RESUMO
Acinetobacter baumannii is highly resistant to antimicrobial agents, and XDR strains have become widespread. A. baumannii has developed resistance to colistin, which is considered the last resort against XDR Gram-negative bacteria, mainly caused by lipooligosaccharide (LOS) phosphoethanolamine (pEtN) and/or galactosamine (GalN) modifications induced by mutations that activate the two-component system (TCS) pmrAB. Although PmrAB of A. baumannii has been recognized as a drug resistance factor, its function as TCS, including its regulatory genes and response factors, has not been fully elucidated. In this study, to clarify the function of PmrAB as TCS, we elucidated the regulatory genes (regulon) of PmrAB via transcriptome analysis using pmrAB-activated mutant strains. We discovered that PmrAB responds to low pH, Fe2+, Zn2+, and Al3+. A. baumannii selectively recognizes Fe2+ rather than Fe3+, and a novel region ExxxE, in addition to the ExxE motif sequence, is involved in the environmental response. Furthermore, PmrAB participates in the phosphoethanolamine modification of LOS on the bacterial surface in response to metal ions such as Al3+, contributing to the attenuation of Al3+ toxicity and development of resistance to colistin and polymyxin B in A. baumannii. This study demonstrates that PmrAB in A. baumannii not only regulates genes that play an important role in drug resistance but is also involved in responses to environmental stimuli such as metal ions and pH, and this stimulation induces LOS modification. This study reveals the importance of PmrAB in the environmental adaptation and antibacterial resistance emergence mechanisms of A. baumannii. IMPORTANCE: Antimicrobial resistance (AMR) is a pressing global issue in human health. Acinetobacter baumannii is notably high on the World Health Organization's list of bacteria for which new antimicrobial agents are urgently needed. Colistin is one of the last-resort drugs used against extensively drug-resistant (XDR) Gram-negative bacteria. However, A. baumannii has become increasingly resistant to colistin, primarily by modifying its lipooligosaccharide (LOS) via activating mutations in the two-component system (TCS) PmrAB. This study comprehensively elucidates the detailed mechanism of drug resistance of PmrAB in A. baumannii as well as its biological functions. Understanding the molecular biology of these molecules, which serve as drug resistance factors and are involved in environmental recognition mechanisms in bacteria, is crucial for developing fundamental solutions to the AMR problem.
Assuntos
Acinetobacter baumannii , Proteínas de Bactérias , Etanolaminas , Regulação Bacteriana da Expressão Gênica , Lipopolissacarídeos , Acinetobacter baumannii/genética , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/metabolismo , Lipopolissacarídeos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Etanolaminas/farmacologia , Etanolaminas/metabolismo , Antibacterianos/farmacologia , Metais/metabolismo , Metais/farmacologia , Fatores de TranscriçãoRESUMO
Subtilase cytotoxin (SubAB) is a major virulence factor produced by eae-negative Shiga-toxigenic Escherichia coli (STEC) that can cause fatal systemic complications. SubAB binds to target cells through multivalent interactions between its B-subunit pentamer and receptor molecules such as glycoproteins with a terminal N-glycolylneuraminic acid (Neu5Gc). We screened randomized multivalent peptide libraries synthesized on a cellulose membrane and identified a series of tetravalent peptides that efficiently bind to the receptor-binding region of the SubAB B-subunit pentamer. These peptides competitively inhibited the binding of the B-subunit to a receptor-mimic molecule containing clustered Neu5Gc (Neu5Gc-polymer). We selected the peptide with the highest inhibitory efficacy, FFP-tet, and covalently bound it to beads to synthesize FFP-tet-beads, a highly clustered SubAB absorber that displayed potency to absorb SubAB cytotoxicity through direct binding to the toxin. The efficacy of FFP-tet-beads to absorb SubAB cytotoxicity in solution was similar to that of Neu5Gc-polymer, suggesting that FFP-tet-beads might be an effective therapeutic agent against complications arising from eae-negative STEC infection.
Assuntos
Proteínas de Escherichia coli , Escherichia coli Shiga Toxigênica , Proteínas de Transporte/metabolismo , Celulose/metabolismo , Citotoxinas , Proteínas de Escherichia coli/metabolismo , Biblioteca de Peptídeos , Polímeros/metabolismo , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/metabolismo , Subtilisinas/toxicidade , Fatores de Virulência/metabolismoRESUMO
Subtilase cytotoxin (SubAB) is a member of bacterial AB5 toxin produced by certain enterohemorrhagic E. coli strains which cleaves host chaperone BiP in endoplasmic reticulum (ER), leading to ER stress-mediated cytotoxicity. Previous study suggested that protein disulfide isomerase (PDI), an enzyme which catalyzes the formation and breakage of disulfide bonds in proteins, regulates AB5 toxin such as cholera toxin by unfolding of A subunit, leading to its translocation into cytosol to induce disease. Although SubAB targets ER and has similar A subunit to that of other AB5 toxins, it is unclear whether PDI can modulate the SubAB function. Here we determined the role of PDI on SubAB-induced BiP cleavage, ER stress response and cytotoxicity in HeLa cells. We found that PDI knockdown significantly suppressed SubAB-induced BiP cleavage and eIF2α phosphorylation. The accumulation of SubAB in ER was perturbed upon PDI knockdown. Finally, cell viability assay showed that PDI knockdown and PDI inhibitor canceled the SubAB-induced cytotoxicity. Present results suggested that SubAB, after cellular uptake, translocates into ER and interacts with BiP that might be modulated by PDI. Identification of pivotal role of host proteins on bacterial toxin to elicit its pathogenesis is necessary basis for development of potential chemotherapy and new diagnostic strategy for control of toxin-producing bacterial infections.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Escherichia coli/toxicidade , Isomerases de Dissulfetos de Proteínas/metabolismo , Subtilisinas/toxicidade , Morte Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Proteínas de Choque Térmico/metabolismo , Interações entre Hospedeiro e Microrganismos/genética , Humanos , MAP Quinase Quinase 4/metabolismo , Fosforilação , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/genética , RNA Interferente PequenoRESUMO
Vibrio cholerae produced-Cholix toxin (Cholix) is a cytotoxin that ADP-ribosylates eukaryotic elongation factor 2, inhibiting protein synthesis, and inducing apoptosis. Here, we identified prohibitin (PHB) 1 and 2 as novel Cholix-interacting membrane proteins in immortalised human hepatocytes and HepG2 cells by Cholix immunoprecipitation assays. The expression level of PHB1 was decreased by Cholix after a 12hr incubation. Cholix-induced poly (ADP-ribose) polymerase (PARP) cleavage was significantly enhanced in PHB (PHB1 or PHB2) knockdown cells. In contrast, transiently overexpressed PHB in hepatocytes attenuated Cholix-induced Bax/Bak conformational changes and PARP cleavage. In addition, Cholix-induced reactive oxygen species production and accumulation of fragmented mitochondria were enhanced in PHB-knockdown cells. Furthermore, Cholix induced activation of Rho-associated coiled coil-containing protein kinase 1 (ROCK1), which was enhanced in PHB-knockdown cells, followed by actin filament depolymerisation and accumulation of tubulin in the blebbing cells. Inhibition of ROCK1 by siRNA or its inhibitor suppressed Cholix-induced PARP cleavage and reactive oxygen species generation. Our findings identify PHB as a new protein that interacts with Cholix and is involved in Cholix-induced mitochondrial dysfunction and cytoskeletal rearrangement by ROCK1 activation during apoptosis.
Assuntos
Fatores de Ribosilação do ADP/química , Toxinas Bacterianas/química , Fator de Iniciação 2 em Eucariotos/metabolismo , Interações Hospedeiro-Patógeno/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/genética , Vibrio cholerae/genética , ADP-Ribosilação , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/microbiologia , Citoesqueleto de Actina/ultraestrutura , Sequência de Aminoácidos , Apoptose/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Linhagem Celular Transformada , Fator de Iniciação 2 em Eucariotos/genética , Regulação da Expressão Gênica , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/microbiologia , Hepatócitos/patologia , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/microbiologia , Mitocôndrias/ultraestrutura , Proibitinas , Ligação Proteica , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Proteólise , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/deficiência , Transdução de Sinais , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Vibrio cholerae/metabolismo , Vibrio cholerae/patogenicidade , Virulência , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
Shiga-toxigenic Escherichia coli (STEC) is a major bacterium responsible for disease resulting from foodborne infection, including bloody diarrhea and hemolytic uremic syndrome. STEC produces important virulence factors such as Shiga toxin (Stx) 1 and/or 2. In the STEC family, some locus of enterocyte effacement-negative STEC produce two different types of cytotoxins, namely, Stx2 and subtilase cytotoxin (SubAB). The Stx2 and SubAB cytotoxins are structurally similar and composed of one A subunit and pentamer of B subunits. The catalytically active A subunit of SubAB is a subtilase-like serine protease and specifically cleaves an endoplasmic reticulum (ER) chaperone 78-kDa glucose-regulated protein (GRP78/BiP), a monomeric ATPase that is crucial in protein folding and quality control. The B subunit binds to cell surface receptors. SubAB recognizes sialic carbohydrate-modified cell surface proteins as a receptor. After translocation into cells, SubAB is delivered to the ER, where it cleaves GRP78/BiP. SubAB-catalyzed BiP cleavage induces ER stress, which causes various cell events including inhibition of protein synthesis, suppression of nuclear factor-kappa B activation, apoptotic cell death, and stress granules formation. In this review, we describe SubAB, the SubAB receptor, and the mechanism of cell response to the toxin.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli Shiga Toxigênica/metabolismo , Subtilisinas/metabolismo , Animais , Apoptose/fisiologia , Retículo Endoplasmático/fisiologia , Chaperona BiP do Retículo Endoplasmático , Proteínas de Escherichia coli/genética , Doenças Transmitidas por Alimentos/microbiologia , Humanos , Camundongos , Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismo , Escherichia coli Shiga Toxigênica/genética , Subtilisinas/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Poly(D,L-lactide-co-glycolic) acid (PLGA) is a synthetic copolymer that has been used to design micro/nanoparticles as a carrier for macromolecules, such as protein and nucleic acids, that can be internalized by the endocytosis pathway. However, it is difficult to control the intracellular delivery to target organelles. Here we report an intracellular delivery system of nanoparticles modified with bacterial cytotoxins to the endoplasmic reticulum (ER) and anti-inflammatory activity of the nanoparticles. Subtilase cytotoxin (SubAB) is a bacterial toxin in certain enterohemorrhagic Escherichia coli (EHEC) strains that cleaves the host ER chaperone BiP and suppresses nuclear factor-kappaB (NF-κB) activation and nitric oxide (NO) generation in macrophages at sub-lethal concentration. PLGA-nanoparticles were modified with oligo histidine-tagged (6 × His-tagged) recombinant SubAB (SubAB-PLGA) through a pH-sensitive linkage, and their translocation to the ER in macrophage cell line J774.1 cells, effects on inducible NO synthase (iNOS), and levels of tumor necrosis factor (TNF)-α cytokine induced by lipopolysaccharide (LPS) were examined. Compared with free SubAB, SubAB-PLGA was significantly effective in BiP cleavage and the induction of the ER stress marker C/EBP homologous protein (CHOP) in J774.1 cells. Furthermore, SubAB-PLGA attenuated LPS-stimulated induction of iNOS and TNF-α. Our findings provide useful information for protein delivery to macrophages and may encourage therapeutic applications of nanoparticles to the treatment of inflammatory diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Toxinas Bacterianas/farmacologia , Sistemas de Liberação de Medicamentos , Macrófagos/efeitos dos fármacos , Nanopartículas/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Animais , Anti-Inflamatórios/química , Toxinas Bacterianas/química , Células Cultivadas , Portadores de Fármacos/química , Escherichia coli/química , Concentração de Íons de Hidrogênio , Camundongos , Estrutura Molecular , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/síntese química , Propriedades de SuperfícieRESUMO
Subtilase cytotoxin (SubAB) is mainly produced by locus of enterocyte effacement (LEE)-negative strains of Shiga-toxigenic Escherichia coli (STEC). SubAB cleaves an endoplasmic reticulum (ER) chaperone, BiP/Grp78, leading to induction of ER stress. This stress causes activation of ER stress sensor proteins and induction of caspase-dependent apoptosis. We found that SubAB induces stress granules (SG) in various cells. Aim of this study was to explore the mechanism by which SubAB induced SG formation. Here, we show that SubAB-induced SG formation is regulated by activation of double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK). The culture supernatant of STEC O113:H21 dramatically induced SG in Caco2 cells, although subAB knockout STEC O113:H21 culture supernatant did not. Treatment with phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, and lysosomal inhibitors, NH4 Cl and chloroquine, suppressed SubAB-induced SG formation, which was enhanced by PKC and PKD inhibitors. SubAB attenuated the level of PKD1 phosphorylation. Depletion of PKCδ and PKD1 by siRNA promoted SG formation in response to SubAB. Furthermore, death-associated protein 1 (DAP1) knockdown increased basal phospho-PKD1(S916) and suppressed SG formation by SubAB. However, SG formation by an ER stress inducer, Thapsigargin, was not inhibited in PMA-treated cells. Our findings show that SubAB-induced SG formation is regulated by the PERK/DAP1 signalling pathway, which may be modulated by PKCδ/PKD1, and different from the signal transduction pathway that results in Thapsigargin-induced SG formation.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli Shiga Toxigênica/metabolismo , Subtilisinas/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Células CACO-2/efeitos dos fármacos , Células CACO-2/metabolismo , Células CACO-2/microbiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cloroquina/farmacologia , Meios de Cultivo Condicionados/farmacologia , DNA Helicases , Chaperona BiP do Retículo Endoplasmático , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/farmacologia , Técnicas de Inativação de Genes , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Proteínas de Ligação a Poli-ADP-Ribose , Proteína Quinase C-delta/metabolismo , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Escherichia coli Shiga Toxigênica/patogenicidade , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Subtilisinas/genética , Subtilisinas/farmacologia , eIF-2 Quinase/metabolismoRESUMO
The novel cytotoxic factor subtilase cytotoxin (SubAB) is produced mainly by non-O157 Shiga-toxigenic Escherichia coli (STEC). SubAB cleaves the molecular chaperone BiP/GRP78 in the endoplasmic reticulum (ER), leading to activation of RNA-dependent protein kinase (PKR)-like ER kinase (PERK), followed by caspase-dependent cell death. However, the SubAB uptake mechanism in HeLa cells is unknown. In this study, a variety of inhibitors and siRNAs were employed to characterize the SubAB uptake process. SubAB-induced BiP cleavage was inhibited by high concentrations of Dynasore, and methyl-ß-cyclodextrin (mßCD) and Filipin III, but not suppressed in clathrin-, dynamin I/II-, caveolin1- and caveolin2-knockdown cells. We observed that SubAB treatment led to dramatic actin rearrangements, e.g. formation of plasma membrane blebs, with a significant increase in fluid uptake. Confocal microscopy analysis showed that SubAB uptake required actin cytoskeleton remodelling and lipid raft cholesterol. Furthermore, internalized SubAB in cells was found in the detergent-resistant domain (DRM) structure. Interestingly, IPA-3, an inhibitor of serine/threonine kinase p21-activated kinase (PAK1), an important protein of macropinocytosis, directly inhibited SubAB-mediated BiP cleavage and SubAB internalization. Thus, our findings suggest that SubAB uses lipid raft- and actin-dependent, but not clathrin-, caveolin- and dynamin-dependent pathways as its major endocytic translocation route.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Microdomínios da Membrana/metabolismo , Escherichia coli Shiga Toxigênica/patogenicidade , Subtilisinas/metabolismo , Antivirais/farmacologia , Transporte Biológico , Caveolinas/genética , Linhagem Celular , Colesterol/metabolismo , Clatrina/genética , Dissulfetos/farmacologia , Dinaminas/genética , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Ativação Enzimática , Filipina/farmacologia , Células HeLa , Humanos , Hidrazonas/farmacologia , Naftóis/farmacologia , Interferência de RNA , RNA Interferente Pequeno , beta-Ciclodextrinas/farmacologia , eIF-2 Quinase/metabolismo , Quinases Ativadas por p21/antagonistas & inibidoresRESUMO
Autophagy and apoptosis play critical roles in cellular homeostasis and survival. Subtilase cytotoxin (SubAB), produced by non-O157 type Shiga-toxigenic Escherichia coli (STEC), is an important virulence factor in disease. SubAB, a protease, cleaves a specific site on the endoplasmic reticulum (ER) chaperone protein BiP/GRP78, leading to ER stress, and induces apoptosis. Here we report that in HeLa cells, activation of a PERK (RNA-dependent protein kinase [PKR]-like ER kinase)-eIF2α (α subunit of eukaryotic initiation factor 2)-dependent pathway by SubAB-mediated BiP cleavage negatively regulates autophagy and induces apoptosis through death-associated protein 1 (DAP1). We found that SubAB treatment decreased the amounts of autophagy markers LC3-II and p62 as well as those of mTOR (mammalian target of rapamycin) signaling proteins ULK1 and S6K. These proteins showed increased expression levels in PERK knockdown or DAP1 knockdown cells. In addition, depletion of DAP1 in HeLa cells dramatically inhibited the SubAB-stimulated apoptotic pathway: SubAB-induced Bax/Bak conformational changes, Bax/Bak oligomerization, cytochrome c release, activation of caspases, and poly(ADP-ribose) polymerase (PARP) cleavage. These results show that DAP1 is a key regulator, through PERK-eIF2α-dependent pathways, of the induction of apoptosis and reduction of autophagy by SubAB.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Autofagia/fisiologia , Proteínas de Escherichia coli/metabolismo , Regulação da Expressão Gênica/imunologia , Subtilisinas/metabolismo , Proteínas Reguladoras de Apoptose/genética , Chaperona BiP do Retículo Endoplasmático , Escherichia coli , Proteínas de Escherichia coli/genética , Células HeLa , Humanos , Subtilisinas/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Endotoxins, or lipopolysaccharides (LPS), are potent immunostimulatory molecules of critical concern in bacterial recombinant protein expression systems. The gram-negative bacterium Acinetobacter baumannii exhibits an interesting and unique phenotype characterized by the complete loss of LPS. In this study, we developed a novel system for producing recombinant proteins completely devoid of endotoxin contamination using LPS-deficient A. baumannii. We purified endotoxin-free functional green fluorescent protein, which reduced endotoxin contamination by approximately three orders of magnitude, and also purified the functional cytokine tumor necrosis factor (TNF)-α. Additionally, utilization of the Omp38 signal peptide of A. baumannii enabled the extracellular production of variable domain of heavy chain of heavy chain (VHH) antibodies. With these advantages, mNb6-tri-20aa, a multivalent VHH that specifically binds to the spike protein of severe acute respiratory syndrome coronavirus 2, was purified from the culture supernatant, and endotoxin contamination was reduced by a factor of approximately 2 × 105 compared with that in conventional expression systems. A virus neutralization assay demonstrated the functionality of the purified antibody in suppressing viral infections. Moreover, we applied our system to produce ozoralizumab, a multispecific VHH that binds to human TNF-α and albumin and are marketed as a rheumatoid arthritis drug. We successfully purified a functional antibody from endotoxin contamination. This system establishes a new, completely endotoxin-free platform for the expression of recombinant proteins, which distinguishes it from other bacterial expression systems, and holds promise for future applications.
RESUMO
In Helicobacter pylori infection, vacuolating cytotoxin (VacA)-induced mitochondrial damage leading to apoptosis is believed to be a major cause of cell death. It has also been proposed that VacA-induced autophagy serves as a host mechanism to limit toxin-induced cellular damage. Apoptosis and autophagy are two dynamic and opposing processes that must be balanced to regulate cell death and survival. Here we identify the low-density lipoprotein receptor-related protein-1 (LRP1) as the VacA receptor for toxin-induced autophagy in the gastric epithelial cell line AZ-521, and show that VacA internalization through binding to LRP1 regulates the autophagic process including generation of LC3-II from LC3-I, which is involved in formation of autophagosomes and autolysosomes. Knockdown of LRP1 and Atg5 inhibited generation of LC3-II as well as cleavage of PARP, a marker of apoptosis, in response to VacA, whereas caspase inhibitor, benzyloxycarbonyl-VAD-fluoromethylketone (Z-VAD-fmk), and necroptosis inhibitor, Necrostatin-1, did not inhibit VacA-induced autophagy, suggesting that VacA-induced autophagy via LRP1 binding precedes apoptosis. Other VacA receptors such as RPTPα, RPTPß, and fibronectin did not affect VacA-induced autophagy or apoptosis. Therefore, we propose that the cell surface receptor, LRP1, mediates VacA-induced autophagy and apoptosis.
Assuntos
Apoptose , Autofagia , Proteínas de Bactérias/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Proteína 5 Relacionada à Autofagia , Linhagem Celular , Inibidores de Cisteína Proteinase/farmacologia , Epitélio/metabolismo , Epitélio/microbiologia , Epitélio/patologia , Fibronectinas/genética , Fibronectinas/metabolismo , Mucosa Gástrica/metabolismo , Infecções por Helicobacter/genética , Infecções por Helicobacter/patologia , Helicobacter pylori/genética , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Estômago/microbiologia , Estômago/patologiaRESUMO
The vacuolating cytotoxin VacA produced by Helicobacter pylori causes massive cellular vacuolation in vitro and gastric tissue damage in vivo, leading to gastric ulcers, when administered intragastrically. Here we report that mice deficient in protein tyrosine phosphatase receptor type Z (Ptprz, also called PTP-zeta or RPTP-beta, encoded by Ptprz) do not show mucosal damage by VacA, although VacA is incorporated into the gastric epithelial cells to the same extent as in wild-type mice. Primary cultures of gastric epithelial cells from Ptprz+/+ and Ptprz-/- mice also showed similar incorporation of VacA, cellular vacuolation and reduction in cellular proliferation, but only Ptprz+/+ cells showed marked detachment from a reconstituted basement membrane 24 h after treatment with VacA. VacA bound to Ptprz, and the levels of tyrosine phosphorylation of the G protein-coupled receptor kinase-interactor 1 (Git1), a Ptprz substrate, were higher after treatment with VacA, indicating that VacA behaves as a ligand for Ptprz. Furthermore, pleiotrophin (PTN), an endogenous ligand of Ptprz, also induced gastritis specifically in Ptprz+/+ mice when administered orally. Taken together, these data indicate that erroneous Ptprz signaling induces gastric ulcers.
Assuntos
Proteínas de Bactérias/fisiologia , Proteínas de Ciclo Celular , Infecções por Helicobacter/etiologia , Fosfoproteínas , Proteínas Tirosina Fosfatases/deficiência , Úlcera Gástrica/etiologia , Animais , Proteínas de Bactérias/toxicidade , Feminino , Proteínas Ativadoras de GTPase/metabolismo , Gastrite/etiologia , Gastrite/patologia , Infecções por Helicobacter/patologia , Helicobacter pylori/patogenicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/fisiologia , Transdução de Sinais , Úlcera Gástrica/patologia , VirulênciaRESUMO
Intracellular bacteria are able to survive and grow in host cells and often cause serious infectious diseases. The B subunit of the subtilase cytotoxin (SubB) found in enterohemorrhagic Escherichia coli O113:H21 recognizes sialoglycans on cell surfaces and triggers the uptake of cytotoxin by the cells, meaning that Sub B is a ligand molecule that is expected to be useful for drug delivery into cells. In this study, we conjugated SubB to silver nanoplates (AgNPLs) for use as an antibacterial drug and examined their antimicrobial activity against intracellularly infecting Salmonella typhimurium (S. typhimurium). The modification of AgNPLs with SubB improved their dispersion stability and antibacterial activity against planktonic S. typhimurium. The SubB modification enhanced the cellular uptake of AgNPLs, and intracellularly infecting S. typhimurium were killed at low concentrations of AgNPLs. Interestingly, larger amounts of SubB-modified AgNPLs were taken up by infected cells compared with uninfected cells. These results suggest that the S. typhimurium infection activated the uptake of the nanoparticles into the cells. SubB-modified AgNPLs are expected to be useful bactericidal systems for intracellularly infecting bacteria.
Assuntos
Anti-Infecciosos , Toxinas Bacterianas , Prata/farmacologia , Prata/química , Escherichia coli/metabolismo , Toxinas Bacterianas/metabolismo , Antibacterianos/farmacologia , Antibacterianos/química , Citotoxinas/química , Citotoxinas/metabolismo , Anti-Infecciosos/metabolismoRESUMO
Cholix toxin (Cholix) is a novel ADP-ribosylating cytotoxin produced by Vibrio cholerae, which utilizes eukaryotic elongation factor 2 as a substrate and acts by a mechanism similar to that of diphtheria toxin and Pseudomonas exotoxin A. First it was found that Cholix-treated HeLa cells exhibited caspase-dependent apoptosis, whereas intestinal cells such as Caco-2, HCT116, and RKO did not. Here we investigated Cholix-induced cell death signaling pathways in HeLa cells. Cholix-induced cytochrome c release into cytosol was initiated by specific conformational changes of pro-apoptotic Bak associated with Bax. Silencing of bak/bax genes or bak gene alone using siRNA significantly suppressed cytochrome c release and caspase-7 activation, but not activation of caspases-3 and -9. Although pretreatment with a caspase-8 inhibitor (Z-IETD-FMK) reduced Cholix-induced cytochrome c release and activation of caspases-3, -7, and -9, cytotoxicity was not decreased. Pretreatment with Z-YVAD-FMK, which inhibits caspase-1, -4, and -5, suppressed not only cytochrome c release, activation of caspase-3, -7, -8, or -9, and PARP cleavage, but also cytotoxicity, indicating that caspase-1, -4, and -5 activation is initiated at an early stage of Cholix-induced apoptosis and promotes caspase-8 activation. These results show that the inflammatory caspases (caspase-1, -4, and -5) and caspase-8 are responsible for both mitochondrial signals and other caspase activation. In conclusion, we showed that Cholix-induced caspase activation plays an essential role in generation of apoptotic signals, which are mediated by both mitochondria-dependent and -independent pathways.
Assuntos
Fatores de Ribosilação do ADP/farmacologia , Apoptose/efeitos dos fármacos , Toxinas Bacterianas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Vibrio cholerae/química , Fatores de Ribosilação do ADP/química , Apoptose/genética , Toxinas Bacterianas/química , Inibidores de Caspase , Caspases/genética , Caspases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Citocromos c/genética , Citocromos c/metabolismo , Ativação Enzimática , Inativação Gênica , Células HeLa , Humanos , Oligopeptídeos/farmacologia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Proteólise/efeitos dos fármacos , RNA Interferente Pequeno/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Shiga-toxigenic Escherichia coli (STEC) produces subtilase cytotoxin (SubAB), which cleaves the molecular chaperone BiP in the endoplasmic reticulum (ER), leading to an ER stress response and then activation of apoptotic signaling pathways. Here, we show that an early event in SubAB-induced apoptosis in HeLa cells is mediated by RNA-dependent protein kinase (PKR)-like ER kinase (PERK), not activating transcription factor 6 (ATF6) or inositol-requiring enzyme 1(Ire1), two other ER stress sensors. PERK knockdown suppressed SubAB-induced eIF2α phosphorylation, activating transcription factor 4 (ATF4) expression, caspase activation, and cytotoxicity. Knockdown of eIF2α by small interfering RNA (siRNA) or inhibition of eIF2α dephosphorylation by Sal003 enhanced SubAB-induced caspase activation. Treatment with proteasome inhibitors (i.e., MG132 and lactacystin), but not a general caspase inhibitor (Z-VAD) or a lysosome inhibitor (chloroquine), suppressed SubAB-induced caspase activation and poly(ADP-ribose) polymerase (PARP) cleavage, suggesting that the ubiquitin-proteasome system controls events leading to caspase activation, i.e., Bax/Bak conformational changes, followed by cytochrome c release from mitochondria. Levels of ubiquitinated proteins in HeLa cells were significantly decreased by SubAB treatment. Further, in an early event, some antiapoptotic proteins, which normally turn over rapidly, have their synthesis inhibited, and show enhanced degradation via the proteasome, resulting in apoptosis. In PERK knockdown cells, SubAB-induced loss of ubiquitinated proteins was inhibited. Thus, SubAB-induced ER stress is caused by BiP cleavage, leading to PERK activation, not by accumulation of ubiquitinated proteins, which undergo PERK-dependent degradation via the ubiquitin-proteasome system.
Assuntos
Apoptose/fisiologia , Proteínas de Escherichia coli/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Subtilisinas/farmacologia , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Degradação Associada com o Retículo Endoplasmático/fisiologia , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Fosforilação , Complexo de Endopeptidases do Proteassoma/genética , Transdução de Sinais , eIF-2 Quinase/genéticaRESUMO
Subtilase cytotoxin (SubAB), which is produced by certain strains of Shiga-toxigenic Escherichia coli (STEC), cleaves an endoplasmic reticulum (ER) chaperone, BiP/Grp78, leading to induction of ER stress and caspase-dependent apoptosis. SubAB alters the innate immune response. SubAB pretreatment of macrophages inhibited lipopolysaccharide (LPS)-induced production of both monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis factor α (TNF-α). We investigated here the mechanism by which SubAB inhibits nitric oxide (NO) production by mouse macrophages. SubAB suppressed LPS-induced NO production through inhibition of inducible NO synthase (iNOS) mRNA and protein expression. Further, SubAB inhibited LPS-induced IκB-α phosphorylation and nuclear localization of the nuclear factor-κB (NF-κB) p65/p50 heterodimer. Reporter gene and chromatin immunoprecipitation (ChIP) assays revealed that SubAB reduced LPS-induced NF-κB p65/p50 heterodimer binding to an NF-κB binding site on the iNOS promoter. In contrast to the native toxin, a catalytically inactivated SubAB mutant slightly enhanced LPS-induced iNOS expression and binding of NF-κB subunits to the iNOS promoter. The SubAB effect on LPS-induced iNOS expression was significantly reduced in macrophages from NF-κB1 (p50)-deficient mice, which lacked a DNA-binding subunit of the p65/p50 heterodimer, suggesting that p50 was involved in SubAB-mediated inhibition of iNOS expression. Treatment of macrophages with an NOS inhibitor or expression of SubAB by E. coli increased E. coli survival in macrophages, suggesting that NO generated by macrophages resulted in efficient killing of the bacteria and SubAB contributed to E. coli survival in macrophages. Thus, we hypothesize that SubAB might represent a novel bacterial strategy to circumvent host defense during STEC infection.
Assuntos
Proteínas de Escherichia coli/farmacologia , Escherichia coli/metabolismo , Macrófagos/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/genética , Subtilisinas/farmacologia , Animais , Sobrevivência Celular , Células Cultivadas , Chaperona BiP do Retículo Endoplasmático , Regulação da Expressão Gênica , Immunoblotting , Imunoprecipitação , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Camundongos , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de SinaisRESUMO
Infection of mice with Citrobacter rodentium is a useful model for studying the pathogenicity of enteropathogenic and enterohemorrhagic Escherichia coli, pathogens that have a close association with humans. Here, we provide a protocol detailing the approaches for non-canonical inflammasome analysis in a mouse model of C. rodentium infection, including preparation of bacteria, oral administration of bacteria to mice, counting colony-forming units to quantify bacterial colonization, and analysis of expression and activation of inflammasome-related factors. For complete details on the use and execution of this protocol, please refer to Tsutsuki et al. (2022).
Assuntos
Citrobacter rodentium , Inflamassomos , Humanos , Animais , Camundongos , VirulênciaRESUMO
Advances in drug delivery systems (DDSs) have enabled the specific delivery of drugs to target cells. Subtilase cytotoxin (SubAB) produced by certain enterohemorrhagic Escherichia coli strains induces endoplasmic reticulum (ER) stress and suppresses nitric oxide generation in macrophages. We previously reported that modification of SubAB with poly(D,L-lactide-co-glycolic) acid (PLGA) nanoparticles (SubAB-PLGA NPs) increased intracellular uptake of SubAB and had an anti-inflammatory effect on macrophages. However, specific delivery of SubAB to macrophages could not be achieved because its effects on other cell types were not negligible. Therefore, to suppress non-specific SubAB binding, we used low-binding mutant SubABS35A (S35A) in which the 35th serine of the B subunit was mutated to alanine. In a macrophage cell line, PLGA NPs modified with S35A (S35A-PLGA NPs) induced ER stress and had anti-inflammatory effects similar to WT-PLGA NPs. However, in an epithelial cell line, S35A-PLGA NPs induced lower ER stress than WT-PLGA NPs. These results suggest that S35A is selectively delivered to macrophages rather than epithelial cells by modification with PLGA NPs and exerts anti-inflammatory effects. Our findings provide a useful technique for protein delivery to macrophages and encourage medical applications of DDSs for the treatment of inflammatory diseases.
RESUMO
Bexarotene selectively activates retinoid X receptor, which is a commonly used anticancer agent for cutaneous T-cell lymphoma. In this study, we aimed to investigate the anticancer effect of bexarotene and its underlying mechanism in ovarian cancer in vitro. The ES2 and NIH:OVACAR3 ovarian cancer cell lines were treated with 0, 5, 10, or 20 µM of bexarotene. After 24 h, cell number measurement and lactate dehydrogenase (LDH) cytotoxicity assay were performed. The effect of bexarotene on CDKN1A expression, cell cycle-related protein, cell cycle, pyroptosis, and apoptosis was evaluated. Bexarotene reduced cell proliferation in all concentrations in both the cells. At concentrations of > 10 µM, extracellular LDH activity increased with cell rupture. Treatment using 10 µM of bexarotene increased CDKN1A mRNA levels, decreased cell cycle-related protein expression, and increased the sub-G1 cell population in both cells. In ES2 cells, caspase-4 and GSDME were activated, whereas caspase-3 was not, indicating that bexarotene-induced cell death might be pyroptosis. A clinical setting concentration of bexarotene induced cell death through caspase-4-mediated pyroptosis in ovarian cancer cell lines. Thus, bexarotene may serve as a novel therapeutic agent for ovarian cancer.
Assuntos
Neoplasias Ovarianas , Neoplasias Cutâneas , Bexaroteno/farmacologia , Carcinoma Epitelial do Ovário , Proteínas de Ciclo Celular , Morte Celular , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Piroptose , Neoplasias Cutâneas/patologiaRESUMO
Subtilase cytotoxin (SubAB) is an AB5 toxin mainly produced by the locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli (STEC) strain such as O113:H21, yet the contribution of SubAB to STEC infectious disease is unclear. We found that SubAB reduced activation of the STEC O113:H21 infection-induced non-canonical NLRP3 inflammasome and interleukin (IL)-1ß and IL-18 production in murine macrophages. Downstream of lipopolysaccharide signaling, SubAB suppressed caspase-11 expression by inhibiting interferon-ß/STAT1 signaling, followed by disrupting formation of the NLRP3/caspase-1 assembly. These inhibitions were regulated by PERK/IRE1α-dependent endoplasmic reticulum (ER) stress signaling initiated by cleavage of the host ER chaperone BiP by SubAB. Our murine model of SubAB-producing Citrobacter rodentium demonstrated that SubAB promoted C. rodentium proliferation and worsened symptoms such as intestinal hyperplasia and diarrhea. These findings highlight the inhibitory effect of SubAB on the NLRP3 inflammasome via ER stress, which may be associated with STEC survival and infectious disease pathogenicity in hosts.