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1.
Mikrobiyol Bul ; 54(1): 144-153, 2020 Jan.
Artigo em Turco | MEDLINE | ID: mdl-32050885

RESUMO

Avian chlamydiosis, is a highly contagious, systemic disease occuring in domestic and wild birds. Chlamydia psittaci, the causative agent of the disease, is a gram-negative bacterium in the Chlamydiaceae family that can only live within the cell. The agent can be transmitted directly to humans by contact with infected animals or feces of infected animals. It can also be transmitted by inhalation of fecal dust. Since the disease has a zoonotic character, it is also important in terms of public health. By using the monoclonal antibodies against cell wall proteins (OMP) of C.psittaci, six (A-F) and two (WC and M56) serotypes were determined in mammals. The aim of this study was to investigate and genotype the presence of C.psittaci ompA gene in domestic pigeon feces grown in family management style in ten different districts in Ankara in winter and summer seasons. Within the scope of the study, 100 pigeon stool samples were collected from birdhouses in 10 different districts of Ankara (Beypazari, Haymana, Kizilcahamam, Cubuk, Pursaklar, Bala, Cankaya, Polatli, Golbasi and city center) in two different seasons. DNA extraction from fecal samples was performed by classical methods. The presence of the agent in the extracted DNA samples was investigated by polymerase chain reaction (PCR) analysis of the ompA gene. Two-way sequence analysis of the ompA gene was performed with the primers used in the study from the target DNA products amplified by PCR. The results of sequence analysis were compared with the international database and serotyping/genotyping was performed. In the study, C.psittaci ompA gene was detected in 6 (6%) samples of 100 pigeon stool samples. Among these positive samples, two were from Bala (one sample from winter, one sample from summer), two were from Haymana (one sample from winter, one sample from summer) and two were from Golbasi (one sample from winter, one sample from summer); where the same agent was isolated in the same aviaries in different seasons. In this study, no difference was found between the presence of C.psittaci in pigeon droppings and season. In addition when the sequence analysis of the isolated samples were compared with the World database; all isolates were found to be 100% genotype B and 99% genotype E. In this study, the sequence analysis of the ompA gene of C.psittaci from domestic pigeon feces was determined for the first time in Turkey. Although the presence of C.psittaci in domestic pigeons is low, it is a zoonotic bacterium and is important for the public health.


Assuntos
Proteínas da Membrana Bacteriana Externa , Doenças das Aves , Chlamydophila psittaci , Columbidae , Fezes , Psitacose , Animais , Proteínas da Membrana Bacteriana Externa/genética , Doenças das Aves/microbiologia , Chlamydophila psittaci/genética , Columbidae/microbiologia , Fezes/microbiologia , Genótipo , Psitacose/microbiologia , Turquia
2.
Nat Commun ; 11(1): 564, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992701

RESUMO

Detergents enable the purification of membrane proteins and are indispensable reagents in structural biology. Even though a large variety of detergents have been developed in the last century, the challenge remains to identify guidelines that allow fine-tuning of detergents for individual applications in membrane protein research. Addressing this challenge, here we introduce the family of oligoglycerol detergents (OGDs). Native mass spectrometry (MS) reveals that the modular OGD architecture offers the ability to control protein purification and to preserve interactions with native membrane lipids during purification. In addition to a broad range of bacterial membrane proteins, OGDs also enable the purification and analysis of a functional G-protein coupled receptor (GPCR). Moreover, given the modular design of these detergents, we anticipate fine-tuning of their properties for specific applications in structural biology. Seen from a broader perspective, this represents a significant advance for the investigation of membrane proteins and their interactions with lipids.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Detergentes/química , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Escherichia coli , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Espectrometria de Massas , Lipídeos de Membrana , Modelos Moleculares , Peptídeo Hidrolases/química , Peptídeo Hidrolases/isolamento & purificação , Redobramento de Proteína , Solubilidade
3.
Acta Virol ; 63(4): 450-458, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31802688

RESUMO

For successful infection, viruses must recognize their respective host cells. A common mechanism of host recognition by viruses is to utilize a portion of the host cell as a receptor. Bacteriophage Sf6, which infects Shigella flexneri, uses lipopolysaccharide as a primary receptor and then requires interaction with a secondary receptor, a role that can be fulfilled by either outer membrane proteins (Omp) A or C. Our previous work showed that specific residues in the loops of OmpA mediate Sf6 infection. To better understand Sf6 interactions with OmpA loop variants, we determined the kinetics of these interactions through the use of biolayer interferometry, an optical biosensing technique that yields data similar to surface plasmon resonance. Here, we successfully tethered whole Sf6 virions, determined the binding constant of Sf6 to OmpA to be 36 nM. Additionally, we showed that Sf6 bound to five variant OmpAs and the resulting kinetic parameters varied only slightly. Based on these data, we propose a model in which Sf6: Omp receptor recognition is not solely based on kinetics, but likely also on the ability of an Omp to induce a conformational change that results in productive infection. Keywords: Sf6; Shigella flexneri; OmpA; biolayer interferometry.


Assuntos
Proteínas da Membrana Bacteriana Externa , Bacteriófagos , Vírion , Proteínas da Membrana Bacteriana Externa/metabolismo , Bacteriófagos/metabolismo , Cinética , Ligação Proteica , Vírion/metabolismo
4.
World J Microbiol Biotechnol ; 36(1): 9, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31858269

RESUMO

Campylobacter jejuni is the one of the leading cause of bacterial food borne gastroenteritis. PglB, a glycosyltransferase, plays a crucial role of mediating glycosylation of numerous periplasmic proteins. It catalyzes N-glycosylation at the sequon D/E-X1-N-X2-S/T in its substrate proteins. Here we report that the PglB itself is a glycoprotein which self-glycosylates at N534 site in its DYNQS sequon by its own catalytic WWDYG motif. Site-directed mutagenesis, lectin Immunoblot, and mobility shift assays confirmed that the DYNQS is an N-glycosylation motif. PglB's N-glycosylation motif is structurally and functionally similar to its widely studied glycosylation substrate, the OMPH1. Its DYNQS motif forms a solvent-exposed crest. This motif is close to a cluster of polar and hydrophilic residues, which form a loop flanked by two α helices. This arrangement extremely apposite for auto-glycosylation at N534. This self-glycosylation ability of PglB could mediate C. jejuni's ability to colonize the intestinal epithelium. Further this capability may also bear significance for the development of novel conjugated vaccines and diagnostic tests.


Assuntos
Campylobacter jejuni/enzimologia , Glicoproteínas/química , Hexosiltransferases/química , Proteínas de Membrana/química , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Glicosilação , Hexosiltransferases/genética , Hexosiltransferases/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Modelos Moleculares , Mutagênese Sítio-Dirigida , Filogenia , Conformação Proteica , Alinhamento de Sequência , Análise de Sequência de Proteína , Vacinas
5.
Int. microbiol ; 22(4): 471-478, dic. 2019. graf, tab
Artigo em Inglês | IBECS | ID: ibc-185065

RESUMO

Chlamydia trachomatis is considered as a public health problem due to its high prevalence and increased rates of gynecological disorders. The major outer membrane protein (MOMP) of this bacterium is the most abundant protein in its membrane and has been evaluated not only as a vaccine development candidate but also is used in many diagnostic tests. The MOMP weighs 69 kDa and contains four variable segments (VS 1-4) separated by constant regions. Several research groups have developed recombinant single-variable segments of MOMP expressed in Escherichia coli cytoplasm. But, all variable segments have been used minimally for the diagnosis of a chlamydial infection. In this experiment, the authors obtained the recombinant MOMP of C. trachomatis (rMOMP) in E. coli rMOMP and extracted, purified, and partially characterized it. This was later used to identify anti-Chlamydia trachomatis antibodies in sera of infertile patients by immunodetection assays, enzyme-linked immunosorbent assay (ELISA), and indirect immunofluorescence tests. The ELISA test showed high sensitivity and low specificity of 100 and 58.3%, respectively. The above results obtained were linked to the cross-reactivity of antibodies against C. pneumoniae or C. psittaci. Hence, an evaluation was performed to obtain an optimized test for the diagnosis of C. trachomatis infection


No disponible


Assuntos
Antígenos de Bactérias/análise , Chlamydia trachomatis/isolamento & purificação , Infecções por Chlamydia/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas Recombinantes/análise , Proteínas da Membrana Bacteriana Externa/metabolismo , Chlamydia trachomatis/genética , Infecções por Chlamydia/microbiologia , Proteínas Recombinantes/genética
6.
Emerg Microbes Infect ; 8(1): 1711-1720, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31769735

RESUMO

Among the factors associated with the resurgence of whooping cough, special emphasis has been given to pathogen adaptation after the introduction of the acellular vaccine (ACV). To assess the impact of the vaccine transition strategy from whole-cell vaccine (WCV) to ACV on population dynamics of Bordetella pertussis in Barcelona (Spain), we studied 339 isolates collected from 1986 to 2015 by PFGE and multi-locus variable-number tandem repeat analysis (MLVA). Additionally, allelic variants for the pertussis toxin and its promoter, pertactin, type 3 fimbriae and fimbrial serotyping were assessed to determine its antigenic drift. A shift was observed in the B. pertussis population as well as in its antigenic profile concurrently with the introduction of ACV in Barcelona. Four out of the five most prevalent PFGE profiles were replaced by new profiles following the ACV introduction. MLVA type 27 was the dominant genotype, and its frequency increased from 25% to 79.3% after WCV replacement. Antigen typing demonstrated the emergence of prn2, ptxP3, fim3-2 and a shift from the fimbriae 3 to the fimbriae 2 serotypes after the ACV introduction. Our findings support the presence of population and antigenic dynamic changes in B. pertussis likely driven by the introduction of ACV.


Assuntos
Variação Antigênica , Vacinas Bacterianas/imunologia , Bordetella pertussis/imunologia , Coqueluche/microbiologia , Proteínas da Membrana Bacteriana Externa/administração & dosagem , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Bordetella pertussis/genética , Genótipo , Humanos , Repetições Minissatélites , Dinâmica Populacional , Espanha , Fatores de Virulência de Bordetella/administração & dosagem , Fatores de Virulência de Bordetella/genética , Fatores de Virulência de Bordetella/imunologia , Coqueluche/prevenção & controle
7.
Zhonghua Yu Fang Yi Xue Za Zhi ; 53(11): 1130-1135, 2019 Nov 06.
Artigo em Chinês | MEDLINE | ID: mdl-31683400

RESUMO

Objective: To understand the situation and genotype distribution of spotted fever group rickettsia (SFGR) in the border area of Tumen River Basin in free ticks in Yanbian Korean Autonomous Prefecture (Yanbian Prefecture), Jilin Province. Methods: From April to September, 2017, ticks were collected using flagging method from Hunchun, Tumen, Helong and Longjing cities in the Tumen River basin of Yanbian Prefecture. Outer membrane protein A (ompA) was detected by Polymerase Chain Reaction (PCR), then, the species were identified by gene sequencing and analyzed systematically. The positive rate of pools and MIR(minimum infection rate per 100 ticks,MIR) of SFGR were calculated, and the difference of positive rate of pools among ticks with different characteristics was compared by Chi-square test. Results: A total of 3 079 ticks were collected and divided into 536 pools. The positive rate of pools of SFGR nucleic acid was 39.7% (213 pools). The MIR of SFGR was 6.9%.The positive rate of pools of SFGR in Dermacentor silvarum, Haemaphysalis concinna, Haemaphysalis japonica, Haemaphysalis longicornis and Ixodes persulcatus were 80.4% (41/51), 14.0% (25/179), 20.2% (18/89), 78.9% (101/128) and 25.9% (21/81), and the difference was statistically significant (P<0.001). There was statistical difference in the positive rate of pools of SFGR in developmental stages of ticks (P<0.001); the positive rate of pools of female adults, male adults, nymph and larvae were 36.4% (95/261), 34.2% (67/196), 56.3% (40/71) and 7/8, and the MIR was 7.9%, 7.7%, 4.9% and 3.5%. The five genotype was detected which was Candidatus Rickettsia longicornii, Rickettsia raoultii, Rickettsia heilongjiangensis, Candidatus Rickettsia tarasevichiae,Rickettsia monacensis and have 98%-100% homology with known gene sequences. Candidatus Rickettsia longicornii, Rickettsia raoultii, Rickettsia heilongjiangensis and Candidatus Rickettsia tarasevichiae showed close evolutionary relationship with known specie (have 98%-100% homology with known gene sequences); Rickettsia monacensis showed Far from evolutionary relationship with known species (have 98% homology with known gene sequences). Conclusion: SFGR infection of ticks is common in the border areas of the Tumen River Basin. There was high diversity in SFGR species and tick species in the areas surveyed.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Ixodidae/microbiologia , Rickettsia/classificação , Rickettsia/isolamento & purificação , Rickettsiose do Grupo da Febre Maculosa/diagnóstico , Carrapatos , Animais , China , Feminino , Ixodidae/classificação , Ixodidae/crescimento & desenvolvimento , Masculino , Reação em Cadeia da Polimerase , Rickettsia/genética , Rios , Análise de Sequência
8.
Infect Immun ; 88(1)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31636136

RESUMO

Streptococcus equi subsp. zooepidemicus is an important pathogen in horses that causes severe diseases such as pneumonia and abortion. Furthermore, it is a zoonotic agent, and contact with horses is a known risk factor. In this study, we investigated the working hypothesis that the zoonotic potential varies among S. equi subsp. zooepidemicus strains in association with differences in M-like protein-mediated binding of host plasma proteins. We demonstrate via in-frame deletion mutagenesis of two different S. equi subsp. zooepidemicus strains that the M-like protein SzM is crucial for the binding of fibrinogen to the bacterial surface and for survival in equine and human blood. S. equi subsp. zooepidemicus isolates of equine and human origins were compared with regard to SzM sequences and binding of equine and human fibrinogens. The N-terminal 216 amino acids of the mature SzM were found to exhibit a high degree of diversity, but the majority of human isolates grouped in three distinct SzM clusters. Plasma protein absorption assays and flow cytometry analysis revealed that pronounced binding of human fibrinogen is a common phenotype of human S. equi subsp. zooepidemicus isolates but much less so in equine S. equi subsp. zooepidemicus isolates. Furthermore, binding of human fibrinogen is associated with specific SzM types. These results suggest that SzM-mediated binding of human fibrinogen is an important virulence mechanism of zoonotic S. equi subsp. zooepidemicus isolates.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/metabolismo , Fibrinogênio/metabolismo , Interações Hospedeiro-Patógeno , Streptococcus equi/metabolismo , Fatores de Virulência/metabolismo , Animais , Antígenos de Bactérias/classificação , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/classificação , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Transporte/classificação , Proteínas de Transporte/genética , Análise por Conglomerados , Variação Genética , Cavalos , Humanos , Fenótipo , Ligação Proteica , Homologia de Sequência , Fatores de Virulência/classificação , Fatores de Virulência/genética
9.
J Chem Theory Comput ; 15(12): 6551-6561, 2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31665603

RESUMO

The plasticity of membranes plays an important functional role in cells, cell components, and micelles, where bending, budding, and remodeling implement numerous recognition and communication processes. Comparatively, molecular simulation methods to induce, control, and quantitatively characterize such deformations remain scarce. This work defines a novel collective coordinate associated with membrane bending, which strives to combine realism (by preserving the notion of local atomic curvatures) and low computational cost (allowing its evaluation at every time step of a molecular dynamics simulation). Enhanced sampling simulations along this conformational coordinate provide convenient access to the underlying bending free energy landscape. To showcase its potential, the method is applied to three state-of-the-art problems: the determination of the bending free energy landscape of a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) bilayer, the formation of a POPE liposome, and the study of the influence of the Pseudomonas quinolone signal on the budding of Gram-negative bacterial outer membranes.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Fosfatidiletanolaminas/química , Lipossomos/química , Conformação Molecular , Quinolonas/química
10.
J Agric Food Chem ; 67(42): 11703-11709, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31578056

RESUMO

Astaxanthin is a carotenoid of high commercial value because of its excellent antioxidative, anti-inflammatory, and anticancer properties. Here, we developed a novel strategy for improving the production of astaxanthin via morphology and oxidative stress engineering. First, we identified the morphology-/membrane- and oxidative stress-related genes, which should be knocked down, using the CRISPRi system. Deleting the morphology-/membrane-related genes (lpp and bamB) and the oxidative stress-related genes (uspE and yggE) generated longer and larger cells with higher reactive oxygen species (ROS) levels, thus enhancing the production of astaxanthin and decreasing cell growth. To not only improve cell growth but also obtain longer and larger cells with higher ROS levels, a complementary expression system using a temperature-sensitive plasmid was established. Complementarily expressing the morphology-/membrane-related genes (lpp and bamB) and the oxidative stress-related genes (uspE and yggE) further improved the production of astaxanthin to 11.92 mg/g dry cell weight in shake flask cultures.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/citologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Engenharia Metabólica , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Xantofilas/biossíntese
11.
APMIS ; 127(12): 753-763, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31512768

RESUMO

Iron uptake system is expressed in early stages of Acinetobacter baumannii infections under iron-restricted conditions. This study is aimed at the evaluation of immuno-protectivity of BfnH in comparison with BauA in both mature and selected fragmental proteins. The study was designed in single and combined forms of antigens. BfnH is presented in 3472 strains of A. baumannii with more than 97% identity. The preliminary immune-informatics analysis of this protein indicated a region from the ß-barrel domain including exposed loops 2-5, with antigenic score comparable to that of BfnH. There was a significant rise in the specific IgG response in all test groups. The bacterial challenge with a lethal dose of A. baumannii demonstrated partial protection of whole proteins which coincides with a significant reduction in the bacterial population colonized in the main organs and an increase in the survival level. Passive immunization of the mice brought about 50% survival in the mice groups immunized with BfnH and with a combination of BfnH and BauA. The protectivity of siderophore receptors suggests their potential immunogenic role that could be considered as a component of multivalent subunit vaccine candidates against A. baumannii.


Assuntos
Infecções por Acinetobacter/prevenção & controle , Acinetobacter baumannii/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Imunização , Receptores de Superfície Celular/imunologia , Infecções por Acinetobacter/imunologia , Infecções por Acinetobacter/microbiologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Carga Bacteriana , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Epitopos de Linfócito B , Feminino , Imunogenicidade da Vacina , Camundongos Endogâmicos BALB C , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética
12.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31548314

RESUMO

Leptospirosis is one of the most widespread zoonoses caused by pathogenic Leptospira spp. In this study, we report that the LIC11966/ErpY-like lipoprotein is a surface-exposed outer membrane protein exclusively present in pathogenic species of Leptospira The recombinant ErpY (rErpY)-like protein is recognized by the immunoglobulins of confirmed leptospirosis sera of diverse hosts (human, bovine, and canine), suggesting the expression of the native leptospiral surface protein during infection. Circular dichroism of pure rErpY-like protein showed the secondary structural integrity to be uncompromised during the purification process. Analysis of the rErpY-like protein by native polyacrylamide gel electrophoresis, chemical cross-linking, dynamic light scattering, and field emission transmission electron microscopy demonstrated it undergoes supramolecular assembly. The rErpY-like protein can bind to diverse host extracellular matrices, and it presented a saturable and strong binding affinity (dissociation constant [KD ] of 70.45 ± 4.13 nM) to fibrinogen, a central host plasma component involved in blood clotting. In the presence of the rErpY-like supramolecule, thrombin-catalyzed fibrin clot formation is inhibited up to 7%, implying its role in inhibiting blood coagulation during Leptospira infection. In addition, binding of the rErpY-like supramolecule to complement factors H and I suggests the protein also contributes to Leptospira evading innate host defense during infection by inactivating alternative complement pathways. This study reveals that rErpY-like protein is functionally active in the supramolecular state and performs moonlighting activity under the given in vitro conditions.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Coagulação Sanguínea/fisiologia , Fator H do Complemento/metabolismo , Fator I do Complemento/metabolismo , Leptospira/imunologia , Leptospirose/diagnóstico , Animais , Dicroísmo Circular , Via Alternativa do Complemento/imunologia , Feminino , Tempo de Lise do Coágulo de Fibrina , Lipoproteínas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Estrutura Secundária de Proteína , Trombina/metabolismo
13.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31548319

RESUMO

Antibodies are essential for immunity against Ehrlichia chaffeensis, and protective mechanisms involve blocking of ehrlichial attachment or complement and Fcγ-receptor-dependent destruction. In this study, we determined that major outer membrane protein 1 (OMP-19) hypervariable region 1 (HVR1)-specific human monoclonal antibodies (huMAbs) are protective through conventional extracellular neutralization and, more significantly, through a novel intracellular TRIM21-mediated mechanism. Addition of OMP-1-specific huMAb EHRL-15 (IgG1) prevented infection by blocking attachment/entry, a mechanism previously reported; conversely, OMP-1-specific huMAb EHRL-4 (IgG3) engaged intracellular TRIM21 and initiated an immediate innate immune response and rapid intracellular degradation of ehrlichiae. EHRL-4-TRIM21-mediated inhibition was significantly impaired in TRIM21 knockout THP-1 cells. EHRL-4 interacted with cytosolic Fc receptor TRIM21, observed by confocal microscopy and confirmed by co-immunoprecipitation. E. chaffeensis-EHRL-4-TRIM21 complexes caused significant upregulation of proinflammatory cytokine/chemokine transcripts and resulted in rapid (<30 min) nuclear accumulation of NF-κB and TRIM21 and ehrlichial destruction. We investigated the role of TRIM21 in the autophagic clearance of ehrlichiae in the presence of EHRL-4. Colocalization between EHRL-4-opsonized ehrlichiae, polyubiquitinated TRIM21, autophagy regulators (ULK1 and beclin 1) and effectors (LC3 and p62), and lysosome-associated membrane protein 2 (LAMP2) was observed. Moreover, autophagic flux defined by conversion of LC3I to LC3II and accumulation and degradation of p62 was detected, and EHRL-4-mediated degradation of E. chaffeensis was abrogated by the autophagy inhibitor 3-methyladenine. Our results demonstrate that huMAbs are capable of inhibiting E. chaffeensis infection by distinct effector mechanisms: extracellularly by neutralization and intracellularly by engaging TRIM21, which mediates a rapid innate immune response that mobilizes the core autophagy components, triggering localized selective autophagic degradation of ehrlichiae.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Ehrlichia chaffeensis/imunologia , Ribonucleoproteínas/genética , Adenina/análogos & derivados , Adenina/farmacologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Bactérias/genética , Autofagia/imunologia , Aderência Bacteriana/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Ehrlichia chaffeensis/genética , Técnicas de Inativação de Genes , Humanos , Imunidade Humoral/imunologia , NF-kappa B/genética , Células THP-1
14.
APMIS ; 127(12): 797-804, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31514254

RESUMO

Brucellosis is a worldwide bacterial zoonosis caused by Brucella spp. No approved vaccine is available for human use against the disease. In this study, outer membrane vesicles (OMVs) from a Brucella melitensis biovar 1 human isolate obtained in Iran were used to immunize BALB/c mice (n = 12) by 2 intramuscular injections with a 2-week interval. Another group of 12 mice was used as non-vaccinated controls. Two weeks after the last vaccination, six mice of each group were sacrificed, and proliferation and interferon gamma (IFNγ) production responses of their splenocytes were evaluated following in vitro stimulation with killed Brucella cells. The other mice were challenged with the virulent B. melitensis isolate. Two weeks later, mice were killed and spleens were cultured to determine the number of the challenge strain. The results showed proliferative response and IFNγ production of splenocytes from vaccinated mice (stimulation index: 2.18 ± 0.57, and 1519.35 ± 10.70 pg/mL, respectively) were significantly higher than those of control mice (stimulation index: 1.02 ± 0.02, and 210.01 ± 17.58 pg/mL, respectively). Numbers of the challenge strain in spleens of vaccinated mice were also significantly less than those in the controls with 1.6 units of protection. Our study revealed vaccination with OMVs of the B. melitensis isolate could induce specific immune responses and protection against infection in the mouse model suggesting their potential application for active immunization against brucellosis.


Assuntos
Vacina contra Brucelose/imunologia , Brucella melitensis/imunologia , Brucelose/imunologia , Brucelose/prevenção & controle , Vesículas Extracelulares/imunologia , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Brucella melitensis/citologia , Brucelose/microbiologia , Modelos Animais de Doenças , Feminino , Humanos , Imunidade Celular , Interferon gama/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Baço/metabolismo , Baço/microbiologia , Vacinação
15.
Int J Nanomedicine ; 14: 6601-6613, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31496701

RESUMO

Purpose: The primary goal of the present study was to explore and evaluate the highly conserved Neisserial surface protein A (NspA) molecule, fused with truncated HBV virus-like particles (VLPs), as a candidate vaccine against the virulent Neisseria meningitidis serogroup B (NMB). Methods: NspA was inserted into the major immunodominant region of the truncated hepatitis B virus core protein (HBc; amino acids 1-144). The chimeric protein, HBc-N144-NspA, was expressed from a prokaryotic vector and generated HBc-like particles, as determined by transmission electron microscopy. Further, the chimeric protein and control proteins were used to immunize mice and the resulting immune responses evaluated by flow cytometry, enzyme-linked immunosorbent assay, and analysis of serum bactericidal activity (SBA) titer. Results: Evaluation of the immunogenicity of the recombinant HBc-N144-NspA protein showed that it elicited the production of high levels of NspA-specific total IgG. The SBA titer of HBc-N144-NspA/F reached 1:16 2 weeks after the last immunization in BALB/c mice, when human serum complement was included in the vaccine. Immunization of HBc-N144-NspA, even without adjuvant, induced high levels of IL-4 and a high IgG1 to IgG2a ratio, confirming induction of an intense Th2 immune response. Levels of IL-17A increased rapidly in mice after the first immunization with HBc-N144-NspA, indicating the potential for this vaccine to induce a mucosal immune response. Meanwhile, the immunization of HBc-N144-NspA without adjuvant induced only mild inflammatory infiltration into the mouse muscle tissue. Conclusion: This study demonstrates that modification using HBc renders NspA a candidate vaccine, which can trigger protective immunity against NMB.


Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Vírus da Hepatite B/metabolismo , Infecções Meningocócicas/imunologia , Infecções Meningocócicas/prevenção & controle , Neisseria meningitidis/patogenicidade , Sorogrupo , Vírion/metabolismo , Adjuvantes Imunológicos/farmacologia , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Citocinas/metabolismo , Escherichia coli/metabolismo , Feminino , Imunidade , Imunização , Inflamação/patologia , Ativação Linfocitária/imunologia , Infecções Meningocócicas/patologia , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/imunologia , Teste Bactericida do Soro , Baço/microbiologia , Linfócitos T/imunologia , Vacinação , Virulência
16.
Vet Microbiol ; 236: 108367, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31500727

RESUMO

Avian pathogenic Escherichia coli (APEC) typically causes colibacillosis and is a major concern for the poultry industry and public health. As a vaccine platform, the outer membrane vesicles (OMVs) derived from various gram-negative bacteria and even some gram-positive bacteria have been reported to be immunogenic in laboratories or upon commercial usage worldwide. Here, we purified OMVs from APEC serotype O78 strain by ultracentrifugation and gradient isolation. By SDS-PAGE and LC-MS/MS analysis, the 20 most abundant proteins located on OMVs were identified and analyzed; the lipopolysaccharide (LPS) profiles of OMVs were not different from those of the bacteria. Moreover, three groups of chickens were immunized with OMV-, outer membrane protein (OMP)- and PBS, with the latter two serving as positive and negative controls, respectively. By analyzing the anti-OMP and anti-LPS IgG titers stimulated by the tested vaccine candidates, the macrophage opsonophagocytic activity and the bactericidal activity mediated by serum antibodies in vaccinated chickens, we found that the OMV-vaccinated chicken group was superior to the two other groups. These findings were confirmed by additional chicken challenge tests, in which all OMV-vaccinated group chickens obtained complete protection but those of the other two groups were barely protected. Our data demonstrate that native APEC O78 OMVs can induce protective immunity in chickens and therefore be used as a candidate vaccine for APEC serotype O78 strain infections.


Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Galinhas , Infecções por Escherichia coli/veterinária , Vacinas contra Escherichia coli/imunologia , Escherichia coli/imunologia , Doenças das Aves Domésticas/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Infecções por Escherichia coli/prevenção & controle , Doenças das Aves Domésticas/microbiologia
17.
J Med Microbiol ; 68(10): 1540-1543, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31483245

RESUMO

Four group A streptococcus (GAS) bacteraemia occurred in a small burn unit within 2 weeks. The GAS patient isolates, characterized as emm89, shared the same PFGE pulsotype with two other strains isolated 2 months later. The outbreak investigation revealed that a nurse was the most likely source of GAS transmission, as she was confirmed to carry the same outbreak strain in her throat and had direct and regular contact with the six outbreak patients in the unit. The outbreak was controlled after the nurse had undergone eradication treatment. This report highlights the emergence of the emm89 clone and its capacity to elicit invasive GAS outbreaks.


Assuntos
Unidades de Queimados/estatística & dados numéricos , Infecção Hospitalar/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/isolamento & purificação , Adulto , Bacteriemia/epidemiologia , Bacteriemia/microbiologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Infecção Hospitalar/epidemiologia , Surtos de Doenças , Feminino , Humanos , Pessoa de Meia-Idade , Epidemiologia Molecular , Infecções Estreptocócicas/epidemiologia , Streptococcus pyogenes/classificação , Streptococcus pyogenes/genética , Tunísia , Adulto Jovem
18.
Top Companion Anim Med ; 36: 12-15, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31472723

RESUMO

Vector-borne rickettsioses represent emerging threats to public health worldwide. The aim of this work was the screening for the presence of Rickettsia spp. in the blood of dogs and cats from southern Portugal. A PCR product of the expected size was amplified from DNA extracts obtained from blood samples of 29 out of 225 (12.9%) cats and in 2 out of 375 (.5%) dogs using genus-specific primers targeting Rickettsia gltA. Rickettsia conorii israelensis was identified by phylogenetical analysis of partial ompB sequences, amplified from blood samples taken from both a cat and a dog. The obtained results reinforce the idea that domestic animals may act as sentinels for the presence of vector-borne Rickettsia spp. in a given geographical area. In addition, rickettsioses should be included in the differential diagnosis of canine and feline vector-borne diseases.


Assuntos
Doenças do Gato/microbiologia , Doenças do Cão/microbiologia , Infecções por Rickettsia/veterinária , Rickettsia conorii/isolamento & purificação , Animais , Proteínas da Membrana Bacteriana Externa/genética , Gatos , Citrato (si)-Sintase/genética , DNA Bacteriano/sangue , Cães , Reação em Cadeia da Polimerase/veterinária , Portugal/epidemiologia , Infecções por Rickettsia/epidemiologia , Rickettsia conorii/genética , Análise de Sequência de DNA
19.
Parasitol Res ; 118(11): 3185-3189, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31473856

RESUMO

A total of 482 bats representing 32 species and two families were captured in the Amazon forests of the Amapá state in northern Brazil. Nineteen Artibeus planirostris bats (3.9 %) were infested with 160 ticks, all identified as Ornithodoros hasei. Three pools of larvae were screened for rickettsial DNA via polymerase chain reaction (PCR) targeting three rickettsial genes: gltA, ompA and htrA. Only one of them yielded an amplicons of the expected size for all three molecular assays. Comparisons of the obtained sequences including a phylogenetic analysis confirmed the occurrence of "Candidatus Rickettsia wissemanii" in Brazil.


Assuntos
Quirópteros/microbiologia , Quirópteros/parasitologia , Ornithodoros/microbiologia , Infecções por Rickettsia/epidemiologia , Rickettsia/isolamento & purificação , Animais , Proteínas da Membrana Bacteriana Externa/genética , Brasil/epidemiologia , DNA Bacteriano/genética , Proteínas de Choque Térmico/genética , Ixodidae/microbiologia , Larva/microbiologia , Proteínas Periplásmicas/genética , Filogenia , Reação em Cadeia da Polimerase , Rickettsia/genética , Serina Endopeptidases/genética
20.
Nat Commun ; 10(1): 3673, 2019 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-31413254

RESUMO

Bacteria use small molecules called siderophores to scavenge iron. Siderophore-Fe3+ complexes are recognised by outer-membrane transporters and imported into the periplasm in a process dependent on the inner-membrane protein TonB. The siderophore enterobactin is secreted by members of the family Enterobacteriaceae, but many other bacteria including Pseudomonas species can use it. Here, we show that the Pseudomonas transporter PfeA recognises enterobactin using extracellular loops distant from the pore. The relevance of this site is supported by in vivo and in vitro analyses. We suggest there is a second binding site deeper inside the structure and propose that correlated changes in hydrogen bonds link binding-induced structural re-arrangements to the structural adjustment of the periplasmic TonB-binding motif.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/metabolismo , Enterobactina/metabolismo , Ferro/metabolismo , Pseudomonas aeruginosa/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Bactérias , Sítios de Ligação , Proteínas de Transporte/química , Cristalização , Cristalografia por Raios X , Enterobactina/química , Escherichia coli , Técnicas In Vitro , Radioisótopos de Ferro , Proteínas de Membrana , Receptores de Superfície Celular/química , Sideróforos/química , Sideróforos/metabolismo
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