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1.
Nat Commun ; 10(1): 4918, 2019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31664022

RESUMO

Nanopore sensing is a powerful single-molecule approach for the detection of biomolecules. Recent studies have demonstrated that aerolysin is a promising candidate to improve the accuracy of DNA sequencing and to develop novel single-molecule proteomic strategies. However, the structure-function relationship between the aerolysin nanopore and its molecular sensing properties remains insufficiently explored. Herein, a set of mutated pores were rationally designed and evaluated in silico by molecular simulations and in vitro by single-channel recording and molecular translocation experiments to study the pore structural variation, ion selectivity, ionic conductance and capabilities for sensing several biomolecules. Our results show that the ion selectivity and sensing ability of aerolysin are mostly controlled by electrostatics and the narrow diameter of the double ß-barrel cap. By engineering single-site mutants, a more accurate molecular detection of nucleic acids and peptides has been achieved. These findings open avenues for developing aerolysin nanopores into powerful sensing devices.


Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Ácidos Nucleicos/química , Peptídeos/química , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Toxinas Bacterianas/metabolismo , Cinética , Mutação , Nanoporos , Nanotecnologia , Ácidos Nucleicos/genética , Peptídeos/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Eletricidade Estática
2.
PLoS Negl Trop Dis ; 13(8): e0007644, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31430284

RESUMO

Bacillus anthracis and Yersinia pestis are zoonotic bacteria capable of causing severe and sometimes fatal infections in animals and humans. Although considered as diseases of antiquity in industrialized countries due to animal and public health improvements, they remain endemic in vast regions of the world disproportionally affecting the poor. These pathogens also remain a serious threat if deployed in biological warfare. A single vaccine capable of stimulating rapid protection against both pathogens would be an extremely advantageous public health tool. We produced multiple-antigen fusion proteins (MaF1 and MaF2) containing protective regions from B. anthracis protective antigen (PA) and lethal factor (LF), and from Y. pestis V antigen (LcrV) and fraction 1 (F1) capsule. The MaF2 sequence was also expressed from a plasmid construct (pDNA-MaF2). Immunogenicity and protective efficacy were investigated in mice following homologous and heterologous prime-boost immunization. Antibody responses were determined by ELISA and anthrax toxin neutralization assay. Vaccine efficacy was determined against lethal challenge with either anthrax toxin or Y. pestis. Both constructs elicited LcrV and LF-specific serum IgG, and MaF2 elicited toxin-neutralizing antibodies. Immunizations with MaF2 conferred 100% and 88% protection against Y. pestis and anthrax toxin, respectively. In contrast, pDNA-MaF2 conferred only 63% protection against Y. pestis and no protection against anthrax toxin challenge. pDNA-MaF2-prime MaF2-boost induced 75% protection against Y. pestis and 25% protection against anthrax toxin. Protection was increased by the molecular adjuvant CARDif. In conclusion, MaF2 is a promising multi-antigen vaccine candidate against anthrax and plague that warrants further investigation.


Assuntos
Antraz/prevenção & controle , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Vacinas Bacterianas/imunologia , Peste/prevenção & controle , Proteínas Recombinantes de Fusão/imunologia , Yersinia pestis/imunologia , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Bacillus anthracis/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Vacinas Bacterianas/isolamento & purificação , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos BALB C , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/imunologia , Proteínas Recombinantes de Fusão/genética , Análise de Sobrevida , Resultado do Tratamento , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação , Yersinia pestis/genética
3.
Chem Commun (Camb) ; 55(63): 9311-9314, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31310244

RESUMO

Discrimination between cysteine and homocysteine at the single-molecule level is achieved within a K238Q mutant aerolysin nanopore, which provides a confined space for high spatial resolution to identify the amino acid difference with a 5'-benzaldehyde poly(dA)4 probe. Our strategy allows potential detection and characterization of various amino acids and their modifications, and provides a crucial step towards developing nanopore protein sequencing devices.


Assuntos
Toxinas Bacterianas/química , Cisteína/análise , Homocisteína/análise , Nanoporos , Proteínas Citotóxicas Formadoras de Poros/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Cromatografia Líquida de Alta Pressão , Mutagênese Sítio-Dirigida , Poli A/química , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Espectrometria de Massas por Ionização por Electrospray
4.
J Food Sci ; 84(8): 2250-2255, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31313323

RESUMO

The ability to produce various extracellular enzymes is considered as an important virulence feature in Aeromonas spp., in addition to producing specific virulence factors such as aerolysin and hemolysin. In this study, the effect of salinity and pH on the production of extracellular virulence factors by Aeromonas was investigated. Aeromonas was obtained from different food sources. A comparative study of the activities of extracellular enzymes secreted by these bacteria at different environmental conditions can widen our understanding on their pathogenicity. The activities of various extracellular enzymes such as amylase, gelatinase, and caseinase, which are implicated as virulence factors, were measured in vitro by calculating the enzymatic activity index (EAI) of each enzyme using standard laboratory protocols. For all enzymes, a significant change (P < 0.05) in the EAI was observed when the concentration of NaCl in the media increased from 0.5% to 3%. Among three enzymes tested, caseinase was found to be affected the most by salinity, with a significant difference in EAI when NaCl concentration in the media increased from 0.5% to 2%. Similarly, amylase was found to be affected the most by acidity. The pH values ranging from 6 to 9 did not exert any significant change in EAI of amylase; however, a pH value of 5 had a significant effect. Overall, compared to salinity, the change in pH was found to be less effective in controlling the extracellular virulence factor production in Aeromonas. PRACTICAL APPLICATIONS: The practical application is to minimize the extracellular virulence factor production by Aeromonas in food commodities by altering the salt content and pH. The results demonstrate that an increase in salinity and a decrease in pH can minimize the extracellular virulence factor production by Aeromonas spp.


Assuntos
Aeromonas/metabolismo , Proteínas de Bactérias/metabolismo , Cloreto de Sódio/metabolismo , Fatores de Virulência/metabolismo , Aeromonas/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Microbiologia de Alimentos , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Concentração de Íons de Hidrogênio , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Salinidade , Cloreto de Sódio/análise , Fatores de Virulência/genética
5.
Infect Immun ; 87(10)2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31331960

RESUMO

In this study, a novel recombinant attenuated Yersinia pseudotuberculosis PB1+ strain (χ10069) engineered with ΔyopK ΔyopJ Δasd triple mutations was used to deliver a Y. pestis fusion protein, YopE amino acid 1 to 138-LcrV (YopENt138-LcrV), to Swiss Webster mice as a protective antigen against infections by yersiniae. χ10069 bacteria harboring the pYA5199 plasmid constitutively synthesized the YopENt138-LcrV fusion protein and secreted it via the type 3 secretion system (T3SS) at 37°C under calcium-deprived conditions. The attenuated strain χ10069(pYA5199) was manifested by the establishment of controlled infection in different tissues without developing conspicuous signs of disease in histopathological analysis of microtome sections. A single-dose oral immunization of χ10069(pYA5199) induced strong serum antibody titers (log10 mean value, 4.2), secretory IgA in bronchoalveolar lavage (BAL) fluid from immunized mice, and Yersinia-specific CD4+ and CD8+ T cells producing high levels of tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and interleukin 2 (IL-2), as well as IL-17, in both lungs and spleens of immunized mice, conferring comprehensive Th1- and Th2-mediated immune responses and protection against bubonic and pneumonic plague challenges, with 80% and 90% survival, respectively. Mice immunized with χ10069(pYA5199) also exhibited complete protection against lethal oral infections by Yersinia enterocolitica WA and Y. pseudotuberculosis PB1+. These findings indicated that χ10069(pYA5199) as an oral vaccine induces protective immunity to prevent bubonic and pneumonic plague, as well as yersiniosis, in mice and would be a promising oral vaccine candidate for protection against plague and yersiniosis for human and veterinary applications.


Assuntos
Anticorpos Antibacterianos/biossíntese , Imunoglobulina A/biossíntese , Vacina contra a Peste/administração & dosagem , Peste/prevenção & controle , Proteínas Recombinantes de Fusão/administração & dosagem , Yersinia pestis/efeitos dos fármacos , Infecções por Yersinia pseudotuberculosis/prevenção & controle , Yersinia pseudotuberculosis/efeitos dos fármacos , Administração Oral , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/microbiologia , Proteção Cruzada , Feminino , Expressão Gênica , Humanos , Imunização , Interferon gama/genética , Interferon gama/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/microbiologia , Masculino , Camundongos , Peste/imunologia , Peste/microbiologia , Peste/mortalidade , Vacina contra a Peste/biossíntese , Vacina contra a Peste/genética , Vacina contra a Peste/imunologia , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Análise de Sobrevida , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Vacinas Sintéticas , Yersinia pestis/imunologia , Yersinia pestis/patogenicidade , Yersinia pseudotuberculosis/imunologia , Yersinia pseudotuberculosis/patogenicidade , Infecções por Yersinia pseudotuberculosis/imunologia , Infecções por Yersinia pseudotuberculosis/microbiologia , Infecções por Yersinia pseudotuberculosis/mortalidade
6.
Nat Commun ; 10(1): 2900, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31263098

RESUMO

The alpha helical CytolysinA family of pore forming toxins (α-PFT) contains single, two, and three component members. Structures of the single component Eschericia coli ClyA and the two component Yersinia enterolytica YaxAB show both undergo conformational changes from soluble to pore forms, and oligomerization to produce the active pore. Here we identify tripartite α-PFTs in pathogenic Gram negative bacteria, including Aeromonas hydrophila (AhlABC). We show that the AhlABC toxin requires all three components for maximal cell lysis. We present structures of pore components which describe a bi-fold hinge mechanism for soluble to pore transition in AhlB and a contrasting tetrameric assembly employed by soluble AhlC to hide their hydrophobic membrane associated residues. We propose a model of pore assembly where the AhlC tetramer dissociates, binds a single membrane leaflet, recruits AhlB promoting soluble to pore transition, prior to AhlA binding to form the active hydrophilic lined pore.


Assuntos
Aeromonas hydrophila/metabolismo , Toxinas Bacterianas/química , Proteínas Hemolisinas/química , Proteínas Citotóxicas Formadoras de Poros/química , Aeromonas hydrophila/química , Aeromonas hydrophila/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Cristalografia por Raios X , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo
7.
mBio ; 10(2)2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015325

RESUMO

The cholesterol-dependent cytolysin (CDC) genes are present in bacterial species that span terrestrial, vertebrate, and invertebrate niches, which suggests that they have evolved to function under widely different environmental conditions. Using a combination of biophysical and crystallographic approaches, we reveal that the relative stability of an intramolecular interface in the archetype CDC perfringolysin O (PFO) plays a central role in regulating its pore-forming properties. The disruption of this interface allows the formation of the membrane spanning ß-barrel pore in all CDCs. We show here that the relative strength of the stabilizing forces at this interface directly impacts the energy barrier posed by the transition state for pore formation, as reflected in the Arrhenius activation energy (Ea) for pore formation. This change directly impacts the kinetics and temperature dependence of pore formation. We further show that the interface structure in a CDC from a terrestrial species enables it to function efficiently across a wide range of temperatures by minimizing changes in the strength of the transition state barrier to pore formation. These studies establish a paradigm that CDCs, and possibly other ß-barrel pore-forming proteins/toxins, can evolve significantly different pore-forming properties by altering the stability of this transitional interface, which impacts the kinetic parameters and temperature dependence of pore formation.IMPORTANCE The cholesterol-dependent cytolysins (CDCs) are the archetype for the superfamily of oligomeric pore-forming proteins that includes the membrane attack complex/perforin (MACPF) family of immune defense proteins and the stonefish venom toxins (SNTX). The CDC/MACPF/SNTX family exhibits a common protein fold, which forms a membrane-spanning ß-barrel pore. We show that changing the relative stability of an extensive intramolecular interface within this fold, which is necessarily disrupted to form the large ß-barrel pore, dramatically alters the kinetic and temperature-dependent properties of CDC pore formation. These studies show that the CDCs and other members of the CDC/MACPF/SNTX superfamily have the capacity to significantly alter their pore-forming properties to function under widely different environmental conditions encountered by these species.


Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Toxinas Bacterianas/genética , Fenômenos Químicos , Cristalografia por Raios X , Análise Mutacional de DNA , Proteínas Hemolisinas/genética , Cinética , Simulação de Dinâmica Molecular , Proteínas Citotóxicas Formadoras de Poros/genética , Temperatura
8.
Proc Natl Acad Sci U S A ; 116(8): 2897-2906, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30728296

RESUMO

The crystal structure of the Gram-negative insecticidal protein, GNIP1Aa, has been solved at 2.5-Å resolution. The protein consists of two structurally distinct domains, a MACPF (membrane attack complex/PerForin) and a previously uncharacterized type of domain. GNIP1Aa is unique in being a prokaryotic MACPF member to have both its structure and function identified. It was isolated from a Chromobacterium piscinae strain and is specifically toxic to Diabrotica virgifera virgifera larvae upon feeding. In members of the MACPF family, the MACPF domain has been shown to be important for protein oligomerization and formation of transmembrane pores, while accompanying domains define the specificity of the target of the toxicity. In GNIP1Aa the accompanying C-terminal domain has a unique fold composed of three pseudosymmetric subdomains with shared sequence similarity, a feature not obvious from the initial sequence examination. Our analysis places this domain into a protein family, named here ß-tripod. Using mutagenesis, we identified functionally important regions in the ß-tripod domain, which may be involved in target recognition.


Assuntos
Proteínas de Bactérias/química , Chromobacterium/química , Besouros/genética , Perforina/química , Sequência de Aminoácidos/genética , Animais , Proteínas de Bactérias/genética , Complexo de Ataque à Membrana do Sistema Complemento/química , Complexo de Ataque à Membrana do Sistema Complemento/genética , Cristalografia por Raios X , Inseticidas/química , Modelos Moleculares , Perforina/genética , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Domínios Proteicos , Estrutura Terciária de Proteína
9.
J Fish Dis ; 42(4): 465-475, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30734315

RESUMO

Aerolysin (aer) is one of the most important and abundant virulence factors in the infection of fish by Aeromonas veronii. A comprehensive study on the molecular characterization and pathogenicity of the aer gene from 34 A. veronii isolates from diseased carp and catfish was carried out and its interactome was analysed to observe the functional correlations between aer and other proteins within the A. veronii network. The PCR-based amplification of aer from the 34 isolates of A. veronii showed more aer-positive isolates from catfish with a high pathogenic potential in the in vivo challenge test than the carp fish. The analysis of aer gene sequence from challenged fish revealed significant sequence divergence according to the types and geographical distribution of the fish. The networking analysis of aer from the model A. veronii B565 revealed histidine kinase (cheA) as the most functional interacting partner. The study of the interaction between aer from the experimental A. veronii and cheA demonstrated that the A chain of cheA plays a more important role than the corresponding B chain during contact, and a linker sequence of 15 residues controlled the entire interaction process. Therefore, cheA could be an excellent drug target for controlling A. veronii infection of fish.


Assuntos
Aeromonas veronii/genética , Aeromonas veronii/patogenicidade , Toxinas Bacterianas/genética , Histidina Quinase/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Animais , Aquicultura , Carpas/microbiologia , Peixes-Gato/microbiologia , Doenças dos Peixes/microbiologia , Virulência/genética , Fatores de Virulência
10.
PLoS One ; 13(12): e0208151, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30517169

RESUMO

The communities of beneficial bacteria that live in our intestines, the gut microbiome, are important for the development and function of the immune system. Bacteroides species make up a significant fraction of the human gut microbiome, and can be probiotic and pathogenic, depending upon various genetic and environmental factors. These can cause disease conditions such as intra-abdominal sepsis, appendicitis, bacteremia, endocarditis, pericarditis, skin infections, brain abscesses and meningitis. In this study, we identify the transport systems and predict their substrates within seven Bacteroides species, all shown to be probiotic; however, four of them (B. thetaiotaomicron, B. vulgatus, B. ovatus, B. fragilis) can be pathogenic (probiotic and pathogenic; PAP), while B. cellulosilyticus, B. salanitronis and B. dorei are believed to play only probiotic roles (only probiotic; OP). The transport system characteristics of the four PAP and three OP strains were identified and tabulated, and results were compared among the seven strains, and with E. coli and Salmonella strains. The Bacteroides strains studied contain similarities and differences in the numbers and types of transport proteins tabulated, but both OP and PAP strains contain similar outer membrane carbohydrate receptors, pore-forming toxins and protein secretion systems, the similarities were noteworthy, but these Bacteroides strains showed striking differences with probiotic and pathogenic enteric bacteria, particularly with respect to their high affinity outer membrane receptors and auxiliary proteins involved in complex carbohydrate utilization. The results reveal striking similarities between the PAP and OP species of Bacteroides, and suggest that OP species may possess currently unrecognized pathogenic potential.


Assuntos
Proteínas de Bactérias/genética , Bacteroides/genética , Proteínas de Transporte/genética , Microbioma Gastrointestinal/genética , Genômica , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Bacteroides/classificação , Bacteroides/metabolismo , Proteínas de Transporte/classificação , Proteínas de Transporte/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Proteínas Citotóxicas Formadoras de Poros/classificação , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Probióticos/classificação , Probióticos/metabolismo , Salmonella/genética , Salmonella/metabolismo
11.
Nat Commun ; 9(1): 4805, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30442932

RESUMO

CD8 T cells protect the liver against viral infection, but can also cause severe liver damage that may even lead to organ failure. Given the lack of mechanistic insights and specific treatment options in patients with acute fulminant hepatitis, we develop a mouse model reflecting a severe acute virus-induced CD8 T cell-mediated hepatitis. Here we show that antigen-specific CD8 T cells induce liver damage in a perforin-dependent manner, yet liver failure is not caused by effector responses targeting virus-infected hepatocytes alone. Additionally, CD8 T cell mediated elimination of cross-presenting liver sinusoidal endothelial cells causes endothelial damage that leads to a dramatically impaired sinusoidal perfusion and indirectly to hepatocyte death. With the identification of perforin-mediated killing as a critical pathophysiologic mechanism of liver failure and the protective function of a new class of perforin inhibitor, our study opens new potential therapeutic angles for fulminant viral hepatitis.


Assuntos
Células Endoteliais/efeitos dos fármacos , Hepatite Viral Animal/tratamento farmacológico , Fígado/efeitos dos fármacos , Proteínas Citotóxicas Formadoras de Poros/antagonistas & inibidores , Substâncias Protetoras/farmacologia , Sulfonamidas/farmacologia , Adenoviridae/genética , Adenoviridae/imunologia , Adenoviridae/patogenicidade , Animais , Anticorpos/administração & dosagem , Antígenos CD40/antagonistas & inibidores , Antígenos CD40/genética , Antígenos CD40/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Capilares/efeitos dos fármacos , Capilares/virologia , Modelos Animais de Doenças , Células Endoteliais/imunologia , Células Endoteliais/virologia , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hepatite Viral Animal/imunologia , Hepatite Viral Animal/virologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Fígado/irrigação sanguínea , Fígado/patologia , Fígado/virologia , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/administração & dosagem , Poli I-C/administração & dosagem , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/imunologia
12.
Microbiol Immunol ; 62(12): 774-785, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30378708

RESUMO

An effective vaccine against Pseudomonas aeruginosa would be hugely beneficial to people who are susceptible to the serious infections it can cause. Vaccination against PcrV of the P. aeruginosa type III secretion system is a potential prophylactic strategy for improving the incidence and prognosis of P. aeruginosa pneumonia. Here, the effect of nasal PcrV adjuvanted with CpG oligodeoxynucleotide (CpG) was compared with a nasal PcrV/aluminum hydroxide gel (alum) vaccine. Seven groups of mice were vaccinated intranasally with one of the following: 1, PcrV-CpG; 2, PcrV-alum; 3, PcrV alone; 4, CpG alone; 5, alum alone; 6 and 7, saline control. Fifty days after the first immunization, anti-PcrV IgG, IgA and IgG isotype titers were measured; significant increases in these titers were detected only in the PcrV-CpG vaccinated mice. The vaccinated mice were then intratracheally infected with a lethal dose of P. aeruginosa and their body temperatures and survival monitored for 24 hr, edema, bacteria, myeloperoxidase activity and lung histology also being evaluated at 24 hr post-infection. It was found that 73% of the PcrV-CpG-vaccinated mice survived, whereas fewer than 30% of the mice vaccinated with PcrV-alum or adjuvant alone survived. Lung edema and other inflammation-related variables were less severe in the PcrV-CpG group. The significant increase in PcrV-specific IgA titers detected following PcrV-CpG vaccination is probably a component of the disease protection mechanism. Overall, our data show that intranasal PcrV-CpG vaccination has potential efficacy for clinical application against P. aeruginosa pneumonia.


Assuntos
Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Oligodesoxirribonucleotídeos/imunologia , Pneumonia/prevenção & controle , Proteínas Citotóxicas Formadoras de Poros/imunologia , Infecções por Pseudomonas/prevenção & controle , Vacinas contra Pseudomonas/imunologia , Pseudomonas aeruginosa/efeitos dos fármacos , Vacinação , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Toxinas Bacterianas/genética , Temperatura Corporal , Modelos Animais de Doenças , Edema , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos , Oligodesoxirribonucleotídeos/genética , Peroxidase/análise , Proteínas Citotóxicas Formadoras de Poros/genética , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Vacinas contra Pseudomonas/administração & dosagem , Pseudomonas aeruginosa/patogenicidade , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Taxa de Sobrevida , Sistemas de Secreção Tipo III/imunologia
13.
ACS Chem Biol ; 13(11): 3153-3160, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30278129

RESUMO

Immunotoxins are proteins containing a cell-targeting element linked to a toxin that are under investigation for next-generation cancer treatment. However, these agents are difficult to synthesize, chemically heterogeneous, expensive, and show toxicity toward healthy cells. In this work, we describe the synthesis and characterization of a new type of immunotoxin that showed exquisite selectivity toward targeted cells. In our construct, targeting molecules were covalently attached or genetically fused to oligomeric pore-forming toxins. The activity of the immunotoxin was then caged by fusing a soluble protein to the transmembrane domain and activated via cleavage with furin, which is a protease that is overexpressed in many cancer cells. During the several coupling steps, directed evolution allowed the efficient synthesis of the molecules in E. coli cells, as well as selection for further specificity toward targeted cells. The final construct showed no off-target activity, while acquiring an additional degree of specificity toward the targeted cells upon activation. The pore-forming toxins described here do not require internalization to operate, while the many protomeric subunits can be individually modified to refine target specificity.


Assuntos
Venenos de Cnidários/farmacologia , Imunotoxinas/farmacologia , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Tetra-Hidrofolato Desidrogenase/farmacologia , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Venenos de Cnidários/genética , Evolução Molecular Direcionada/métodos , Desenho de Fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Fólico/química , Furina/metabolismo , Humanos , Imunotoxinas/química , Imunotoxinas/genética , Imunotoxinas/metabolismo , Mutagênese , Proteínas Citotóxicas Formadoras de Poros/genética , Proteólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Salmonella typhi/química , Anêmonas-do-Mar/química , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo
14.
Virulence ; 9(1): 1112-1125, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30067143

RESUMO

Trueperella pyogenes (T. pyogenes) is an important opportunistic pathogen. Pyolysin (PLO) importantly contributes to the pathogenicity of T. pyogenes. However, the relationship between the structure and function and the virulence of PLO is not well documented. In the current study, recombinant PLO (rPLO) and three rPLO mutants were prepared. rPLO D238R, a mutant with the 238th aspartic acid replaced with an arginine, showed impairment in oligomerization activity on cholesterol-containing liposome and pore-forming activity on sheep red blood cell membrane. Further study employing the prepared mutants confirmed that the pore-forming activity of PLO is essential for inducing excessive inflammation responses in mice by upregulating the expression levels of IL-1ß, TNF-α, and IL-6. By contrast, rPLO P499F, another mutant with impaired cell membrane binding capacity, elicited an inflammation response that was dependent on pathogen-associated molecular pattern (PAMP) activity, given that the mutant significantly upregulated the expression of IL-10 in macrophages and in mice, whereas rPLO did not. Results indicated that domain 1 of the PLO molecule plays an important role in maintaining pore-forming activity. Moreover, the PLO pore-forming activity and not PAMP activity is responsible for the inflammation-inducing effect of PLO. The results of this study provided new information for research field on the structure, function, and virulence of PLO. ABBREVIATIONS: T. pyogenes: Trueperella pyogenes; PLO: Pyolysin; rPLO: recombinant PLO; PAMP: pathogen-associated molecular pattern; CDCs: cholesterol-dependent cytolysins; PLY: pneumolysin; NLRP3: NLR family pyrin domain containing protein 3; PRRs: pattern recognition receptors; Asp: aspartic acid; TLR4: Toll-like receptor 4; Arg: arginine; Asn: asparagine; IPTG: Isopropyl-ß-d-thiogalactoside; PBS: phosphate-buffered saline; sRBCs: sheep red blood cells; TEM: Transmission electron microscopy; RBCM: red blood cell membrane; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; NC membrane: nitrocellulose membrane; SDS-AGE: dodecyl sulfate agarose gel electrophoresis; MDBK cells: Madin-Darby bovine kidney cells; RPMI-1640 medium: Roswell Park Memorial Institute-1640 medium; FBS: fetal bovine serum; BMDMs: bone marrow-derived macrophages; TNF-α: tumor necrosis factor α; IL-1ß: interleukin-1ß; IFN-γ: interferon-γ; TGF-ß: transforming growth factor-ß; ELISA: enzyme-linked immunosorbent assay.


Assuntos
Actinomycetaceae/genética , Arginina/genética , Ácido Aspártico/genética , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Proteínas Hemolisinas/química , Inflamação/induzido quimicamente , Actinomycetaceae/química , Actinomycetaceae/patogenicidade , Substituição de Aminoácidos , Animais , Arginina/química , Ácido Aspártico/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Bovinos , Linhagem Celular , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/ultraestrutura , Feminino , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/toxicidade , Hemólise , Interleucina-10/genética , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Distribuição Aleatória , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidade , Ovinos , Fator de Necrose Tumoral alfa/genética , Virulência
15.
Environ Microbiol ; 20(9): 3442-3456, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30136361

RESUMO

Aeromonas species are ubiquitous inhabitants of freshwater environments, and are responsible for fish motile aeromonad septicemia (MAS). A. hydrophila is implicated as the primary etiologic agent of MAS. Here, we analysed MAS epidemiological data for cyprinid fish in southern China, and found that A. veronii infections dominated. Consistent with this observation, A. veronii isolates were generally more virulent than A. hydrophila isolates when infecting germ-free zebrafish larvae via continuous immersion challenge. Through in vivo screening of the transposon library of the A. veronii strain Hm091, aerolysin was identified as the key virulence factor. Further results indicated that A. veronii Hm091 aerolysin disrupts the intestinal barrier of zebrafish, enabling systematic invasion by not only A. veronii Hm091 in a mono-infection, but also A. hydrophila NJ-1 in a mixed infection. Moreover, the differences in aerolysin expression and activity were the major contributor to the observed differences between the A. veronii and A. hydrophila strains regarding invasion efficacy via intestine. Together, our results provide new insights into the aetiology and pathogenesis of Aeromonas infections, and highlight the importance of A. veronii-targeted treatments in future efforts against MAS.


Assuntos
Aeromonas veronii/metabolismo , Aeromonas veronii/patogenicidade , Toxinas Bacterianas/metabolismo , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Sepse/veterinária , Aeromonas/isolamento & purificação , Aeromonas veronii/genética , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , China , Infecções por Bactérias Gram-Negativas/microbiologia , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/toxicidade , Sepse/microbiologia , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Fatores de Virulência/toxicidade , Peixe-Zebra/microbiologia
16.
Mar Drugs ; 16(6)2018 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-29794988

RESUMO

Sea anemones produce pore-forming toxins, actinoporins, which are interesting as tools for cytoplasmic membranes study, as well as being potential therapeutic agents for cancer therapy. This investigation is devoted to structural and functional study of the Heteractis crispa actinoporins diversity. Here, we described a multigene family consisting of 47 representatives expressed in the sea anemone tentacles as prepropeptide-coding transcripts. The phylogenetic analysis revealed that actinoporin clustering is consistent with the division of sea anemones into superfamilies and families. The transcriptomes of both H. crispa and Heteractis magnifica appear to contain a large repertoire of similar genes representing a rapid expansion of the actinoporin family due to gene duplication and sequence divergence. The presence of the most abundant specific group of actinoporins in H. crispa is the major difference between these species. The functional analysis of six recombinant actinoporins revealed that H. crispa actinoporin grouping was consistent with the different hemolytic activity of their representatives. According to molecular modeling data, we assume that the direction of the N-terminal dipole moment tightly reflects the actinoporins' ability to possess hemolytic activity.


Assuntos
Venenos de Cnidários/farmacologia , Hemólise/efeitos dos fármacos , Família Multigênica/genética , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Anêmonas-do-Mar/genética , Sequência de Aminoácidos , Animais , Membrana Celular/efeitos dos fármacos , Venenos de Cnidários/química , Venenos de Cnidários/genética , Simulação por Computador , Duplicação Gênica , Simulação de Dinâmica Molecular , Filogenia , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Anêmonas-do-Mar/metabolismo , Transcriptoma/genética
17.
Proteins ; 86(9): 897-911, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29722060

RESUMO

We report the characterization of the dimeric protein AB21 from Agaricus bisporus, one of the most commonly and widely consumed mushrooms in the world. The protein shares no significant sequence similarity with any protein of known function, and it is the first characterized member of its protein family. The coding sequence of the ab21 gene was determined and the protein was expressed in E. coli in a recombinant form. We demonstrated a high thermal and pH stability of AB21 and proved the weak affinity of the protein to divalent ions of some transition metals (nickel, zinc, cadmium, and cobalt). The reported crystallographic structure exhibits an interesting rod-like helical bundle fold with structural similarity to bacterial toxins of the ClyA superfamily. By immunostaining, we demonstrated an abundance of AB21 in the fruiting bodies of A. bisporus.


Assuntos
Agaricus/química , Toxinas Bacterianas/química , Proteínas Fúngicas/biossíntese , Proteínas Citotóxicas Formadoras de Poros/biossíntese , Cátions Bivalentes/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Elementos de Transição/química
18.
ACS Sens ; 3(4): 779-783, 2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29619834

RESUMO

Selectivity and sensitivity are two key parameters utilized to describe the performance of a sensor. In order to investigate selectivity and sensitivity of the aerolysin nanosensor, we manipulated its surface charge at different locations via single site-directed mutagenesis. To study the selectivity, we replaced the positively charged R220 at the entrance of the pore with negatively charged glutamic acid, resulting in barely no current blockages for sensing negatively charged oligonucleotides. For the sensitivity, we substituted the positively charged lumen-exposed amino acid K238 located at trans-ward third of the ß-barrel stem with glutamic acid. This leads to a surprisingly longer duration time at +140 mV, which is about 20 times slower in translocation speed for Poly(dA)4 compared to that of wild-type aerolysin, indicating the stronger pore-analyte interactions and enhanced sensitivity. Therefore, it is both feasible and understandable to rationally design confined biological nanosensors for single molecule detection with high selectivity and sensitivity.


Assuntos
Toxinas Bacterianas/análise , Toxinas Bacterianas/genética , Mutagênese Sítio-Dirigida , Nanoporos , Proteínas Citotóxicas Formadoras de Poros/análise , Proteínas Citotóxicas Formadoras de Poros/genética , Toxinas Bacterianas/química , Modelos Moleculares , Proteínas Citotóxicas Formadoras de Poros/química
19.
Toxicon ; 148: 40-49, 2018 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-29649486

RESUMO

Actinoporins are pore-forming proteins found in sea anemones. Although we now have a large collection of data on actinoporins, our knowledge is based heavily on those identified in shallow-water anemones. Because the deep sea differs considerably from shallow waters in hydrostatic pressures, temperatures, and the prey composition, the deep-sea actinoporin may have evolved in unique ways. This study, therefore, aimed to obtain new actinoporins in the deep-sea anemone Cribrinopis japonica (Actiniaria, Actiniidae). An actinoporin-like sequence was identified from the previously established C. japonica RNA-Seq database, and the complete length (663 bp) of the deep-sea actinoporin gene, Cjtox I, was obtained. In addition, a similar gene, Cjtox II (666 bp), was also identified from RNA of actinopharynx. CJTOX I and CJTOX II were similar in their primary structures, but CJTOX I lacked one residue in the middle of the protein. There was also a difference in the gene expression in live animals, where only Cjtox I was expressed in tentacles of C. japonica. In the heterologous expression where BL21 (DE3) strain was retransformed with the plasmid containing either Cjtox I or Cjtox II gene, the supernatants of both cell lysates showed hemolytic activity on the equine erythrocytes. Preincubation of the supernatants with sphingomyelin caused reduced activity, implying that the CJTOX I and II would target sphingomyelin as with other actinoporins. Because of the structures similarity to the known actinoporins and the sphingomyelin-inhibitable hemolytic activity, both CJTOX I and II were concluded to be new actinoporins, which were identified for the first time from a deep-sea anemone.


Assuntos
Venenos de Cnidários/química , Proteínas Citotóxicas Formadoras de Poros/química , Anêmonas-do-Mar , Sequência de Aminoácidos , Animais , Sequência de Bases , Venenos de Cnidários/genética , Venenos de Cnidários/metabolismo , Eritrócitos/efeitos dos fármacos , Expressão Gênica , Hemólise/efeitos dos fármacos , Cavalos , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Esfingomielinas/antagonistas & inibidores
20.
Dev Comp Immunol ; 82: 49-54, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29317232

RESUMO

Following the Aeromonas hydrophila aerolysin, various aerolysin-like pore-forming proteins have been identified from bacteria to vertebrates. We have recently reported the mechanism of receptor recognition and in vitro pore-formation of a zebrafish aerolysin-like protein Dln1/Aep1. However, the physiological function of Aep1 remains unknown. Here we detected that aep1 gene is constitutively expressed in various immune-related tissues of adult zebrafish; and moreover, its expression is significantly up-regulated upon bacterial challenge, indicating its involvement in antimicrobial infection. Pre-injection of recombinant Aep1 into the infected zebrafish greatly accelerated the clearance of bacteria, resulting in significantly increased survival rate. Meanwhile, the induced expression of cytokines such as interleukin IL-1ß and tumor necrosis factor TNF-α in zebrafish upon injection of recombinant Aep1 suggested that Aep1 may be a pro-inflammatory protein that triggers the antimicrobial immune responses. However, compared to the overproduction of these cytokines in the infected zebrafish, pre-injection of Aep1 could significantly reduce the expression level of these cytokines, accompanying with a reduced bacterial load. Moreover, the expression profiles through the developmental stages of zebrafish demonstrated that aep1 is activated at the very early stage prior to the maturation of adaptive immune system. Altogether, our findings proved that Aep1 is an innate immune molecule that prevents the bacterial infection.


Assuntos
Toxinas Bacterianas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/imunologia , Imunidade Adaptativa/genética , Aeromonas hydrophila/genética , Animais , Toxinas Bacterianas/genética , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Imunidade Inata , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Filogenia , Proteínas Citotóxicas Formadoras de Poros/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas de Peixe-Zebra/genética
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