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1.
Carbohydr Polym ; 232: 115813, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31952611

RESUMO

Shigella flexneri is a gram-negative pathogen that causes shigellosis in humans and primates. MxiH antigen is known as one of the invasive factors in most Gram-negative bacteria consisting of a needle-like structure in the main backbone of the type 3 secretory system. Recombinant MxiH antigen was produced by E. coli BL21 and purified antigen was loaded into chitosan nanoparticles (CS-MxiH). After 20thand 55th of intranasal vaccinations, the titers of IgG, IgA, IL-4, and IFN-γ were evaluated. The results indicated the successful synthesis of CS nanoparticles followed by the effective loading of MxiH antigen. The results of animal experiments showed that the intranasal administration of CS-MxiH increased IgG and IgA compared to control groups. Increased levels of IL-4 and IFN-γ in groups immunized with CS-MxiH are probably due to the activation of plasmacytoid and myeloid cells presenting antigen in nasal epithelial mucosa and stimulating B cells.


Assuntos
Antígenos de Bactérias/química , Quitosana/química , Nanopartículas/química , Shigella flexneri/química , Vacinas/química , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Vacinas/imunologia
2.
Mol Biotechnol ; 62(3): 177-184, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31894514

RESUMO

The purpose of this study was to develop an efficient and inexpensive method for the useful production of recombinant protein V antigen, an important virulence factor for Yersinia pestis. To this end, the synthetic gene encoding the V antigen was subcloned into the downstream of the intein (INT) and chitin-binding domain (CBD) from the pTXB1 vector using specific primers. In the following, the produced new plasmid, pTX-V, was transformed into E. coli ER2566 strain, and the expression accuracy was confirmed using electrophoresis and Western blotting. In addition, the effects of medium, inducer, and temperature on the enhancement of protein production were studied using the Taguchi method. Finally, the V antigen was purified by a chitin affinity column using INT and CBD tag. The expression was induced by 0.05 mM IPTG at 25 °C under optimal conditions including TB medium. It was observed that the expression of the V-INT-CBD fusion protein was successfully increased to more than 40% of the total protein. The purity of V antigen was as high as 90%. This result indicates that V antigen can be produced at low cost and subjected to one-step purification using a self-cleaving INT tag.


Assuntos
Antígenos de Bactérias/isolamento & purificação , Cromatografia de Afinidade , Proteínas Citotóxicas Formadoras de Poros/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Yersinia pestis , Antígenos de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Recombinantes de Fusão/genética
3.
BMC Vet Res ; 15(1): 388, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676013

RESUMO

BACKGROUND: Clinical cases of Erysipelothrix rhusiopathiae, a zoonotic gram-positive bacterium, have been reported in many ruminant species, including in cattle, deer, moose and muskoxen. Fatal cases have been repeatedly reported in cattle over the years but to date there is only one Japanese study investigating the seroprevalence of this bacterium in cattle using the growth agglutination test (GAT). This technique is subjective, time-consuming, expensive and hazardous compared to modern serological tests such as enzyme-linked immunosorbent assays (ELISA) or the newly developed fluorescent microbead-based immunoassays (FMIA). RESULTS: The FMIA based on the surface protein SpaA (rSpaA415) antigen of E. rhusiopathiae developed in this study had an almost perfect agreement with the GAT (k = 0.83) and showed a sensitivity of 89.7% and a specificity of 92.9% when compared to the GAT. Overall, detection rates of E. rhusiopathiae antibody positive samples were 13.8% (51/370) in British herds and 6% (12/200) in US herds. Positive cattle were present in 34.3% (24/70) of the investigated British farms and in 34.7% (8/23) of the US farms with an on-farm prevalence of 7.1 to 100% for the British farms and 8.3-30% for the US farms. CONCLUSIONS: FMIA is a fast, safe and economic alternative to the GAT for the diagnosis of E. rhusiopathiae in cattle. This work is the first seroprevalence study of E. rhusiopathiae in healthy farmed cattle in Great Britain and the US and revealed that infection occurs at a low level. Further investigations to evaluate risks of zoonotic transmission when handling cattle are needed.


Assuntos
Antígenos de Bactérias/isolamento & purificação , Doenças dos Bovinos/microbiologia , Infecções por Erysipelothrix/microbiologia , Erysipelothrix , Animais , Anticorpos Antibacterianos , Bovinos , Doenças dos Bovinos/epidemiologia , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Infecções por Erysipelothrix/epidemiologia , Fluorescência , Imunoensaio/métodos , Imunoensaio/veterinária , Microesferas , Reino Unido/epidemiologia , Estados Unidos/epidemiologia
4.
Microb Pathog ; 137: 103705, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31487535

RESUMO

Nocardia farcinica is the etiological agent of nocardiosis, leading to serious pulmonary or systemic infections. To uncover virulence factors and early diagnostic markers, secreted proteins of N. farcinica IFM 10152 were analyzed using an immunoproteome-based approach. A total of 5 proteins were identified by matrix-assisted laser desorption (MALDI-TOF-MS). Bioinformatic analyses showed that the identified proteins were involved in defense against the host innate immune system and required for pathogenesis. All proteins were expressed in E. coli and antigenicity was analyzed with Western blot. To our knowledge, these proteins with antigenicity were identified for the first time in N. farcinica and they may help elucidate the pathogenesis underlying Nocardia and provide potential future diagnostic markers.


Assuntos
Antígenos de Bactérias/imunologia , Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/isolamento & purificação , Nocardia/imunologia , Proteômica , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Escherichia coli/genética , Feminino , Regulação Bacteriana da Expressão Gênica , Imunidade Inata , Camundongos , Camundongos Endogâmicos BALB C , Nocardia/genética , Nocardiose/diagnóstico , Nocardiose/imunologia , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Fatores de Virulência/genética , Fatores de Virulência/imunologia
5.
Biosens Bioelectron ; 143: 111625, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31476595

RESUMO

The first serum diagnosis of leprosy based on the detection of antibodies of patients using a recombinant mimetic peptide (PGL1M3R) as recognition element and exploiting a photoelectrochemical sensor is presented in this work. The photoeletrochemical platform consists of cadmium sulphide and nickel hydroxide electrodeposited on fluorine-doped tin oxide coated glass slide (CdS/Ni(OH)2/FTO). The optical band gap and flat band potential of the photoelectroactive materials were evaluated by UV-Vis spectroscopy and electrochemical impedance spectroscopy. The spatial photoelectrochemical response of the platform was evaluated by Scanning Electrochemical Microscopy and the morphology of the films was investigated by Scanning Electron Microscopy (SEM). The photoelectrochemical response of the CdS/Ni(OH)2/FTO platform was optimized by evaluating the effects of the kind, concentration, and pH of the buffer. Furthermore, the applied potential to the CdS/Ni(OH)2/FTO platform was also investigated. The CdS/Ni(OH)2/FTO photoelectrochemical platform was modified with a synthetic peptide by using glutaraldehyde as cross-linking reagent and chitosan (CS) for the covalent coupling of the peptide to the photoelectrochemical platform (PGL1M3R/CdS/Ni(OH)2/FTO). The photoelectrochemical immunosensor is able to distinguishing between positive and negative leprosy human sera samples diluted from 1:640 up to 1:10240. Furthermore, to test the specificity of the sensor, samples from tuberculosis and leishmaniasis patients were analyzed using the proposed photoelectrochemical immunosensor.


Assuntos
Antígenos de Bactérias/isolamento & purificação , Técnicas Biossensoriais , Hanseníase/diagnóstico , Mycobacterium leprae/isolamento & purificação , Biomimética , Humanos , Hanseníase/microbiologia , Mycobacterium leprae/patogenicidade , Proteínas Recombinantes/química
6.
Ann Agric Environ Med ; 26(3): 392-395, 2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31559791

RESUMO

Existing research for using the protective antigen (PA) of Bacillus anthracis as a vaccine component shows that protection against anthrax may be obtained using fragments of this protein. The aim of the research is to check whether the selected protein fragment of the protective antigen (domain 4) encoded by an appropriate nucleotide sequence of gene pag of B. anthracis, was expressed in the bacterial system of E. coli. In order to examine the selected sequence of the pag gene, a PCR reaction and a highly effective TOPO cloning strategy were used, followed by purification of the recombinant proteins and their detection by a western-blot method. In the planning of the PA4 antigen expression a higher level of effectiveness in production of small protein - domain 4 - was anticipated. As a result, the 139 amino acids protein fragment of B. anthracis PA (domain 4) was isolated. The research may have found the basis for in vivo research aimed at finding potential anthrax vaccine components.


Assuntos
Vacinas contra Antraz/imunologia , Antraz/microbiologia , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Toxinas Bacterianas/imunologia , Animais , Antraz/imunologia , Antraz/prevenção & controle , Vacinas contra Antraz/administração & dosagem , Vacinas contra Antraz/genética , Vacinas contra Antraz/isolamento & purificação , Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Bacillus anthracis/química , Bacillus anthracis/genética , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Western Blotting , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Domínios Proteicos
7.
Mikrobiyol Bul ; 53(3): 274-284, 2019 Jul.
Artigo em Turco | MEDLINE | ID: mdl-31414629

RESUMO

Coxiella burnetii is the causative agent of Q fever, a zoonotic infection. The bacteria is a gram-negative, pleomorphic, coccobacilli and capable to survive and proliferate within the host cell's phagolysosome. There are two morphological cell types of C.burnetii including small and large cell variants. C.burnetii is divided into phase I and phase II serologically variants according to LPS structure in the cell wall. Phase I is the natural phase found in infected animals or humans and is highly infectious. Phase II is not very infectious and could be obtained only in laboratories after serial passages in cell cultures or embryonated egg cultures. Q fever can be asymptomatic (in 50% of the cases), acute or chronic. Major presentations of acute Q fever are flu-like illness, pneumonia, and hepatitis, whereas the chronic form presents mainly as infective endocarditis. The aim of this study was to obtain C.burnetii phase II variant from C.burnetii phase I variant by a phase change study. In this study, C.burnetii was isolated by cell culture method from the heart valve tissue of a Q fever endocarditis case. C.burnetii phase I antigen for the indirect fluorescent antibody test (IFAT) was prepared from the isolated strain. For the isolation and identification of C.burnetii, heart valve tissue of the patient was homogenized and DNA was extracted by tissue extraction kit. C.burnetii DNA in the valve tissue was determined by real-time PCR (Rt-PCR). This C.burnetii DNA positive specimen was inoculated into Vero cells by shell vial centrifugation method. The scraped Vero cells were fixed on the slides after one week of incubation and IFAT was performed using C.burnetii phase I IgG positive sera, bacteria that were grown in and surrounding the Vero cells stained apple green were determined microscopically. Infected cells were disrupted by freeze and thaw method to obtain bacterial suspension. The DNA obtained from the bacterial suspension was again found to be positive for C.burnetii by Rt-PCR. Isolation sample was found to be positive in PCR at an earlier cycle compared to heart tissue sample, thus the bacterial growth was also confirmed with PCR. 16S ribosomal RNA gene of our isolate was amplified by PCR using 27F and 1492 primers and then sequenced. The DNA sequences were compared with reference DNA sequences of GeneBank; and the nucleotide sequence of the 16S ribosomal RNA gene of our isolate was found to be 99% similar to C.burnetii strain ATCC VR-615 an accession number NR104916. Serial cell culture passages of the isolated strain were performed to obtain C.burnetii phase II variant from C.burnetii phase I variant. After each passage, presence of phase change was investigated by IFAT using C.burnetii phase I and phase II IgG positive sera. At the end of 17 cell culture passages, phase change could not be observed. C.burnetii phase I IFAT antigen was prepared from the obtained bacterial suspension. In this study, we presented the isolation and identification of C.burnetii by cell culture, molecular and serological methods from the heart valve of a patient with endocarditis for the first time in our country.


Assuntos
Coxiella burnetii , Endocardite , Valvas Cardíacas , Febre Q , Animais , Antígenos de Bactérias/isolamento & purificação , Antígenos de Bactérias/metabolismo , Chlorocebus aethiops , Coxiella burnetii/genética , Coxiella burnetii/isolamento & purificação , Endocardite/microbiologia , Valvas Cardíacas/microbiologia , Humanos , Febre Q/microbiologia , RNA Ribossômico 16S/genética , Turquia , Células Vero
8.
World J Microbiol Biotechnol ; 35(8): 120, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332578

RESUMO

Mycobacterium avium subsp. paratuberculosis (MAP) is responsible for Johne's disease (JD) or paratuberculosis. Diagnosis of MAP infection by measuring host cell-mediated and humoral immune responses has been a major focus in MAP research. For this purpose, several MAP antigens such as secreted protein, cell envelope protein, cell-mediated immune and lipoprotein antigens have been identified and tested to measure their diagnostic utility with varying degree of success. Identifying the optimal antigen or antigen combinations for diagnosis of infected animals is hindered by the complex nature of the disease, prolonged subclinical infection, the differential expression of antigens and scarcity of well characterized MAP-specific epitopes making selection of a single MAP antigen very difficult. Thus, multiplexing of antigens with larger scale and longitudinal studies may lead to development of cost-effective next generation serodiagnostics. This mini review focuses on the role of different MAP antigens in the diagnosis of JD.


Assuntos
Doenças dos Bovinos/diagnóstico , Mycobacterium avium subsp. paratuberculosis/imunologia , Paratuberculose/diagnóstico , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/isolamento & purificação , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/microbiologia , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/imunologia
9.
Glycoconj J ; 36(5): 429-438, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31230165

RESUMO

Enterococcus faecium (E. faecium) has emerged as one of today's leading causes of health care-associated infections that is difficult to treat with the available antibiotics. These pathogens produce capsular polysaccharides on the cell surface which play a significant role in adhesion, virulence and evasion. Therefore, we aimed at the identification and characterization of bacterial polysaccharide antigens which are central for the development of vaccine-based prophylactic approaches. The crude cell wall-associated polysaccharides from E. faecium, its mutant and complemented strains were purified and analyzed by a primary antibody raised against lipoteichoic acid (LTA) and diheteroglycan (DHG). The resistant E. faecium strains presumably possess novel capsular polysaccharides that allow them to avoid the evasion from opsonic killing. The E. faecium U0317 strain was very well opsonized by anti-U0317 (~95%), an antibody against the whole bacterial cell. The deletion mutant showed a significantly increased susceptibility to opsonophagocytic killing (90-95%) against the penicillin binding protein (anti-PBP-5). By comparison, in a mouse urinary tract and rat endocarditis infection model, respectively, there were no significant differences in virulence. In this study we explored the biological role of the capsule of E. faecium. Our findings showed that the U0317 strain is not only sensitive to anti-LTA but also to antibodies against other enterococcal surface proteins. Our findings demonstrate that polysaccharides capsule mediated-resistance to opsonophagocytosis. We also found that the capsular polysaccharides do not play an important role in bacterial virulence in urinary tract and infective endocarditis in vivo models.


Assuntos
Anticorpos Antibacterianos/farmacologia , Antígenos de Bactérias/isolamento & purificação , Parede Celular/química , Enterococcus faecium/química , Lipopolissacarídeos/isolamento & purificação , Polissacarídeos Bacterianos/isolamento & purificação , Ácidos Teicoicos/isolamento & purificação , Animais , Antibacterianos/farmacologia , Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Cápsulas Bacterianas/química , Cápsulas Bacterianas/imunologia , Parede Celular/imunologia , Modelos Animais de Doenças , Farmacorresistência Bacteriana , Endocardite Bacteriana/tratamento farmacológico , Endocardite Bacteriana/microbiologia , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/imunologia , Enterococcus faecium/patogenicidade , Feminino , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Opsonizantes/farmacologia , Proteínas de Ligação às Penicilinas/química , Proteínas de Ligação às Penicilinas/imunologia , Proteínas de Ligação às Penicilinas/isolamento & purificação , Proteínas de Ligação às Penicilinas/farmacologia , Fagocitose/efeitos dos fármacos , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/imunologia , Cultura Primária de Células , Ratos , Ratos Wistar , Ácidos Teicoicos/química , Ácidos Teicoicos/imunologia , Ácidos Teicoicos/farmacologia , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologia
10.
mSphere ; 4(3)2019 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-31167949

RESUMO

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the most successful human pathogens. One reason for its success is that Mtb can reside within host macrophages, a cell type that normally functions to phagocytose and destroy infectious bacteria. However, Mtb is able to evade macrophage defenses in order to survive for prolonged periods of time. Many intracellular pathogens secrete virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. We hypothesized that Mtb secreted proteins that target host membranes are vital for Mtb to adapt to and manipulate the host environment for survival. Thus, we characterized 200 secreted proteins from Mtb for their ability to associate with eukaryotic membranes using a unique temperature-sensitive yeast screen and to manipulate host trafficking pathways using a modified inducible secretion screen. We identified five Mtb secreted proteins that both associated with eukaryotic membranes and altered the host secretory pathway. One of these secreted proteins, Mpt64, localized to the endoplasmic reticulum during Mtb infection of murine and human macrophages and impaired the unfolded protein response in macrophages. These data highlight the importance of secreted proteins in Mtb pathogenesis and provide a basis for further investigation into their molecular mechanisms.IMPORTANCE Advances have been made to identify secreted proteins of Mycobacterium tuberculosis during animal infections. These data, combined with transposon screens identifying genes important for M. tuberculosis virulence, have generated a vast resource of potential M. tuberculosis virulence proteins. However, the function of many of these proteins in M. tuberculosis pathogenesis remains elusive. We have integrated three cell biological screens to characterize nearly 200 M. tuberculosis secreted proteins for eukaryotic membrane binding, host subcellular localization, and interactions with host vesicular trafficking. In addition, we observed the localization of one secreted protein, Mpt64, to the endoplasmic reticulum (ER) during M. tuberculosis infection of macrophages. Interestingly, although Mpt64 is exported by the Sec pathway, its delivery into host cells was dependent upon the action of the type VII secretion system. Finally, we observed that Mpt64 impairs the ER-mediated unfolded protein response in macrophages.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Mycobacterium tuberculosis/metabolismo , Fatores de Virulência/metabolismo , Animais , Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Membrana Celular/metabolismo , Células Cultivadas , Retículo Endoplasmático/metabolismo , Feminino , Células HeLa , Humanos , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Células RAW 264.7 , Tuberculose/microbiologia
11.
Artigo em Inglês | MEDLINE | ID: mdl-31069177

RESUMO

Flagella are expressed on the surface of a wide range of bacteria, conferring motility and contributing to virulence and innate immune stimulation. Host-pathogen interaction studies of the roles of flagella in infection, including due to uropathogenic Escherichia coli (UPEC), have used various methods to purify and examine the biology of the major flagella subunit protein, FliC. These studies have offered insight into the ways in which flagella proteins interact with host cells. However, previous methods used to extract and purify FliC, such as mechanical shearing, ultracentrifugation, heterologous expression in laboratory E. coli strains, and precipitation-inducing chemical treatments have various limitations; as a result, there are few observations based on highly purified, non-denatured FliC in the literature. This is especially relevant to host-pathogen interaction studies such as immune assays that are designed to parallel, as closely as possible, naturally-occurring interactions between host cells and flagella. In this study, we sought to establish a new, carefully optimized method to extract and purify non-denatured, native FliC from the reference UPEC strain CFT073 to be suitable for immune assays. To achieve purification of FliC to homogeneity, we used a mutant CFT073 strain containing deletions in four major chaperone-usher fimbriae operons (type 1, F1C and two P fimbrial gene clusters; CFT073Δ4). A sequential flagella extraction method based on mechanical shearing, ultracentrifugation, size exclusion chromatography, protein concentration and endotoxin removal was applied to CFT073Δ4. Protein purity and integrity was assessed using SDS-PAGE, Western blots with anti-flagellin antisera, and native-PAGE. We also generated a fliC-deficient strain, CFT073Δ4ΔfliC, to enable the concurrent preparation of a suitable carrier control to be applied in downstream assays. Innate immune stimulation was examined by exposing J774A.1 macrophages to 0.05-1 µg of purified FliC for 5 h; the supernatants were analyzed for cytokines known to be induced by flagella, including TNF-α, IL-6, and IL-12; the results were assessed in the context of prior literature. Macrophage responses to purified FliC encompassed significant levels of several cytokines consistent with prior literature reports. The purification method described here establishes a new approach to examine highly purified FliC in the context of host-pathogen interaction model systems.


Assuntos
Antígenos de Bactérias/isolamento & purificação , Cromatografia Líquida/métodos , Proteínas de Escherichia coli/isolamento & purificação , Flagelos/química , Flagelina/isolamento & purificação , Escherichia coli Uropatogênica/química , Animais , Linhagem Celular , Citocinas/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Ultracentrifugação/métodos
12.
Methods Mol Biol ; 1997: 77-85, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31119618

RESUMO

Gonococcal colony typing is part a science and part an art that has been central to studies which have identified crucial virulence antigens and also demonstrated the ability of the bacteria to undergo rapid phase and antigenic variation. Without this fundamental work, modern molecular biological studies of gonococcal pathogenesis would not have been possible. Indeed colony typing is still essential when performing biological experiments with clinical and laboratory isolates and for monitoring their outcome. In this chapter, the methods for performing colony typing and techniques to optimize the process are described.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Neisseria gonorrhoeae/classificação , Variação Antigênica/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/isolamento & purificação , DNA Bacteriano/isolamento & purificação , Neisseria gonorrhoeae/patogenicidade , Fatores de Virulência/imunologia , Fatores de Virulência/isolamento & purificação
13.
Methods Mol Biol ; 1997: 87-96, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31119619

RESUMO

Neisseria gonorrhoeae is a gram-negative obligate human pathogen that contains lipooligosaccharide (LOS) as a major constituent within the outer membrane. LOS plays a major role in pathogenesis by inducing host inflammatory responses and also enabling evasion of host innate immunity through sialylation. Epitopes within LOS are also potential vaccine candidates. In this chapter, we describe a general method based on the Westphal hot phenol extraction process to purify whole LOS from N. gonorrhoeae for structural analyses and for use in in vivo and in vitro biological assays.


Assuntos
Antígenos de Bactérias/isolamento & purificação , Fracionamento Químico/métodos , Lipopolissacarídeos/isolamento & purificação , Neisseria gonorrhoeae/química , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Neisseria gonorrhoeae/imunologia , Neisseria gonorrhoeae/patogenicidade , Fenol/química
14.
Methods Mol Biol ; 1997: 121-141, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31119622

RESUMO

The emergence and spread of fully antimicrobial resistant Neisseria gonorrhoeae (GC) highlights a clear need for next-generation antigonococcal therapeutics. A broadly reactive anti-GC vaccine would best address this global public health threat. Polyantigenic outer membrane vesicles (OMVs) derived from GC can overcome the challenges posed by GC's high rate of phase and antigen variation. In fact, GC OMVs have already shown promise as a vaccine antigen; however, all previous studies have utilized vesicles contaminated by RMP, a bacterioprotective antigen known to entirely abrogate vaccine-induced bactericidal activity in vivo. Additionally, these studies primarily utilized vesicles isolated through techniques like membrane disruption with detergents, which are known to increase contamination of cytoplasmic components as compared to naturally released OMVs (nOMVs). This chapter describes the isolation and characterization of naturally released nOMVs through sequential size and weight restrictive filtration. nOMVs are characterized by morphology, proteomics, and bioactivity via various methods. Herein we also describe methods for further evaluation of the innate and induced immunogenicity of rmp-deficient GC nOMVs by cell stimulation and murine vaccination. Per these methods, nOMVs are found to be largely homogenous spherical structures approximately 70 nm in diameter containing a consistent subset of GC outer membrane proteins. The rmp-deficient vesicles demonstrate a morphology and, with the exception of RMP, antigenic profile consistent with that of nOMVs derived from wild time N. gonorrhoeae. Additionally, vesicles lacking RMP are able to engage and strongly activate a diverse array of pattern recognition receptors in vitro. These methods lay the groundwork for future experiments examining the in vivo protective efficacy of the anti-GC response induced by these nOMVs as well as studies examining the mechanism of vaccine induced female genital tract immunity.


Assuntos
Antígenos de Bactérias/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Vacinas Bacterianas/imunologia , Neisseria gonorrhoeae/imunologia , Vesículas Secretórias/imunologia , Animais , Antígenos de Bactérias/imunologia , Membrana Externa Bacteriana/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/isolamento & purificação , Vacinas Bacterianas/uso terapêutico , Feminino , Filtração/instrumentação , Filtração/métodos , Gonorreia/imunologia , Gonorreia/microbiologia , Gonorreia/terapia , Humanos , Imunogenicidade da Vacina , Camundongos , Modelos Animais , Neisseria gonorrhoeae/citologia , Proteômica , Vacinação , Vagina/microbiologia
15.
Bull Exp Biol Med ; 166(6): 759-765, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31028588

RESUMO

Campylobacter genus bacteria causing campylobacteriasis are difficult to culture. This fact necessitates creation of special approaches to studies of the behavior of these pathogens during the manufacture and storage of foodstuffs. The regularities of Campylobacter jejuni transition into an uncultivable state are studied under conditions simulating the process of immersion cooling of fresh poultry products. The proportion of viable colony-forming (CFU) cells to the total count of planktonic and uncultivable cells in the population was calculated by the level of genomic DNA in the samples evaluated by quantitative real-time PCR with intercalating dyes. PCR was carried out with primers detecting the cytolethal toxin subunit B gene cdtB and invasion gene ciaB in C. jejuni strains. The count of detected cells was 5-10-fold higher than the count of CFU; the cultural method failed to detect the agent in 40% analyzed samples of superficially infected products, while the level of uncultivable cells detected by PCR was significantly higher. The relationship between culturing conditions and formation of C. jejuni biofilms was studied. The most intensive formation of film exomatrix was observed under unfavorable conditions for this microorganism at 25oC. In microaerophilic gaseous medium, weak formation of films and intensive growth of C. jejuni populations were observed. Culturing at higher temperatures (37-42oC) was in fact inessential for the film formation process.


Assuntos
Antígenos de Bactérias/genética , Toxinas Bacterianas/genética , Biofilmes/crescimento & desenvolvimento , Campylobacter jejuni/crescimento & desenvolvimento , Produtos Avícolas/microbiologia , Animais , Antígenos de Bactérias/isolamento & purificação , Antígenos de Bactérias/metabolismo , Carga Bacteriana , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/metabolismo , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Galinhas , Contagem de Colônia Microbiana , Contaminação de Alimentos/análise , Expressão Gênica , Humanos , Reação em Cadeia da Polimerase , Temperatura
16.
Biotechniques ; 66(5): 240-242, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30955344

RESUMO

We propose a new detection method for a tuberculosis-specific protein, MPB64, obtained from active bacillus Calmette-Guérin (BCG) by heating. When BCG was included in solution at a concentration >2.75 × 104 CFU/ml, our method for collecting MPB64 through heating active BCG combined with an immunochromatographic assay detected active bacilli within 2.5 h. By contrast, a culture test, which is the gold standard for tuberculosis diagnosis, does not provide results for between 1 week and 2 months. The rapid tests based on PCR have some drawbacks, for example they detect DNA from both active and latent (or even dead) tubercle bacilli. Therefore, our method may pave the way toward detecting only active tubercle bacilli at a reasonable cost and providing same-day diagnosis.


Assuntos
Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Mycobacterium bovis/genética , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/diagnóstico , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Cromatografia de Afinidade , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Tuberculose/genética , Tuberculose/microbiologia
17.
Front Immunol ; 10: 113, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30837982

RESUMO

Reverse Vaccinology (RV) is a widely used approach to identify potential vaccine candidates (PVCs) by screening the proteome of a pathogen through computational analyses. Since its first application in Group B meningococcus (MenB) vaccine in early 1990's, several software programs have been developed implementing different flavors of the first RV protocol. However, there has been no comprehensive review to date on these different RV tools. We have compared six of these applications designed for bacterial vaccines (NERVE, Vaxign, VaxiJen, Jenner-predict, Bowman-Heinson, and VacSol) against a set of 11 pathogens for which a curated list of known bacterial protective antigens (BPAs) was available. We present results on: (1) the comparison of criteria and programs used for the selection of PVCs (2) computational runtime and (3) performances in terms of fraction of proteome identified as PVC, fraction and enrichment of BPA identified in the set of PVCs. This review demonstrates that none of the programs was able to recall 100% of the tested set of BPAs and that the output lists of proteins are in poor agreement suggesting in the process of prioritize vaccine candidates not to rely on a single RV tool response. Singularly the best balance in terms of fraction of a proteome predicted as good candidate and recall of BPAs has been observed by the machine-learning approach proposed by Bowman (1) and enhanced by Heinson (2). Even though more performing than the other approaches it shows the disadvantage of limited accessibility to non-experts users and strong dependence between results and a-priori training dataset composition. In conclusion we believe that to significantly enhance the performances of next RV methods further studies should focus on the enhancement of accuracy of the existing protein annotation tools and should leverage on the assets of machine-learning techniques applied to biological datasets expanded also through the incorporation and curation of bacterial proteins characterized by negative experimental results.


Assuntos
Antígenos de Bactérias/imunologia , Infecções Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Software , Vacinologia/tendências , Animais , Antígenos de Bactérias/isolamento & purificação , Biologia Computacional , Conjuntos de Dados como Assunto , Ensaios de Triagem em Larga Escala , Humanos , Aprendizado de Máquina , Proteômica
18.
J Infect Chemother ; 25(4): 267-272, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30642770

RESUMO

The application and clinical impact of rapid antigen detection test (RADT) in the treatment of acute pharyngitis is unknown in Japan. We aimed to examine the proportions of RADT usage to identify Group A ß-hemolytic Streptococcus (GAS) in outpatients with acute pharyngitis and evaluate the association between RADT and antibiotic treatment. We analyzed health insurance claims data from 2013 to 2015. Logistic regression models were used to analyze associated factors with RADT, overall antibiotic prescription, or penicillin use. We analyzed 1.27 million outpatient visits with acute pharyngitis, in which antibiotics were prescribed in 59.3% of visits. Of the total visits, 5.6% of patients received RADT, and 10.8% of the antibiotics were penicillin. Penicillin selection rates were higher in cases with RADT (25.4%) than those without RADT (9.7%). Compared to large-scale facilities, antibiotic prescription rates were higher in physicians' offices. For factor analysis, age (3-15 years), diagnosis code (streptococcal pharyngitis), size of the medical facility (large-scale hospitals), and physician's specialty (pediatrics) were associated with RADT use. Penicillin selection rate increased with RADT implementation (25.4% vs. 9.7%: adjusted odds ratio 1.55; 95% CI, 1.50-1.60). At 63% of the facilities, the RADT implementation rate was <5% of acute pharyngitis visits prescribed antibiotics. In conclusion, the proportion of RADT usage for outpatients with acute pharyngitis was low in Japan. With appropriate indication and evaluation, we expect that more utilization of RADT can help promote antimicrobial stewardship for outpatients with acute pharyngitis by prompting penicillin therapy. Further investigation with detailed clinical data are warranted.


Assuntos
Antibacterianos/uso terapêutico , Antígenos de Bactérias/isolamento & purificação , Testes Imunológicos/estatística & dados numéricos , Faringite/tratamento farmacológico , Infecções Estreptocócicas/tratamento farmacológico , Doença Aguda/terapia , Adolescente , Adulto , Idoso , Antibacterianos/farmacologia , Gestão de Antimicrobianos/normas , Gestão de Antimicrobianos/estatística & dados numéricos , Criança , Pré-Escolar , Humanos , Testes Imunológicos/instrumentação , Lactente , Recém-Nascido , Japão , Pessoa de Meia-Idade , Penicilinas/farmacologia , Penicilinas/uso terapêutico , Faringite/imunologia , Faringite/microbiologia , Guias de Prática Clínica como Assunto , Kit de Reagentes para Diagnóstico/estatística & dados numéricos , Estudos Retrospectivos , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/imunologia , Streptococcus pyogenes/isolamento & purificação , Adulto Jovem
19.
Artigo em Inglês | MEDLINE | ID: mdl-30648911

RESUMO

Helicobacter pylori causes one of the most common infections in human populations. The role of this bacterium in chronic gastritis, gastric ulcer, gastric cancer, as well as extra-digestive diseases such as ischemic heart disease and chronic obstructive pulmonary diseases, is well known. Prevention and control of these diseases can occur by early diagnosis and eradication of H. pylori infection. At present, different methods have been established to detect H. pylori infection. The biopsy-based tests, which are known as invasive methods, such as rapid urease test and histology, have the highest specificity among the others. Similarly, culture of biopsy samples is used for diagnosis of H. pylori infection. It has a high specificity value, and also allows us to perform antibiotic sensitivity testing. On the contrary, polymerase chain reaction and other molecular methods have good sensitivity and specificity, and can be used for detection of H. pylori infection, its virulence factors, and eradication success after treatment. While serological tests are more appropriate for epidemiological studies, their main weakness for clinical use is low specificity. Overall, specificity and sensitivity, cost, usefulness, and limitation of tests should be considered for selection of detection methods of H. pylori in each country.


Assuntos
Antígenos de Bactérias/isolamento & purificação , Testes Diagnósticos de Rotina/métodos , Infecções por Helicobacter/diagnóstico , Helicobacter pylori/isolamento & purificação , Antígenos de Bactérias/genética , Biópsia , Análise Custo-Benefício , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/química , Humanos , Sensibilidade e Especificidade , Urease/química , Urease/isolamento & purificação
20.
Future Microbiol ; 14: 223-233, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30663893

RESUMO

AIM: There is an urgent need to design a reliable diagnostic test for tuberculosis (TB). METHODS: Real-time immuno-PCR (RT-I-PCR) assay was devised for the quantitative detection of a cocktail of mycobacterial MPT64 (Rv1980c) and PstS1 (Rv0934) in TB patients. RESULTS: A broad dynamic range of 0.95 pg/ml-95 ng/ml of MPT64+PstS1 was detected in TB patients. In smear-positive (n = 59) and smear-negative (n = 42) pulmonary TB cases, sensitivities of 93.2 and 83.3% were observed, respectively with 92.8% specificity, whereas a sensitivity of 77.9% and a specificity of 91.3% were observed in extrapulmonary TB cases (n = 86). Furthermore, significantly reduced MPT64+PstS1 concentrations (p < 0.001) were noticed in patients on therapy by RT-I-PCR as compared with untreated patients. CONCLUSION: Our RT-I-PCR assay revealed high sensitivity especially for the rapid diagnosis of smear-negative pulmonary TB and paucibacillary extrapulmonary TB samples, which could also monitor the dynamics of disease in patients on therapy.


Assuntos
Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Tuberculose/diagnóstico , Transportadores de Cassetes de Ligação de ATP/genética , Adolescente , Adulto , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Sensibilidade e Especificidade , Tuberculose/microbiologia , Tuberculose Pulmonar/diagnóstico , Adulto Jovem
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