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1.
Nat Struct Mol Biol ; 27(3): 288-296, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32123390

RESUMO

The iota toxin produced by Clostridium perfringens type E is a binary toxin comprising two independent polypeptides: Ia, an ADP-ribosyltransferase, and Ib, which is involved in cell binding and translocation of Ia across the cell membrane. Here we report cryo-EM structures of the translocation channel Ib-pore and its complex with Ia. The high-resolution Ib-pore structure demonstrates a similar structural framework to that of the catalytic ϕ-clamp of the anthrax protective antigen pore. However, the Ia-bound Ib-pore structure shows a unique binding mode of Ia: one Ia binds to the Ib-pore, and the Ia amino-terminal domain forms multiple weak interactions with two additional Ib-pore constriction sites. Furthermore, Ib-binding induces tilting and partial unfolding of the Ia N-terminal α-helix, permitting its extension to the ϕ-clamp gate. This new mechanism of N-terminal unfolding is crucial for protein translocation.


Assuntos
ADP Ribose Transferases/química , Antígenos de Bactérias/química , Toxinas Bacterianas/química , Clostridium perfringens/química , Subunidades Proteicas/química , ADP Ribose Transferases/genética , ADP Ribose Transferases/metabolismo , Sequência de Aminoácidos , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Sítios de Ligação , Clonagem Molecular , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Clostridium perfringens/patogenicidade , Microscopia Crioeletrônica , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
2.
Proc Natl Acad Sci U S A ; 117(2): 1049-1058, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31896582

RESUMO

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


Assuntos
ADP Ribose Transferases/química , ADP Ribose Transferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Clostridium difficile/metabolismo , Enterotoxinas/química , Enterotoxinas/metabolismo , ADP Ribose Transferases/genética , Animais , Proteínas de Bactérias/genética , Sítios de Ligação , Fenômenos Biofísicos , Chlorocebus aethiops , Microscopia Crioeletrônica , Cristalografia por Raios X , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Domínios Proteicos , Células Vero
3.
Int J Mol Sci ; 20(20)2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31615004

RESUMO

Plant pathogens secrete proteins called effectors into the cells of their host to modulate the host immune response against colonization. Effectors can either modify or arrest host target proteins to sabotage the signaling pathway, and therefore are considered potential drug targets for crop disease control. In earlier research, the Xanthomonas type III effector XopAI was predicted to be a member of the arginine-specific mono-ADP-ribosyltransferase family. However, the crystal structure of XopAI revealed an altered active site that is unsuitable to bind the cofactor NAD+, but with the capability to capture an arginine-containing peptide from XopAI itself. The arginine peptide consists of residues 60 through 69 of XopAI, and residue 62 (R62) is key to determining the protein-peptide interaction. The crystal structure and the molecular dynamics simulation results indicate that specific arginine recognition is mediated by hydrogen bonds provided by the backbone oxygen atoms from residues W154, T155, and T156, and a salt bridge provided by the E265 sidechain. In addition, a protruding loop of XopAI adopts dynamic conformations in response to arginine peptide binding and is probably involved in target protein recognition. These data suggest that XopAI binds to its target protein by the peptide-binding ability, and therefore, it promotes disease progression. Our findings reveal an unexpected and intriguing function of XopAI and pave the way for further investigation on the role of XopAI in pathogen invasion.


Assuntos
ADP Ribose Transferases/química , Arginina/química , Peptídeos/química , Xanthomonas/química , ADP Ribose Transferases/genética , Sequência de Aminoácidos/genética , Arginina/genética , Domínio Catalítico/genética , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Oxigênio/química , Peptídeos/genética , Plantas/genética , Plantas/microbiologia , Ligação Proteica , Conformação Proteica , Transdução de Sinais/genética , Xanthomonas/enzimologia , Xanthomonas/patogenicidade
4.
DNA Cell Biol ; 38(8): 773-785, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31339741

RESUMO

Pierisin-5 protein (pie-5) belongs to a family of proteins possessing DNA-dependent ADP-ribosyltransferase activity, which can induce apoptotic cell death. The baculovirus-mediated expression vector system (BEVS) has been commonly used for in vitro expression of heterologous protein subunits for basic scientific research, in addition to the development and production of diagnostics and vaccines. In this study, a new method for the in vitro expression of the cytotoxic protein was established using the baculovirus expression system. The antiproliferative and apoptotic effect of the novel recombinant pierisin-5 protein (rpie-5) was investigated in different human cancer cell lines, such as HeLa, HepG2, and AGS. Cloning, in vitro overexpression, and purification of the rpie-5 protein were performed by using BEVS in Sf21 (Spodoptera frugiperda) insect cell line. The rpie-5 protein exhibits cytotoxicity in all the cell lines, but HeLa (IC50 0.6 µg/mL) was more sensitive when compared with HepG2 (IC50 1.9 µg/mL) and AGS (IC50 3.7 µg/mL) cell lines. The cytotoxic effects of rpie-5 lead to apoptotic cell death in cancer cells and resulted in nuclear fragmentation, enlargement of the nucleus, loss of mitochondrial membrane potential, and finally release of lactose dehydrogenase (LDH) enzyme from the cell membrane. This study reports the molecular mechanism of apoptotic cell death through the upregulation of Bax (Bcl-2 family activating protein-X), Bad, APAF-1 (apoptotic protease activating factor-1), Cyt-c, and caspase-3/9 and the downregulation of Bcl-2 (B-cell lymphoma 2) in rpie-5-treated cancer cells. The study concludes that rpie-5 has p53-independent apoptosis in HepG2 cells and p53-dependent apoptosis in HeLa and AGS cell lines. In the future, this study helps to understand the molecular mechanism of rpie-5 to induction of apoptosis and cell death.


Assuntos
ADP Ribose Transferases/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Insetos/farmacologia , Proteínas Recombinantes/farmacologia , ADP Ribose Transferases/genética , Animais , Apoptose/fisiologia , Baculoviridae/genética , Linhagem Celular Tumoral , Clonagem Molecular , Humanos , Proteínas de Insetos/genética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Engenharia de Proteínas/métodos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes/genética , Células Sf9 , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/genética
5.
Vet Microbiol ; 235: 63-70, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31282380

RESUMO

Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum (SG) causes fowl typhoid (FT) and substantial economic loss in Korea due to egg drop syndrome and mortality. Despite the extensive use of vaccines, FT still occurs in the field. Therefore, the emergence of more pathogenic SG or the recovered pathogenicity of a vaccine strain has been suspected. SpvB, an ADP-ribosyl transferase, is a major pathogenesis determinant, and the length of the polyproline linker (PPL) of SpvB affects pathogenic potency. SG strains accumulate pseudogenes in their genomes during host adaptation, and pseudogene profiling may provide evolutionary information. In this study, we found that the PPL length of Korean SG isolates varied from 11 to 21 prolines and was longer than that of a live vaccine strain, SG 9R (9 prolines). According to growth competition in chickens, the growth of an SG isolate with a PPL length of 17 prolines exceeded that of an SG isolate with a PPL length of 15 prolines. We investigated the pseudogenes of the field isolates, SG 9R and reference strains in GenBank by resequencing and comparative genomics. The pseudogene profiles of the field isolates were notably different from those of the foreign SG strains, and they were subdivided into 7 pseudogene subgroups. Collectively, the field isolates had gradually evolved by changing PPL length and acquiring additional pseudogenes. Thus, the characterization of PPL length and pseudogene profiling may be useful to understand the molecular evolution of SG and the epidemiology of FT.


Assuntos
Galinhas/microbiologia , Evolução Molecular , Doenças das Aves Domésticas/microbiologia , Salmonelose Animal/microbiologia , Salmonella enterica/genética , ADP Ribose Transferases/genética , Animais , Surtos de Doenças , Ligantes , Peptídeos/genética , Pseudogenes , República da Coreia , Salmonella enterica/isolamento & purificação , Sorogrupo
6.
Comp Immunol Microbiol Infect Dis ; 65: 207-212, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31300115

RESUMO

C2 toxin produced from Clostridium botulinum serotypes C and D has a potential role in many pathophysiological mechanisms in birds and animals. It has encompassed an ADP ribosyltransferase subunit (C2I) and a translocation/binding subunit (C2II). In the present study, we intended to produce C2I mutant proteins as recombinant subunit vaccines by using glutathione-S-transferase-gene fusion system. The mutants of this study were previously evaluated from their evolutionary imprints and suggested as suitable candidates for subunit vaccines. A synthetic C2 gene was inserted in a pGEX-2T vector, cloned and expressed in Escherichia coli BL21 host. The expressed mutant proteins were purified by using glutathione-agarose column and then examined for their ADP ribosyltransferase activity and vaccinogenic characteristics. The pGEX-2T-C2I constructs with Y298F, S347A and S350A substitutions have shown effective transformation efficiencies in E. coli XL10 competent cells but their mutagenesis efficiency was relatively low. Gene expression analysis indicated the rate of gene expression was depended on the fused mutant genes. A high-level expression was achieved for Y298F, S347A and S350A mutant proteins. All purified protein exhibited a molecular mass of 49 kDa. C2I mutant proteins exhibited a reduced ADP ribosyltransferase activity with retained immunogenic and vaccinogenic characteristics compared to the wild-type C2I subunit. The overall analysis of our study suggested the recombinant C2I proteins (S197A and Y298F) are the most promising candidates for the development of subunit vaccine or immunogen for C2 mutants mediated diseases in birds and animals.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Clostridium botulinum/genética , Clostridium botulinum/imunologia , Proteínas Mutantes/imunologia , ADP Ribose Transferases/genética , ADP Ribose Transferases/imunologia , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos , Mutagênese , Vacinas de Subunidades/genética , Vacinas de Subunidades/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
7.
Nucleic Acids Res ; 47(11): 5658-5669, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31216043

RESUMO

ADP-ribosylation is a reversible chemical modification catalysed by ADP-ribosyltransferases such as PARPs that utilize nicotinamide adenine dinucleotide (NAD+) as a cofactor to transfer monomer or polymers of ADP-ribose nucleotide onto macromolecular targets such as proteins and DNA. ADP-ribosylation plays an important role in several biological processes such as DNA repair, transcription, chromatin remodelling, host-virus interactions, cellular stress response and many more. Using biochemical methods we identify RNA as a novel target of reversible mono-ADP-ribosylation. We demonstrate that the human PARPs - PARP10, PARP11 and PARP15 as well as a highly diverged PARP homologue TRPT1, ADP-ribosylate phosphorylated ends of RNA. We further reveal that ADP-ribosylation of RNA mediated by PARP10 and TRPT1 can be efficiently reversed by several cellular ADP-ribosylhydrolases (PARG, TARG1, MACROD1, MACROD2 and ARH3), as well as by MACROD-like hydrolases from VEEV and SARS viruses. Finally, we show that TRPT1 and MACROD homologues in bacteria possess activities equivalent to the human proteins. Our data suggest that RNA ADP-ribosylation may represent a widespread and physiologically relevant form of reversible ADP-ribosylation signalling.


Assuntos
ADP-Ribosilação , Difosfato de Adenosina/química , RNA/metabolismo , ADP Ribose Transferases/genética , Adenosina Difosfato Ribose , Animais , Catálise , Cromatina/química , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Escherichia coli/metabolismo , Humanos , Hidrolases/metabolismo , Camundongos , NAD/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/química , Plasmídeos/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais
8.
PLoS Pathog ; 15(6): e1007812, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31220187

RESUMO

While considered solely an extracellular pathogen, increasing evidence indicates that Pseudomonas aeruginosa encounters intracellular environment in diverse mammalian cell types, including macrophages. In the present study, we have deciphered the intramacrophage fate of wild-type P. aeruginosa PAO1 strain by live and electron microscopy. P. aeruginosa first resided in phagosomal vacuoles and subsequently could be detected in the cytoplasm, indicating phagosomal escape of the pathogen, a finding also supported by vacuolar rupture assay. The intracellular bacteria could eventually induce cell lysis, both in a macrophage cell line and primary human macrophages. Two bacterial factors, MgtC and OprF, recently identified to be important for survival of P. aeruginosa in macrophages, were found to be involved in bacterial escape from the phagosome as well as in cell lysis caused by intracellular bacteria. Strikingly, type III secretion system (T3SS) genes of P. aeruginosa were down-regulated within macrophages in both mgtC and oprF mutants. Concordantly, cyclic di-GMP (c-di-GMP) level was increased in both mutants, providing a clue for negative regulation of T3SS inside macrophages. Consistent with the phenotypes and gene expression pattern of mgtC and oprF mutants, a T3SS mutant (ΔpscN) exhibited defect in phagosomal escape and macrophage lysis driven by internalized bacteria. Importantly, these effects appeared to be largely dependent on the ExoS effector, in contrast with the known T3SS-dependent, but ExoS independent, cytotoxicity caused by extracellular P. aeruginosa towards macrophages. Moreover, this macrophage damage caused by intracellular P. aeruginosa was found to be dependent on GTPase Activating Protein (GAP) domain of ExoS. Hence, our work highlights T3SS and ExoS, whose expression is modulated by MgtC and OprF, as key players in the intramacrophage life of P. aeruginosa which allow internalized bacteria to lyse macrophages.


Assuntos
Proteínas de Bactérias/biossíntese , Regulação para Baixo , Regulação Bacteriana da Expressão Gênica , Macrófagos/microbiologia , Pseudomonas aeruginosa , Sistemas de Secreção Tipo III/metabolismo , ADP Ribose Transferases/genética , ADP Ribose Transferases/metabolismo , Animais , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Linhagem Celular , Humanos , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Mutação , Fagossomos/microbiologia , Fagossomos/ultraestrutura , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Sistemas de Secreção Tipo III/genética
9.
Genome Biol Evol ; 11(1): 1780-1796, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31173069

RESUMO

The diversification of microbial populations may be driven by many factors including adaptation to distinct ecological niches and barriers to recombination. We examined the population structure of the bacterial pathogen Pseudomonas aeruginosa by analyzing whole-genome sequences of 739 isolates from diverse sources. We confirmed that the population structure of P. aeruginosa consists of two major groups (referred to as Groups A and B) and at least two minor groups (Groups C1 and C2). Evidence for frequent intragroup but limited intergroup recombination in the core genome was observed, consistent with sexual isolation of the groups. Likewise, accessory genome analysis demonstrated more gene flow within Groups A and B than between these groups, and a few accessory genomic elements were nearly specific to one or the other group. In particular, the exoS gene was highly overrepresented in Group A compared with Group B isolates (99.4% vs. 1.1%) and the exoU gene was highly overrepresented in Group B compared with Group A isolates (95.2% vs. 1.8%). The exoS and exoU genes encode effector proteins secreted by the P. aeruginosa type III secretion system. Together these results suggest that the major P. aeruginosa groups defined in part by the exoS and exoU genes are divergent from each other, and that these groups are genetically isolated and may be ecologically distinct. Although both groups were globally distributed and caused human infections, certain groups predominated in some clinical contexts.


Assuntos
ADP Ribose Transferases/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Genética Populacional , Pseudomonas aeruginosa/genética , Fluxo Gênico , Genoma Bacteriano , Filogenia , Recombinação Genética
10.
Int J Oncol ; 55(1): 309-319, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31180549

RESUMO

Fusion toxins consisting of an affinity protein fused to toxic polypeptides derived from Pseudomonas exotoxin A (ETA) are promising agents for targeted cancer therapy. In this study, we examined whether fusion toxins consisting of an albumin binding domain­derived affinity protein (ADAPT) interacting with human epidermal growth factor receptor 2 (HER2), coupled to the ETA­derived polypeptides PE38X8 or PE25, with or without an albumin binding domain (ABD) for half­life extension, can be used for specific killing of HER2­expressing cells. The fusion toxins could easily be expressed in a soluble form in Escherichia coli and purified to homogeneity. All constructs had strong affinity for HER2 (KD 10 to 26 nM) and no tendency for aggregation could be detected. The fusion toxins including the ABD showed strong interaction with human and mouse serum albumin [equilibrium dissociation constant (KD) 1 to 3 nM and 2 to 10 nM, respectively]. The in vitro investigation of the cytotoxic potential revealed IC50­values in the picomolar range for cells expressing high levels of HER2. The specificity was also demonstrated, by showing that free HER2 receptors on the target cells are required for fusion toxin activity. In mice, the fusion toxins containing the ABD exhibited an appreciably longer time in circulation. The uptake was highest in liver and kidney. Fusion with PE25 was associated with the highest hepatic uptake. Collectively, the results suggest that fusion toxins consisting of ADAPTs and ETA­derivatives are promising agents for targeted cancer therapy.


Assuntos
ADP Ribose Transferases/administração & dosagem , Toxinas Bacterianas/administração & dosagem , Exotoxinas/administração & dosagem , Neoplasias/tratamento farmacológico , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Fatores de Virulência/administração & dosagem , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , ADP Ribose Transferases/farmacocinética , Albuminas/administração & dosagem , Albuminas/química , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacocinética , Linhagem Celular Tumoral , Exotoxinas/química , Exotoxinas/genética , Exotoxinas/farmacocinética , Feminino , Humanos , Camundongos , Neoplasias/metabolismo , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacocinética , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinética , Ressonância de Plasmônio de Superfície , Distribuição Tecidual , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/farmacocinética
11.
Mol Ther ; 27(7): 1327-1338, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31129118

RESUMO

Primary open-angle glaucoma (POAG) is considered a lifelong disease characterized by optic nerve deterioration and visual field damage. Although the disease progression can usually be controlled by lowering the intraocular pressure (IOP), therapeutic effects of current approaches do not last long. Gene therapy could be a promising method for persistent treatment of the disease. Our previous study demonstrated that gene transfer of exoenzyme C3 transferase (C3) to the trabecular meshwork (TM) to inhibit Rho GTPase (Rho), the upstream signal molecule of Rho-associated kinase (ROCK), resulted in lowered IOP in normal rodent eyes. In the present study, we show that the lentiviral vector (LV)-mediated C3 expression inactivates RhoA in human TM cells by ADP ribosylation, resulting in disruption of the actin cytoskeleton and altered cell morphology. In addition, intracameral delivery of the C3 vector to monkey eyes leads to persistently lowered IOP without obvious signs of inflammation. This is the first report of using a vector to transduce the TM of an alive non-human primate with a gene that alters cellular machinery and physiology. Our results in non-human primates support that LV-mediated C3 expression in the TM may have therapeutic potential for glaucoma, the leading cause of irreversible blindness in humans.


Assuntos
ADP Ribose Transferases/genética , ADP Ribose Transferases/metabolismo , Toxinas Botulínicas/genética , Toxinas Botulínicas/metabolismo , Terapia Genética/métodos , Vetores Genéticos/uso terapêutico , Pressão Intraocular , ADP-Ribosilação/genética , Citoesqueleto de Actina/metabolismo , Animais , Câmara Anterior/metabolismo , Células Cultivadas , Vetores Genéticos/administração & dosagem , Glaucoma de Ângulo Aberto/terapia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lentivirus , Macaca mulatta , Masculino , Distribuição Tecidual , Malha Trabecular/citologia , Malha Trabecular/metabolismo , Transdução Genética , Proteína rhoA de Ligação ao GTP/metabolismo
12.
Artigo em Inglês | MEDLINE | ID: mdl-31085514

RESUMO

In 2011, we initiated a sentinel surveillance network to assess changes in Clostridioides (formerly Clostridium) difficile antimicrobial susceptibility to fidaxomicin from 6 geographically dispersed medical centers in the United States. This report summarizes data from 2013 to 2016. C. difficile isolates or toxin-positive stools from patients were referred to a central laboratory. Antimicrobial susceptibility was determined by agar dilution. CLSI, EUCAST, or FDA breakpoints were used, where applicable. Toxin gene profiles were characterized by multiplex PCR on each isolate. A random sample of approximately 40% of isolates, stratified by institution and year, was typed by restriction endonuclease analysis (REA). Among 1,889 isolates from 2013 to 2016, the fidaxomicin MIC90 was 0.5 µg/ml; all isolates were inhibited at ≤1 µg/ml. There were decreases in metronidazole and vancomycin MICs over time. Clindamycin resistance remained unchanged (27.3%). An increase in imipenem resistance was observed. By 2015 to 2016, moxifloxacin resistance decreased in all centers. The proportion of BI isolates decreased from 25.5% in 2011 to 2012 to 12.8% in 2015 to 2016 (P < 0.001). The BI REA group correlated with moxifloxacin resistance (BI 84% resistant versus non-BI 12.5% resistant). Fidaxomicin MICs have not changed among C. difficile isolates of U.S. origin over 5 years post licensure. There has been an overall decrease in MICs for vancomycin, metronidazole, moxifloxacin, and rifampin and an increase in isolates resistant to imipenem. Moxifloxacin resistance remained high among the BI REA group, but the proportion of BI isolates has decreased. Continued geographic variations in REA groups and antimicrobial resistance persist.


Assuntos
Antibacterianos/farmacologia , Infecções por Clostridium/microbiologia , Clostridium difficile/efeitos dos fármacos , Diarreia/microbiologia , Fidaxomicina/farmacologia , ADP Ribose Transferases/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Clindamicina/farmacologia , Clostridium difficile/genética , Clostridium difficile/isolamento & purificação , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Enterotoxinas/genética , Humanos , Imipenem/farmacologia , Testes de Sensibilidade Microbiana , Vigilância de Evento Sentinela , Estados Unidos
13.
Infect Genet Evol ; 71: 205-210, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30902742

RESUMO

BACKGROUND & AIMS: Clostridioides difficile (C. difficile) has been identified as the leading cause of antibiotic associated diarrhea (AAD). Co-carriage of an intact pathogenicity locus (PaLoc) with binary toxin genes in C. difficile strains seems to be linked with severe disease outcomes in the infected patients. Epidemiology of C. difficile infection (CDI) in hospital setting and knowledge about their genetic context help us to decrease the morbidity, mortality, and costs associated with Clostridioides difficile infection. In the present study was aimed to characterize genetic diversity of PaLoc among different C. difficile strains isolated from hospitalized patients and carriage of cytolethal distending toxin gene (cdt) in different hospitals. METHOD: C. difficile strains were isolated from stool samples of inpatients referred to a reference laboratory from different hospitals and also outpatients with diarrhea, during 2008-2011. DNA was extracted from pure culture of the bacterium and PCR was performed for tcdA, tcdB, tcdE, tcdC, tcdD, and cdu2 genes. Carriage of two binary toxin genes cdtA, cdtB was also determined in these strains. To find clonal strains, similarity of genotypes and integrity of PaLoc among the isolates was compared in each hospital. RESULTS: The intact PaLoc was found most frequently among the isolates in the outpatients (19/51, 37.2%, Group I), while incomplete PaLoc found mostly in patients who were hospitalized in the infectious diseases and internal diagnosis wards. tcdA and tcdB genes were detected in different combinations among the studied strains. These strains showed tcdA+B+, tcdA+B-, and tcdA-B+ genotypes in a frequency of 76.4% (39/51), 7.8% (4/51), and 17.6% (9/51), respectively. Analysis of gene composition of the PaLoc showed 19 distinct genotypes among the 51 strains. Accordingly, 38 strains were classified mainly into 6 regular groups, while the remaining strains showed heterogeneous patterns. tcdC-/tcdD- constituted the most common genotypic group among the strains with partial PaLoc (7/51, 13.7%). A hypertoxigenic genotype, tcdC-/tcdA+/tcdB+, was detected in 2 strains (2/51, 3.9%). The intact genotype was also detected in a C. difficile isolate from outpatients. Cdt encoding genes toxins was observed in low numbers of the strains (7/52, 13.5%). All of cdtA+B+ strains were belonged to PaLoc group 1 (intact genotype). Statistical analyses showed no correlation between particular genotypes and special wards of the hospitals (p value>0.05). CONCLUSION: Collectively, our results showed diversity of C. difficile strains in most wards of the studied hospitals. Diversity of PaLoc genotypes in the strains that isolated from the same wards proposed endogenous routes of the infection, as common cause of CDI in these patients.


Assuntos
ADP Ribose Transferases/genética , Proteínas de Bactérias/genética , Clostridium difficile/genética , Clostridium difficile/patogenicidade , Enterocolite Pseudomembranosa/epidemiologia , Fatores de Virulência/genética , Toxinas Bacterianas/genética , Proteínas de Ligação a DNA/genética , Enterotoxinas/genética , Feminino , Técnicas de Genotipagem , Hospitais , Humanos , Irã (Geográfico)/epidemiologia , Masculino , Epidemiologia Molecular , Proteínas Repressoras/genética
14.
Anaerobe ; 57: 35-38, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30880150

RESUMO

Clostridium (Clostridioides) difficile has been identified in humans and a wide range of animal species, but there has been little study of remote animal populations with limited human contact. The objective of this study was to determine the prevalence and types of C. difficile in wild and captive polar bears (Ursus maritimus). Fecal samples were collected from two populations of wild polar bears in Nunavut Canada; M'Clintock Channel and Hudson Strait (Davis Strait or Foxe Basin), as well as from a facility (PBJ) in Churchill, Manitoba that temporarily houses nuisance polar bears and from captive bears in a zoological park. Enrichment culture was performed and isolates were characterized by ribotyping and toxinotyping. Clostridium difficile was isolated from 24/143 (16.8%) of samples; 18/120 (15%) wild bear samples, 4/7 (57%) from the PBJ and 2/16 (13%) samples from three zoo bears. The prevalence of C. difficile was significantly higher in bears that were housed at the PBJ vs wild bears (P = 0.0042), but there was no difference between wild bears from M'Clintock Channel (14/100, 14%) and those from Hudson Strait (4/20, 20%) (P = 0.50). Fourteen of the 24 (58%) isolates were toxigenic; 13/18 (72%) wild bear isolates, 0/4 PBJ isolate and 1/2 zoo isolates. Four toxigenic ribotypes were identified, with one that possessed tcdB and cdtA predominating. None of the toxigenic isolates were ribotypes that have been identified previously by the authors. There was no overlap in toxigenic ribotypes between the different populations. Clostridium difficile was not uncommonly identified in polar bears, with differences in type distribution amongst the different regions. The presence of strains that have not been identified in humans or domestic animals suggests that polar bears may be a natural reservoir of unique strains of this important bacterium.


Assuntos
Derrame de Bactérias , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/veterinária , Clostridium difficile/isolamento & purificação , Ursidae/microbiologia , ADP Ribose Transferases/genética , Animais , Regiões Árticas/epidemiologia , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Clostridium difficile/classificação , Clostridium difficile/genética , Fezes/microbiologia , Doenças dos Peixes , Manitoba/epidemiologia , Nunavut/epidemiologia , Prevalência , Ribotipagem
15.
Immunotherapy ; 11(6): 483-496, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30860437

RESUMO

AIM: We have shown that IL-4 fused to Pseudomonas exotoxin (IL-4-PE) is cytotoxic to ovarian cancer cell lines. The antineoplastic properties of IFN-α, IFN-γ and IL-4-PE have been studied and showed some promise in the clinical trials. Here, we investigated whether the combination of IL-4-PE, IFN-α and IFN-γ will result in increased ovarian cancer cell death in vitro and in vivo. MATERIALS & METHODS: Ovarian cancer cells were tested in vitro to analyze the cytotoxic effects of IL-4-PE, IFN-α and IFN-γ, and the combination of all three. Tumor-bearing xenograft mice were treated with the combination of IL-4-PE, IFN-α and IFN-γ to monitor their overall survival. The JAK/STAT phosphorylation signaling pathways were studied to delineate the mechanism of synergistic antitumor activity. RESULTS: The combination of IL-4-PE with IFN-α and IFN-γ resulted in increased ovarian cancer cell death in vitro and in vivo. Mechanistically, the synergistic antitumor effect was dependent on interferon signaling, but not IL-4-PE signaling as determined by signaling specific chemical inhibitors. The combination therapy induced the activation of critical mediators of apoptosis. CONCLUSION: The combination of IL-4-PE with interferons increased overall survival of mice with human ovarian cancer xenograft. These data suggest that this novel combination could provide a unique approach to treating ovarian cancer.


Assuntos
Imunoterapia/métodos , Interferon-alfa/uso terapêutico , Interferon gama/uso terapêutico , Neoplasias Ovarianas/terapia , Proteínas Recombinantes de Fusão/uso terapêutico , ADP Ribose Transferases/genética , Animais , Apoptose , Toxinas Bacterianas/genética , Linhagem Celular Tumoral , Terapia Combinada , Modelos Animais de Doenças , Exotoxinas/genética , Feminino , Humanos , Interleucina-4/genética , Camundongos , Camundongos Nus , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais , Fatores de Virulência/genética , Ensaios Antitumorais Modelo de Xenoenxerto
16.
BMC Res Notes ; 12(1): 28, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30646938

RESUMO

OBJECTIVE: The present study aimed to determine the prevalence of virulence factors and antimicrobial resistance profile of Pseudomonas aeruginosa strains isolated from Iranian burn patients. RESULTS: This cross-sectional study performed on 100 P. aeruginosa isolates which were recovered from burn wound specimens in 2014-2015. All presumptive isolates were identified by standard microbiologic tests. Antimicrobial susceptibility test was carried out by disk diffusion method. The presence of virulence genes was determined by PCR method. Antibiotic susceptibility results revealed that the isolates were mostly susceptible to amikacin (61%), ceftazidime (60%), and imipenem (55%). Moreover, 59% of the isolates were multi-drug resistance (MDR). The most prevalent MDR pattern was aminoglycosides-penicillins-fluoroquinolones-carbapenems (15%). The presence of exoT, exoY, exoS and exoU genes was detected in 100%, 100%, 59%, and 41% of the tested isolates, respectively. Results points out the pattern of MDR and genetic diversity of type III secretion system among P. aeruginosa strains isolated from the burn population. Overall, the association of MDR and the presence of the specific virulence genes can be a predictive marker for the persistence of these isolates in the hospitals and subsequently a worse clinical condition for the affected patients.


Assuntos
ADP Ribose Transferases/genética , Antibacterianos , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Unidades de Queimados , Queimaduras/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Proteínas Ativadoras de GTPase/genética , Glucosiltransferases/genética , Pseudomonas aeruginosa/genética , Sistemas de Secreção Tipo III , Fatores de Virulência/genética , Estudos Transversais , Humanos , Irã (Geográfico) , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/isolamento & purificação
17.
J Infect Chemother ; 25(4): 262-266, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30642771

RESUMO

In this study, we investigated all Clostridioides difficile strains isolated from stool samples in Nagasaki University Hospital between January 2012 and December 2014. Toxin genes (tcdA, tcdB and cdtA/cdtB) were analyzed for multiplex PCR in a total of 213 strains. In the toxin gene-positive strain, PCR ribotyping was conducted using capillary gel electrophoresis-based PCR and the Webribo database. Patients' backgrounds were analyzed by departments, disorders, antimicrobials, and clinical dates. The positive rates of tcdA, tcdB, and cdtA/cdtB genes were 62.9%, 63.4%, and 2.8%, respectively. The most frequent PCR ribotype was 047 (14.1%), followed by 014/0 (11.1%) and 002/0 (8.2%). In univariate analysis, the risk factors for the detection of toxin gene-positive strains in patients were older age (p = 0.0036), over ≥ 65 years old (p = 0.0175), the patients hospitalized at Department of Digestive Surgery (P = 0.0059), higher CRP level (P = 0.0395), and lower albumin level (p = 0.0014). In the multivariate analysis, the risk factor for detection of toxin gene-positive strains was the patients hospitalized at Department of Digestive Surgery (OR; 4.62, 95% CI; 1.18-18.0, p = 0.0274). In this study, the percentage of toxin gene-positive and cdtA/cdtB gene-positive strains was almost the same as that reported in previous studies, but the ribotype was different. In addition, we revealed that the risk factor associated with the detection of toxin gene-positive strains was the patients hospitalized at Department of digestive surgery.


Assuntos
Infecções por Clostridium/epidemiologia , Clostridium difficile/genética , Ribotipagem/métodos , ADP Ribose Transferases/genética , ADP Ribose Transferases/isolamento & purificação , Adolescente , Adulto , Fatores Etários , Idoso , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/microbiologia , Clostridium difficile/isolamento & purificação , Fezes/microbiologia , Feminino , Hospitais Universitários/estatística & dados numéricos , Humanos , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Fatores de Risco , Adulto Jovem
18.
FEBS J ; 286(6): 1214-1229, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30633437

RESUMO

The ammonium-dependent posttranslational regulation of nitrogenase activity in Azospirillum brasilense requires dinitrogenase reductase ADP-ribosyl transferase (DraT) and dinitrogenase reductase ADP-glycohydrolase (DraG). These enzymes are reciprocally regulated by interaction with the PII proteins, GlnB and GlnZ. In this study, purified ADP-ribosylated Fe-protein was used as substrate to study the mechanism involved in the regulation of A. brasilense DraG in vitro. The data show that DraG is partially inhibited by GlnZ and that DraG inhibition is further enhanced by the simultaneous presence of GlnZ and AmtB. These results are the first to demonstrate experimentally that DraG inactivation requires the formation of a ternary DraG-GlnZ-AmtB complex in vitro. Previous structural data have revealed that when the DraG-GlnZ complex associates with AmtB, the flexible T-loops of the trimeric GlnZ bind to AmtB and become rigid; these molecular events stabilize the DraG-GlnZ complex, resulting in DraG inactivation. To determine whether restraining the flexibility of the GlnZ T-loops is a limiting factor in DraG inhibition, we used a GlnZ variant that carries a partial deletion of the T-loop (GlnZΔ42-54). However, although the GlnZΔ42-54 variant was more effective in inhibiting DraG in vitro, it bound to DraG with a slightly lower affinity than does wild-type GlnZ and was not competent to completely inhibit DraG activity either in vitro or in vivo. We, therefore, conclude that the formation of a ternary complex between DraG-GlnZ-AmtB is necessary for the inactivation of DraG.


Assuntos
ADP Ribose Transferases/metabolismo , Compostos de Amônio/metabolismo , Azospirillum brasilense/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , N-Glicosil Hidrolases/metabolismo , Proteínas PII Reguladoras de Nitrogênio/metabolismo , ADP Ribose Transferases/genética , Azospirillum brasilense/genética , Azospirillum brasilense/crescimento & desenvolvimento , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Transporte de Cátions/genética , Regulação Bacteriana da Expressão Gênica , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/genética , Proteínas PII Reguladoras de Nitrogênio/genética , Ligação Proteica , Conformação Proteica , Transdução de Sinais
19.
Microb Drug Resist ; 25(4): 594-602, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30461338

RESUMO

Over the last decade, nanotechnology-based therapeutic platforms have been directed toward developing nanoparticles with unique properties to combat biofilms. In this study, we evaluated the antibiofilm activity of the sulfur-functionalized fullerene nanoparticles (SFF Nps) against Pseudomonas aeruginosa and also analyzed the effect of this nanoparticle on the expression of exotoxin A (toxA) gene. The functionalized fullerenes were prepared by chemical vapor deposition method. We assessed the potential of SFF Nps to inhibit biofilm formation and eradicate preformed biofilms. Also, the effect of this nanoparticle on the expression of toxA gene was investigated by real-time PCR. The minimum biofilm inhibitory concentration of SFF Nps was 1 mg/mL. The minimum biofilm-eradication concentration of SFF Nps on the young (24- and 48-hr old) and older (72- and 96-hr old) biofilms was 2 and 4 mg/mL, respectively. Field emission electron scanning microscopy images confirmed the potent ability of SFF Nps to eradicate biofilm of P. aeruginosa. The expression of toxA was downregulated in the presence of SFF Nps. In conclusion, considering the ability of SFF Nps to kill P. aeruginosa biofilm and downregulate the expression of exotoxin A, this nanoparticle can be used for treatment of both chronic and acute P. aeruginosa infections.


Assuntos
ADP Ribose Transferases/genética , Antibacterianos/farmacologia , Toxinas Bacterianas/genética , Biofilmes/efeitos dos fármacos , Exotoxinas/genética , Fulerenos/farmacologia , Nanopartículas/química , Pseudomonas aeruginosa/efeitos dos fármacos , Enxofre/farmacologia , Fatores de Virulência/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Testes de Sensibilidade Microbiana/métodos , Pseudomonas aeruginosa/genética
20.
Biochem J ; 475(23): 3827-3846, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30373764

RESUMO

Here, we report the biochemical characterization of the mono-ADP-ribosyltransferase 2,3,7,8-tetrachlorodibenzo-p-dioxin poly-ADP-ribose polymerase (TIPARP/ARTD14/PARP7), which is known to repress aryl hydrocarbon receptor (AHR)-dependent transcription. We found that the nuclear localization of TIPARP was dependent on a short N-terminal sequence and its zinc finger domain. Deletion and in vitro ADP-ribosylation studies identified amino acids 400-657 as the minimum catalytically active region, which retained its ability to mono-ADP-ribosylate AHR. However, the ability of TIPARP to ADP-ribosylate and repress AHR in cells was dependent on both its catalytic activity and zinc finger domain. The catalytic activity of TIPARP was resistant to meta-iodobenzylguanidine but sensitive to iodoacetamide and hydroxylamine, implicating cysteines and acidic side chains as ADP-ribosylated target residues. Mass spectrometry identified multiple ADP-ribosylated peptides in TIPARP and AHR. Electron transfer dissociation analysis of the TIPARP peptide 33ITPLKTCFK41 revealed cysteine 39 as a site for mono-ADP-ribosylation. Mutation of cysteine 39 to alanine resulted in a small, but significant, reduction in TIPARP autoribosylation activity, suggesting that additional amino acid residues are modified, but loss of cysteine 39 did not prevent its ability to repress AHR. Our findings characterize the subcellular localization and mono-ADP-ribosyltransferase activity of TIPARP, identify cysteine as a mono-ADP-ribosylated residue targeted by this enzyme, and confirm the TIPARP-dependent mono-ADP-ribosylation of other protein targets, such as AHR.


Assuntos
ADP Ribose Transferases/genética , Cisteína/genética , Mutação de Sentido Incorreto , Poli(ADP-Ribose) Polimerases/genética , ADP Ribose Transferases/metabolismo , ADP-Ribosilação/efeitos dos fármacos , Animais , Biocatálise/efeitos dos fármacos , Células COS , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Chlorocebus aethiops , Cisteína/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Células MCF-7 , Poli(ADP-Ribose) Polimerases/metabolismo , Dibenzodioxinas Policloradas/farmacologia , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Dedos de Zinco/genética
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