efeitos dos fármacos , RNA Ribossômico 23S/efeitos dos fármacos , Tioestreptona/química , Tioestreptona/farmacologia
5.
Toxins (Basel) ; 13(6)2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071730

RESUMO

The clinically highly relevant Clostridioides (C.) difficile releases several AB-type toxins that cause diseases such as diarrhea and pseudomembranous colitis. In addition to the main virulence factors Rho/Ras-glycosylating toxins TcdA and TcdB, hypervirulent strains produce the binary AB-type toxin CDT. CDT consists of two separate proteins. The binding/translocation B-component CDTb facilitates uptake and translocation of the enzyme A-component CDTa to the cytosol of cells. Here, CDTa ADP-ribosylates G-actin, resulting in depolymerization of the actin cytoskeleton. We previously showed that CDTb exhibits cytotoxicity in the absence of CDTa, which is most likely due to pore formation in the cytoplasmic membrane. Here, we further investigated this cytotoxic effect and showed that CDTb impairs CaCo-2 cell viability and leads to redistribution of F-actin without affecting tubulin structures. CDTb was detected at the cytoplasmic membrane in addition to its endosomal localization if CDTb was applied alone. Chloroquine and several of its derivatives, which were previously identified as toxin pore blockers, inhibited intoxication of Vero, HCT116, and CaCo-2 cells by CDTb and CDTb pores in vitro. These results further strengthen pore formation by CDTb in the cytoplasmic membrane as the underlying cytotoxic mechanism and identify pharmacological pore blockers as potent inhibitors of cytotoxicity induced by CDTb and CDTa plus CDTb.


Assuntos
Toxinas Bacterianas/antagonistas & inibidores , Clostridioides difficile/patogenicidade , Actinas/metabolismo , Animais , Toxinas Bacterianas/farmacologia , Células CACO-2 , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Cloroquina/farmacologia , Humanos , Células Vero
6.
Mol Cell ; 81(15): 3160-3170.e9, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34174184

RESUMO

RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes.


Assuntos
Toxinas Bacterianas/metabolismo , Guanosina Pentafosfato/metabolismo , Ligases/metabolismo , RNA de Transferência/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacologia , Bacilos Gram-Positivos Asporogênicos/química , Bacilos Gram-Positivos Asporogênicos/metabolismo , Guanosina Pentafosfato/química , Ligases/química , Ligases/genética , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , Inibidores da Síntese de Proteínas/farmacologia , Pirofosfatases , Ribossomos/metabolismo
7.
Biosci Biotechnol Biochem ; 85(6): 1371-1382, 2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-33851985

RESUMO

Oscillatoxins (OTXs) and aplysiatoxins are biosynthetically related polyketides produced by marine cyanobacteria. We previously developed a synthetic route to phenolic O-methyl analogs of OTX-D and 30-methyl-OTX-D during collective synthesis of these natural products. According to our synthetic strategy, we achieved total synthesis of OTX-D, 30-methyl-OTX-D, OTX-E, and OTX-F by deprotecting the O-methyl group in an earlier intermediate, and determined their biological activities. Although OTX-D and 30-methyl-OTX-D have been reported to show antileukemic activity against L1210 cell line, we found that their cytotoxicity in vitro against this cell line is relatively weak (IC50: 29-52 µm). In contrast, OTX-F demonstrated cell line-selective antiproliferative activity against DMS-114 lung cancer cells, which implies that OTXs target as yet unknown target molecules as part of this unique activity.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Toxinas Bacterianas/síntese química , Toxinas Bacterianas/farmacologia , Antineoplásicos/química , Toxinas Bacterianas/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Química Sintética , Humanos
8.
Toxins (Basel) ; 13(4)2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33921797

RESUMO

Binary toxin (Bin toxin), BinA and BinB, produced by Lysinibacillus sphaericus has been used as a mosquito-control agent due to its high toxicity against the mosquito larvae. The crystal structures of Bin toxin and non-insecticidal but cytotoxic parasporin-2 toxin share some common structural features with those of the aerolysin-like toxin family, thus suggesting a common mechanism of pore formation of these toxins. Here we explored the possible cytotoxicity of Bin proteins (BinA, BinB and BinA + BinB) against Hs68 and HepG2 cell lines. The cytotoxicity of Bin proteins was evaluated using the trypan blue exclusion assay, MTT assay, morphological analysis and LDH efflux assay. The intracellular localization of Bin toxin in HepG2 cells was assessed by confocal laser scanning microscope. HepG2 cells treated with BinA and BinB (50 µg/mL) showed modified cell morphological features and reduced cell viability. Bin toxin showed no toxicity against Hs68 cells. The EC50 values against HepG2 at 24 h were 24 ng/mL for PS2 and 46.56 and 39.72 µg/mL for BinA and BinB, respectively. The induction of apoptosis in treated HepG2 cells was confirmed by upregulation of caspase levels. The results indicated that BinB mediates the translocation of BinA in HepG2 cells and subsequently associates with mitochondria. The study supports the possible development of Bin toxin as either an anticancer agent or a selective delivery vehicle of anticancer agents to target mitochondria of human cancer cells in the future.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Bacillaceae/metabolismo , Toxinas Bacterianas/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Antineoplásicos/metabolismo , Toxinas Bacterianas/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Caspases/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Receptores de Superfície Celular/metabolismo
9.
J Med Microbiol ; 70(4)2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33830910

RESUMO

Introduction. Clostridioides difficile infection (CDI) causes toxin-mediated enteropathy, such as antibiotic-associated diarrhoea and pseudomembranous colitis. Rho-glucosylating toxin A (TcdA) and toxin B (TcdB) have been clearly implicated in pathogenesis, whereas the virulence of binary toxin (CDT) is still debated.Hypothesis statement. We hypothesized that CDT is involved in the host immune response and plays a pivotal role in establishing virulence by modulating pro-inflammatory cytokine production; this is achieved through the integral Toll-like receptor (TLR) signalling pathways.Aim. The aim of the present study was to determine whether and how CDT impacts macrophages compared to TcdA or TcdB by examining the induction of CXC chemokine ligand 2 (CXCL2) and tumour necrosis factor-α (TNF-α), both of which are crucial in mediating local and systematic inflammatory responses.Methodology. RAW264.7 cells or transfected human embryonic kidney (HEK) 293 T cells were incubated with TcdA, TcdB, or CDT. In some experiments, a neutralizing antibody against TLR2 or TLR4, or myeloid differentiation 88 inhibitory peptide were added. The amount of CXCL2 and TNF-α secreted was then measured.Results. In RAW264.7 macrophages, CXCL2 and TNF-α were produced via the Toll-like receptor 2 (TLR2) or Toll-like receptor 4 (TLR4) pathway in a TcdA, TcdB, or CDT dose-dependent manner. Interleukin-8 secretion was induced in TLR4/MD2/CD14-transfected, but not in TLR2-transfected, HEK 293 T cells following TcdB or CDT exposure.Conclusion. Our results showed that C. difficile toxins, including CDT, enhanced macrophage-mediated CXCL2 and TNF-α production via TLR2 and TLR4, indicating that CDT affects host immune responses.


Assuntos
Toxinas Bacterianas/farmacologia , Quimiocina CXCL2/metabolismo , Clostridioides difficile/patogenicidade , Macrófagos/efeitos dos fármacos , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células HEK293 , Humanos , Macrófagos/metabolismo , Camundongos , Fator 88 de Diferenciação Mieloide/antagonistas & inibidores , Fator 88 de Diferenciação Mieloide/metabolismo , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/antagonistas & inibidores , Virulência
10.
Toxins (Basel) ; 13(3)2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33800135

RESUMO

Current strategies for glioma treatment are only partly effective because of the poor selectivity for tumoral cells. Hence, the necessity to identify novel approaches is urgent. Recent studies highlighted the effectiveness of the bacterial protein cytotoxic necrotizing factor 1 (CNF1) in reducing tumoral mass, increasing survival of glioma-bearing mice and protecting peritumoral neural tissue from dysfunction. However, native CNF1 needs to be delivered into the brain, because of its incapacity to cross the blood-brain barrier (BBB) per se, thus hampering its clinical translation. To allow a non-invasive administration of CNF1, we here developed a chimeric protein (CTX-CNF1) conjugating CNF1 with chlorotoxin (CTX), a peptide already employed in clinics due to its ability of passing the BBB and selectively binding glioma cells. After systemic administration, we found that CTX-CNF1 is able to target glioma cells and significantly prolong survival of glioma-bearing mice. Our data point out the potentiality of CTX-CNF1 as a novel effective tool to treat gliomas.


Assuntos
Antineoplásicos/farmacologia , Toxinas Bacterianas/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Proteínas de Escherichia coli/farmacologia , Glioma/tratamento farmacológico , Venenos de Escorpião/farmacologia , Animais , Antineoplásicos/metabolismo , Toxinas Bacterianas/metabolismo , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Proteínas de Escherichia coli/metabolismo , Glioma/metabolismo , Glioma/patologia , Humanos , Injeções Intravenosas , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/farmacologia , Venenos de Escorpião/metabolismo
11.
PLoS Pathog ; 17(3): e1009320, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33662035

RESUMO

Humans are frequently exposed to bacterial genotoxins of the gut microbiota, such as colibactin and cytolethal distending toxin (CDT). In the present study, whole genome microarray-based identification of differentially expressed genes was performed in vitro on HT29 intestinal cells while following the ectopic expression of the active CdtB subunit of Helicobacter hepaticus CDT. Microarray data showed a CdtB-dependent upregulation of transcripts involved in positive regulation of autophagy concomitant with the downregulation of transcripts involved in negative regulation of autophagy. CdtB promotes the activation of autophagy in intestinal and hepatic cell lines. Experiments with cells lacking autophagy related genes, ATG5 and ATG7 infected with CDT- and colibactin-producing bacteria revealed that autophagy protects cells against the genotoxin-induced apoptotic cell death. Autophagy induction could also be associated with nucleoplasmic reticulum (NR) formation following DNA damage induced by these bacterial genotoxins. In addition, both genotoxins promote the accumulation of the autophagic receptor P62/SQSTM1 aggregates, which colocalized with foci concentrating the RNA binding protein UNR/CSDE1. Some of these aggregates were deeply invaginated in NR in distended nuclei together or in the vicinity of UNR-rich foci. Interestingly, micronuclei-like structures and some vesicles containing chromatin and γH2AX foci were found surrounded with P62/SQSTM1 and/or the autophagosome marker LC3. This study suggests that autophagy and P62/SQSTM1 regulate the abundance of micronuclei-like structures and are involved in cell survival following the DNA damage induced by CDT and colibactin. Similar effects were observed in response to DNA damaging chemotherapeutic agents, offering new insights into the context of resistance of cancer cells to therapies inducing DNA damage.


Assuntos
Autofagia/efeitos dos fármacos , Toxinas Bacterianas/farmacologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/efeitos dos fármacos , Proteína Sequestossoma-1/metabolismo , Autofagia/fisiologia , Núcleo Celular/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Helicobacter hepaticus/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Mutagênicos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína Sequestossoma-1/genética
12.
PLoS Pathog ; 17(2): e1009244, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33539469

RESUMO

Tc toxin is an exotoxin composed of three subunits named TcA, TcB and TcC. Structural analysis revealed that TcA can form homopentamer that mediates the cellular recognition and delivery processes, thus contributing to the host tropism of Tc toxin. N-glycans and heparan sulfates have been shown to act as receptors for several Tc toxins. Here, we performed two independent genome-wide CRISPR-Cas9 screens, and have validated glycans and sulfated glycosaminoglycans (sGAGs) as Tc toxin receptors also for previously uncharacterized Tc toxins. We found that TcdA1 form Photorhabdus luminescens W14 (TcdA1W14) can recognize N-glycans via the RBD-D domain, corroborating previous findings. Knockout of N-glycan processing enzymes specifically blocks the intoxication of TcdA1W14-assembled Tc toxin. On the other hand, our results showed that sGAG biosynthesis pathway is involved in the cell surface binding of TcdA2TT01 (TcdA2 from P. luminescens TT01). Competition assays and biolayer interferometry demonstrated that the sulfation group in sGAGs is required for the binding of TcdA2TT01. Finally, based on the conserved domains of representative TcA proteins, we have identified 1,189 putative TcAs from 1,039 bacterial genomes. These TcAs are categorized into five subfamilies. Each subfamily shows a good correlation with both genetic organization of the TcA protein(s) and taxonomic origin of the genomes, suggesting these subfamilies may utilize different mechanisms for cellular recognition. Taken together, our results support the previously described two different binding modalities of Tc toxins, leading to unique host targeting properties. We also present the bioinformatics data and receptor screening strategies for TcA proteins, provide new insights into understanding host specificity and biomedical applications of Tc toxins.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/farmacologia , Glicosaminoglicanos/química , Photorhabdus/metabolismo , Polissacarídeos/química , Compostos de Sulfidrila/química , Proteínas de Bactérias/genética , Células HeLa , Humanos , Photorhabdus/efeitos dos fármacos
13.
Toxins (Basel) ; 13(1)2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33466571

RESUMO

Since its introduction as a treatment for strabismus, botulinum toxin (BoNT) has had a phenomenal journey and is now recommended as first-line treatment for focal dystonia, despite short-term clinical benefits and the risks of adverse effects. To cater for the high demand across various medical specialties, at least six US Food and Drug Administration (FDA)-approved formulations of BoNT are currently available for diverse labelled indications. The toxo-pharmacological properties of these formulations are not uniform and thus should not be used interchangeably. Synthetic BoNTs and BoNTs from non-clostridial sources are not far from clinical use. Moreover, the study of mutations in naturally occurring toxins has led to modulation in the toxo-pharmacokinetic properties of BoNTs, including the duration and potency. We present an overview of the toxo-pharmacology of conventional and novel BoNT preparations, including those awaiting imminent translation from the laboratory to the clinic.


Assuntos
Toxinas Botulínicas/farmacologia , Toxinas Botulínicas/uso terapêutico , Composição de Medicamentos , Desenvolvimento de Medicamentos , Toxinas Bacterianas/farmacologia , Toxinas Bacterianas/uso terapêutico , Toxinas Botulínicas/química , Prescrições de Medicamentos , Humanos , Doenças do Sistema Nervoso/tratamento farmacológico , Fármacos Neuromusculares/farmacologia , Fármacos Neuromusculares/uso terapêutico , Junção Neuromuscular/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico
14.
Toxins (Basel) ; 13(1)2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33418946

RESUMO

Over the last few decades, proteins and peptides have become increasingly more common as FDA-approved drugs, despite their inefficient delivery due to their inability to cross the plasma membrane. In this context, bacterial two-component systems, termed AB toxins, use various protein-based membrane translocation mechanisms to deliver toxins into cells, and these mechanisms could provide new insights into the development of bio-based drug delivery systems. These toxins have great potential as therapies both because of their intrinsic properties as well as the modular characteristics of both subunits, which make them highly amenable to conjugation with various drug classes. This review focuses on the therapeutical approaches involving the internalization mechanisms of three representative AB toxins: botulinum toxin type A, anthrax toxin, and cholera toxin. We showcase several specific examples of the use of these toxins to develop new therapeutic strategies for numerous diseases and explain what makes these toxins promising tools in the development of drugs and drug delivery systems.


Assuntos
Toxinas Bacterianas/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia
15.
Invest Ophthalmol Vis Sci ; 62(1): 4, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33393970

RESUMO

Purpose: Endophthalmitis models have reported the virulent role of Panton-Valentine leucocidin (PVL) secreted by Staphylococcus aureus on the retina. PVL targets retinal ganglion cells (RGCs), expressing PVL membrane receptor C5aR. Interactions between PVL and retinal cells lead to glial activation, retinal inflammation, and apoptosis. In this study, we explored oxidative stress and retinal neurotransmitters in a rabbit retinal explant model incubated with PVL. Methods: Reactive oxygen species (ROS) production in RGCs has been assessed with fluorescent probes and immunohistochemistry. Nuclear magnetic resonance (NMR) spectroscopy quantified retinal concentrations of antioxidant molecules and neurotransmitters, and concentrations of neurotransmitters released in the culture medium. Quantifying the expression of some pro-inflammatory and anti-inflammatory factors was performed using RT-qPCR. Results: PVL induced a mitochondrial ROS production in RGCs after four hours' incubation with the toxin. Enzymatic sources of ROS, involving nicotinamide adenine dinucleotide phosphate-oxidase and xanthine oxidase, were also activated after four hours in PVL-treated retinal explants. Retinal antioxidants defenses, that is, glutathione, ascorbate and taurine, decreased after two hours' incubation with PVL. Glutamate retinal concentrations and glutamate release in the culture medium remained unaltered in PVL-treated retinas. GABA, glycine, and acetylcholine (Ach) retinal concentrations decreased after PVL treatment. Glycine release in the culture medium decreased, whereas Ach release increased after PVL treatment. Expression of proinflammatory and anti-inflammatory cytokines remained unchanged in PVL-treated explants. Conclusions: PVL activates oxidative pathways and alters neurotransmitter retinal concentrations and release, supporting the hypothesis that PVL could induce a neurogenic inflammation in the retina.


Assuntos
Toxinas Bacterianas/farmacologia , Exotoxinas/farmacologia , Leucocidinas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptor da Anafilatoxina C5a/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Staphylococcus aureus/química , Acetilcolina/metabolismo , Animais , Células Cultivadas , Meios de Cultura , Citocinas/metabolismo , Corantes Fluorescentes , Glicina/metabolismo , Espectroscopia de Ressonância Magnética , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NADP/metabolismo , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Células Ganglionares da Retina/metabolismo , Xantina Oxidase/metabolismo , Ácido gama-Aminobutírico/metabolismo
16.
Braz J Microbiol ; 52(2): 939-952, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33454924

RESUMO

Trueperella pyogenes (T. pyogenes) is a common opportunistic pathogen of many livestock and play an important regulation role during multibacterial infection and interaction with the host by its primary virulence factor pyolysin (PLO). The purpose of this study was to investigate the regulation role of PLO which serve as a combinational pathogen with lipopolysaccharide (LPS) during endometritis. In this study, the expression of bioactive recombinant PLO (rPLO) in a prokaryotic expression system and its purification are described. Moreover, we observed that rPLO inhibited the innate immune response triggered by LPS and that methyl-ß-cyclodextrin (MBCD) abrogated this inhibitory effect in goat endometrium stromal cells (gESCs). Additionally, we show from pharmacological and genetic studies that rPLO-induced autophagy represses gene expression by inhibiting NLRP3 inflammasome activation. Importantly, this study reported that ATF6 serves as a primary regulator of the cellular inflammatory reaction to rPLO. Overall, these observations suggest that T. pyogenes PLO could create an immunosuppressive environment for other pathogens invasion by regulating cellular signaling pathways.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Autofagia/efeitos dos fármacos , Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Endométrio/citologia , Proteínas Hemolisinas/farmacologia , Lipopolissacarídeos/farmacologia , Actinomycetaceae/patogenicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Feminino , Cabras , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Inflamação , Mediadores da Inflamação/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo
17.
Prep Biochem Biotechnol ; 51(1): 9-15, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32393098

RESUMO

Cancer is considered as a disease with high rates of mortality and morbidity. The limitations and side effects of common treatments have prompted the need for innovative cancer therapies. Furthermore, selectivity and targeting of cancer cells are crucial factors to successful treatment of cancer. One of these methods is the use of bacterial toxins including Bacillus anthracis toxin to aid cancer therapy. This toxin is composed of three polypeptides: protective factor (PA), lethal factor (LF), and edema factor (EF). PA can bind to various surface receptors of all types of human cells and it internalizes the lethal factor and edema factor subunits of the toxin in the cytosol. In the present study, we cloned and expressed the lef gene of B. anthracis as the lethal part of the toxin in Bacillus subtilis WB600 by a shuttle expression vector PHT4. The rLF made in B. subtilis is efficiently secreted by the host into the culture medium which facilitates downstream processing. The rLF can be used to study cancer treatment. Abbreviations: EF: edema factor; LF: lethal factor; PA: protective factor; rLF: recombinant lethal factor; rPAm: recombinant protective factor mutants; uPA: urokinase-type plasminogen activator; uPAR: urokinase-type plasminogen activator receptor.


Assuntos
Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/genética , Bacillus anthracis/genética , Bacillus anthracis/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/genética , Neoplasias/metabolismo , Antígenos de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica , Genes Bacterianos , Vetores Genéticos , Células HeLa , Humanos , Neoplasias/patologia , Plasmídeos/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
18.
Microbiol Res ; 242: 126642, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33191102

RESUMO

The nematode-bacterium pair Heterorhabditis indica-Photorhabdus akhurstii is a malleable model system to investigate mutualistic relations. A number of toxins produced by P. akhurstii allow the bacterium to kill the insect host. However, a few of these heterologously expressed toxins are orally active against different insects which possibly caused neglected attention to Photorhabdus toxins compared to Bt (Bacillus thuringiensis). In the current study, a functional subunit of orally active toxin complex (Tc) protein, TcaB (63 kDa), isolated from two strains of P. akhurstii namely IARI-SGHR2 and IARI-SGMS1, was tested for biological activity against Galleria mellonella. A force feeding-based administration of the toxin translated into LD50 values of 45.63-58.90 ng/g which was even lower compared to injection LD50 values (51.48-64.30 ng/g) at 48 h after inoculation. An oral uptake of 500 ng toxin caused extensive gut damage in G. mellonella during 6-24 h incubation period coupled with a gradual disruption of gut integrity leading to escape of TcaB into the hemocoel. This finding was supported by the cytotoxic and immune-stimulatory effect of TcaB in the insect hemocoel at 6-24 h after force feeding. The circulatory hemocyte numbers and cell viability was markedly reduced to 0.66-0.68 × 106 ml-1 and 49-52 %, respectively, in TcaB force fed insect at 24 h, compared to control (2.55 × 106 ml-1; 100 %). The hemolymph phenoloxidase (PO) activity was elevated by 10.2-fold in force fed larvae than control at 24 h. An in silico docking study revealed that TcaB putatively interacts with a number of G. mellonella receptor proteins in order to become a gut-active toxin. Present research reinforces the potential of gut-active Photorhabdus toxins for their inclusion in sustainable insect management tactics and strengthens the existing Bt-dominated management repository.


Assuntos
Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/farmacologia , Inseticidas/metabolismo , Inseticidas/farmacologia , Photorhabdus/metabolismo , Animais , Bacillus thuringiensis/metabolismo , Proteínas de Bactérias , Agentes de Controle Biológico/metabolismo , Agentes de Controle Biológico/farmacologia , Epitélio/efeitos dos fármacos , Epitélio/microbiologia , Controle de Insetos , Insetos , Larva , Mariposas
19.
Toxins (Basel) ; 13(1)2020 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-33374361

RESUMO

Pathogenic microorganisms produce various virulence factors, e.g., enzymes, cytotoxins, effectors, which trigger development of pathologies in infectious diseases. Cholera toxin (CT) produced by O1 and O139 serotypes of Vibrio cholerae (V. cholerae) is a major cytotoxin causing severe diarrhea. Cholix cytotoxin (Cholix) was identified as a novel eukaryotic elongation factor 2 (eEF2) adenosine-diphosphate (ADP)-ribosyltransferase produced mainly in non-O1/non-O139 V. cholerae. The function and role of Cholix in infectious disease caused by V. cholerae remain unknown. The crystal structure of Cholix is similar to Pseudomonas exotoxin A (PEA) which is composed of an N-terminal receptor-recognition domain and a C-terminal ADP-ribosyltransferase domain. The endocytosed Cholix catalyzes ADP-ribosylation of eEF2 in host cells and inhibits protein synthesis, resulting in cell death. In a mouse model, Cholix caused lethality with severe liver damage. In this review, we describe the mechanism underlying Cholix-induced cytotoxicity. Cholix-induced apoptosis was regulated by mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways, which dramatically enhanced tumor necrosis factor-α (TNF-α) production in human liver, as well as the amount of epithelial-like HepG2 cancer cells. In contrast, Cholix induced apoptosis in hepatocytes through a mitochondrial-dependent pathway, which was not stimulated by TNF-α. These findings suggest that sensitivity to Cholix depends on the target cell. A substantial amount of information on PEA is provided in order to compare/contrast this well-characterized mono-ADP-ribosyltransferase (mART) with Cholix.


Assuntos
Fatores de Ribosilação do ADP/farmacologia , Toxinas Bacterianas/farmacologia , Morte Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Vibrio cholerae/metabolismo , Animais , Linhagem Celular , Humanos
20.
Front Immunol ; 11: 527310, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33193301

RESUMO

Adjuvants enhance magnitude and duration of immune responses induced by vaccines. In this study we assessed in neonatal mice if and how the adjuvant LT-K63 given with a pneumococcal conjugate vaccine, Pnc1-TT, could affect the expression of tumor necrosis factor receptor (TNF-R) superfamily members, known to be involved in the initiation and maintenance of antibody responses; B cell activating factor receptor (BAFF-R) and B cell maturation antigen (BCMA) and their ligands, BAFF, and a proliferation inducing ligand (APRIL). Initially we assessed the maturation status of different B cell populations and their expression of BAFF-R and BCMA. Neonatal mice had dramatically fewer B cells than adult mice and the composition of different subsets within the B cell pool differed greatly. Proportionally newly formed B cells were most abundant, but they had diminished BAFF-R expression which could explain low proportions of marginal zone and follicular B cells observed. Limited BCMA expression was also detected in neonatal pre-plasmablasts/plasmablasts. LT-K63 enhanced vaccine-induced BAFF-R expression in splenic marginal zone, follicular and newly formed B cells, leading to increased plasmablast/plasma cells, and their enhanced expression of BCMA in spleen and bone marrow. Additionally, the induction of BAFF and APRIL expression occurred early in neonatal mice immunized with Pnc1-TT either with or without LT-K63. However, BAFF+ and APRIL+ cells in spleens were maintained at a higher level in mice that received the adjuvant. Furthermore, the early increase of APRIL+ cells in bone marrow was more profound in mice immunized with vaccine and adjuvant. Finally, we assessed, for the first time in neonatal mice, accessory cells of the plasma cell niche in bone marrow and their secretion of APRIL. We found that LT-K63 enhanced the frequency and APRIL expression of eosinophils, macrophages, and megakaryocytes, which likely contributed to plasma cell survival, even though APRIL+ cells showed a fast decline. All this was associated with enhanced, sustained vaccine-specific antibody-secreting cells in bone marrow and persisting vaccine-specific serum antibodies. Our study sheds light on the mechanisms behind the adjuvanticity of LT-K63 and identifies molecular pathways that should be triggered by vaccine adjuvants to induce sustained humoral immunity in early life.


Assuntos
Linfócitos B/imunologia , Toxinas Bacterianas/farmacologia , Enterotoxinas/farmacologia , Proteínas de Escherichia coli/farmacologia , Imunidade Humoral/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Linfócitos B/citologia , Camundongos
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