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1.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(7): 589-594, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31537242

RESUMO

Objective To construct and identify Bifidobacterium bifidum-vectored outer membrane protein F-I[rBb(pGEX-OprF-I)] vaccine of Pseudomonas aeruginosa and observe its protection against Pseudomonas aeruginosa infection in mice. Methods OprF and OprI genes were amplified by PCR, then the OprF-I fusion gene obtained by gene SOEing was digested and ligated into the vector pGEX-1λT to construct the recombinant plasmid pGEX-OprF-I. The plasmid was transformed into Bifidobacterium bifidum (Bb) by electroporation, and the rBb(pGEX-OprF-I) vaccine was constructed and identified by double enzyme digestion and PCR. Expression products of the vaccine induced by IPTG were analyzed and identified by SDS-PAGE and Western blot analysis. Twenty-one BALB/c mice were randomly divided into rBb(pGEX-OprF-I) vaccine group, Bb-pGEX-1λT empty vector group and Bb control group. The 5×108 CFUs vaccine was intragastrically administered for 3 consecutive days per week for 3 weeks. All mice were challenged intranasally with 5×107 CFUs of PA01 strain at the 4th week after the first immunization. At the 2nd week after the challenge, all mice were sacrificed to count the lung bacteria loads. IgG levels in sera from the mice before immunization, 4th week after the first immunization and 2nd week after the challenge were detected by routine ELISA. Results A total of 1289 bp OprF-I fusion gene was amplified by PCR. Double enzyme digestion and PCR identification confirmed that the gene was ligated into pGEX-1λT and transformed into Bb, and the rBb(pGEX-OprF-I) vaccine was successfully constructed. SDS-PAGE showed that the fusion protein with a relative molecular mass (Mr) of about 68 000 could be expressed by IPTG-induced vaccine. Western blot analysis indicated that the protein could be specifically recognized by the sera of Pseudomonas aeruginosa-infected mice. The number of bacteria colonies in the lung of the mice immunized with rBb(pGEX-OprF-I) vaccine was significantly lower than that of the control group. The IgG levels in the sera of the immunized mice increased successively at 4th week after the first immunization and 2nd week after the challenge, and higher than that in the other control groups at the same time point. Conclusion The rBb(pGEX-OprF-I) vaccine has been successfully constructed, and it may take a certain protective effect on the mice against Pseudomonas aeruginosa infection.


Assuntos
Proteínas de Bactérias/imunologia , Bifidobacterium bifidum , Lipoproteínas/imunologia , Infecções por Pseudomonas/prevenção & controle , Vacinas contra Pseudomonas/imunologia , Animais , Camundongos , Camundongos Endogâmicos BALB C , Pseudomonas aeruginosa , Distribuição Aleatória , Proteínas Recombinantes de Fusão/imunologia
2.
J Med Microbiol ; 68(11): 1629-1640, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31553301

RESUMO

Introduction. ML1899 is conserved in all mycobacterium sp. and is a middle member of mle-ML1898 operon involved in mycolic acid modification.Aim. In the present study attempts were made to characterize ML1899 in detail.Methodology. Bioinformatics tools were used for prediction of active-site residues, antigenic epitopes and a three-dimensional model of protein. The gene was cloned, expressed and purified as His-tagged protein in Escherichia coli for biophysical/biochemical characterization. Recombinant protein was used to treat THP-1 cells to study change in production of nitric oxide (NO), reactive oxygen species (ROS), cytokines and chemokines using flowcytometry/ELISA.Results. In silico analysis predicted ML1899 as a member of α/ß hydrolase family with GXSXG-motif and Ser126, His282, Asp254 as active-site residues that were confirmed by site-directed mutagensis. ML1899 exhibited esterase activity. It hydrolysed pNP-butyrate as optimum substrate at pH 8.0 and 50 °C with 5.56 µM-1 min-1 catalytic efficiency. The enzyme exhibited stability up to 60 °C temperature and between pH 6.0 to 9.0. K m, V max and specific activity of ML1899 were calculated to be 400 µM, 40 µmoles min-1 ml-1 and 27 U mg- 1, respectively. ML1899 also exhibited phospholipase activity. The protein affected the survival of macrophages when treated at higher concentration. ML1899 enhanced ROS/NO production and up-regulated pro-inflammatory cytokines and chemokine including TNF-α, IFN-γ, IL-6 and IL-8 in macrophages. ML1899 was also observed to elicit humoral response in 69 % of leprosy patients.Conclusion. These results suggested that ML1899, an esterase could up-regulate the immune responses in favour of macrophages at a low concentration but kills the THP-1 macrophages cells at a higher concentration.


Assuntos
Proteínas de Bactérias/imunologia , Esterases/imunologia , Hanseníase/microbiologia , Mycobacterium leprae/enzimologia , Sequência de Aminoácidos , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Citocinas/genética , Citocinas/imunologia , Estabilidade Enzimática , Esterases/química , Esterases/genética , Feminino , Humanos , Concentração de Íons de Hidrogênio , Cinética , Hanseníase/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Mycobacterium leprae/química , Mycobacterium leprae/genética , Mycobacterium leprae/imunologia , Óxido Nítrico/imunologia , Espécies Reativas de Oxigênio/imunologia , Alinhamento de Sequência
3.
Braz J Infect Dis ; 23(4): 246-253, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31421107

RESUMO

Accurate and rapid diagnostic tools are important aspects of managing tuberculosis (TB) cases appropriately. However, the sensitivity and specificity of diagnostic kits based on immune response such as the tuberculin skin test (TST) and interferon gamma release assay (IGRA) are still debated. Thus, the exploration and assessment of specific biomarker-targeted antibodies are needed for the development of an accurate and rapid diagnostic tool. The present study was conducted in patients with a respiratory problem suspected to be TB at Dr. Soetomo Hospital, Surabaya, Indonesia. Among 102 patients tested by GeneXpert and AFB, 59 serum samples were from cases retrospectively determined to have active TB. A total of 102 serum of healthy controls (HC) was also collected. The PPD antigen and the recombinant CFP-10 and ESAT-6 proteins were prepared. Antibody responses against these proteins were evaluated by ELISA. All samples were also screened for the possibility of Mycobacterium avium-intracellulare complex (MAC) infection using Capilla MaC kit. The results showed that TB patients had a significantly higher concentration of IgG antibody in response to PPD than the HC. In addition, the receiver operating characteristic (ROC) curve analysis showed that PPD was acceptable for diagnostic purposes with an AUC value of 0.835 (95% CI 0.770-0.900, p < 0.0001). However, ESAT-6 and CFP-10 had low AUCs, and 32 samples from both groups showed a low concentration of IgA antibody against all antigens. The MAC detection results also showed that the concentration of IgA in the HC group was the highest. The current results indicate that PPD is a better antigen for antibody-based detection of TB than ESAT-6 and CFP-10. Based on the MAC detection assay, 53 people in the HC group were probably infected with rapidly growing nontuberculous mycobacteria (NTM), although antibody response to PPD was low.


Assuntos
Formação de Anticorpos/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculina/imunologia , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/microbiologia , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Criança , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Indonésia , Masculino , Pessoa de Meia-Idade , Valores de Referência , Estudos Retrospectivos , Sensibilidade e Especificidade , Estatísticas não Paramétricas , Teste Tuberculínico , Tuberculose Pulmonar/sangue , Adulto Jovem
4.
Vet Microbiol ; 235: 53-62, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31282379

RESUMO

Nucleases are ubiquitously recognized as essential proteins in mycoplasmas because these organisms lack most capacities for de novo synthesis of nucleotides. Some of these proteins were proved to be important pathogenic factors during the infection of mycoplasms. In this study, the protein Mhp597 from M. hyopneumoniae was expressed and purified in Escherichia coli. Analysis of nuclease activity showed that recombinant Mhp597 (rMhp597) was a Ca2+ or Mg2+ dependent thermostable nuclease with very high activity and neutrophil extracellular traps (NETs) induced by M. hyopneumoniae were completely degraded by this nuclease. In addition, when PK15 cells were incubated with different concentrations of rMhp597, their viability was reduced and cell apoptosis was observed in a dose-dependent manner. To further investigate the host immune system response, we report that rMhp597 up-regulated the exression of inflammatory genes showing that TLR4/MyD88/NF-κB signal pathway was involved. On the other hand, rMhp597 down-regulated the expression of type I IFN (IFN-α/ß) and promoted the multiplication of porcine reproductive and respiratory syndrome virus (PRRSV). Recombinant rMhp597δ315-377 lacking C-terminal 63 amino acids exhibited all biological functions mentioned above except for nuclease activity. In summary, Mhp597 is a dynamic secreted nuclease involved in cytotoxicity, inflammation and immunosuppression.


Assuntos
Proteínas de Bactérias/imunologia , Inflamação/genética , Nuclease do Micrococo/imunologia , Mycoplasma hyopneumoniae/enzimologia , Mycoplasma hyopneumoniae/imunologia , Animais , Apoptose , Proteínas de Bactérias/genética , Sobrevivência Celular/efeitos dos fármacos , Escherichia coli/genética , Interferon Tipo I/genética , Nuclease do Micrococo/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Transdução de Sinais , Suínos , Replicação Viral
5.
Nat Commun ; 10(1): 2727, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227708

RESUMO

A fundamental challenge in medical microbiology is to characterize the dynamic protein-protein interaction networks formed at the host-pathogen interface. Here, we generate a quantitative interaction map between the significant human pathogen, Streptococcus pyogenes, and proteins from human saliva and plasma obtained via complementary affinity-purification and bacterial-surface centered enrichment strategies and quantitative mass spectrometry. Perturbation of the network using immunoglobulin protease cleavage, mixtures of different concentrations of saliva and plasma, and different S. pyogenes serotypes and their isogenic mutants, reveals how changing microenvironments alter the interconnectivity of the interaction map. The importance of host immunoglobulins for the interaction with human complement proteins is demonstrated and potential protective epitopes of importance for phagocytosis of S. pyogenes cells are localized. The interaction map confirms several previously described protein-protein interactions; however, it also reveals a multitude of additional interactions, with possible implications for host-pathogen interactions involving other bacterial species.


Assuntos
Anticorpos Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/imunologia , Cromatografia de Afinidade , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Mapeamento de Epitopos , Voluntários Saudáveis , Humanos , Espectrometria de Massas , Proteínas Opsonizantes/imunologia , Proteínas Opsonizantes/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/imunologia , Infecções Estreptocócicas/sangue , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/metabolismo
6.
BMC Genomics ; 20(1): 394, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31113361

RESUMO

BACKGROUND: Mycobacterium tuberculosis (MTB) is a common bacterium causing tuberculosis and remains a major pathogen for mortality. Although the MTB genome has been extensively explored for two decades, the functions of 27% (1051/3906) of encoded proteins have yet to be determined and these proteins are annotated as hypothetical proteins. METHODS: We assigned functions to these hypothetical proteins using SSEalign, a newly designed algorithm utilizing structural information. A set of rigorous criteria was applied to these annotations in order to examine whether they were supported by each parameter. Virulence factors and potential drug targets were also screened among the annotated proteins. RESULTS: For 78% (823/1051) of the hypothetical proteins, we could identify homologs in Escherichia coli and Salmonella typhimurium by using SSEalign. Functional classification analysis indicated that 62.2% (512/823) of these annotated proteins were enzymes with catalytic activities and most of these annotations were supported by at least two other independent parameters. A relatively high proportion of transporter was identified in MTB genome, indicating the potential frequent transportation of frequent absorbing essential metabolites and excreting toxic materials in MTB. Twelve virulence factors and ten vaccine candidates were identified within these MTB hypothetical proteins, including two genes (rpoS and pspA) related to stress response to the host immune system. Furthermore, we have identified six novel drug target candidates among our annotated proteins, including Rv0817 and Rv2927c, which could be used for treating MTB infection. CONCLUSIONS: Our annotation of the MTB hypothetical proteins will probably serve as a useful dataset for future MTB studies.


Assuntos
Proteínas de Bactérias/fisiologia , Mycobacterium tuberculosis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Genoma Bacteriano , Anotação de Sequência Molecular , Mycobacterium tuberculosis/imunologia , Homologia de Sequência de Aminoácidos , Fatores de Virulência
7.
mSphere ; 4(3)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31043513

RESUMO

BBK32 is a multifunctional surface lipoprotein expressed by Borrelia burgdorferi sensu lato, the causative agent of Lyme disease. Previous studies suggested that BBK32 could be a sensitive antigen target of new, more effective, serodiagnostic assays for the laboratory diagnosis of Lyme disease. However, nonspecific antibody binding to full-length BBK32 has hampered its use as a target in clinical assays. Specificity can be improved by the use of peptides composed of linear B cell epitopes that are unique to B. burgdorferi, eliminating cross-reactive epitopes that bind to antibodies generated by non-B. burgdorferi antigens. In this study, we identified linear B cell epitopes in 2 regions, BBK32 amino acids 16 to 30 [BBK32(16-30)] and BBK32 amino acids 51 to 80 [BBK32(51-80)], by probing overlapping peptide libraries of BBK32 with serum from patients with early Lyme disease. We screened synthetic peptides containing these epitopes using a large panel of serum (n = 355) obtained from patients with erythema migrans lesions (early Lyme disease), Lyme arthritis, syphilis, rheumatoid arthritis, or healthy volunteers. BBK32(16-30) demonstrated a nearly universal antibody binding in serum from all patients, indicating that regions of BBK32 are highly cross-reactive. BBK32(51-80) was less cross-reactive, being able to distinguish serum from Lyme disease patients from control patient serum; however, an unacceptable level of antibody binding was still observed in control samples, resulting in a reduced specificity (94.7%). These results indicate that BBK32 contains cross-reactive epitopes that make it a poor antigen target for inclusion in a serodiagnostic assay for Lyme disease and highlight the difficulties in identifying highly sensitive and specific seroassay targets.IMPORTANCE Lyme disease is an infectious disease that has the potential to cause significant morbidity with damage to nervous and musculoskeletal systems if left untreated. Appropriate antibiotic treatment during early infection prevents disease progression. Unfortunately, currently available diagnostics are suboptimal in the detection of early disease. The inability to confirm Borrelia infection using laboratory methods during early disease is, in part, responsible for much of the controversy surrounding Lyme disease today. As a result, there has been significant investment in the identification of new antigen targets to generate diagnostic assays that are more sensitive for the detection of early infection. The importance of our research is that in our evaluation of BBK32, an antigen that was previously identified as a promising target for use in serodiagnostics, we found a high degree of cross-reactivity that could compromise the specificity of assays that utilize this antigen, leading to false-positive diagnoses.


Assuntos
Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/química , Borrelia burgdorferi/imunologia , Epitopos de Linfócito B/imunologia , Doença de Lyme/diagnóstico , Afinidade de Anticorpos , Antígenos de Bactérias/sangue , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Borrelia burgdorferi/química , Humanos , Doença de Lyme/imunologia , Sensibilidade e Especificidade , Testes Sorológicos/métodos
8.
Microb Pathog ; 133: 103554, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31121271

RESUMO

We have previously shown that Listeria monocytogenes, a causative agent of listeriosis, can produce membrane vesicles (MVs) during in vitro culture. The aim of this study was to investigate the ability of MVs from L. monocytogenes cultured with or without salt stress to induce cytotoxicity and pro-inflammatory responses in colon epithelial Caco-2 cells. MVs were purified from wild-type L. monocytogenes 10403S strain and an isogenic ΔsigB mutant strain. MVs from both wild-type and ΔsigB mutant strains increased viability of Caco-2 cells regardless of salt stress. Both MVs from wild-type and ΔsigB mutant strains stimulated expression of pro-inflammatory cytokine and chemokine genes in Caco-2 cells. Expression levels of pro-inflammatory cytokine genes in cells treated with MVs from bacteria cultured without salt stress were significantly higher than those in cells treated with MVs from bacteria cultured with salt stress. However, expression levels of chemokine genes in cells treated with MVs from bacteria cultured with salt stress were significantly higher than those in cells treated with MVs from bacteria cultured without salt stress. In addition, expression levels of interleukin (IL)-1ß and IL-8 genes were partially inhibited by either lysozyme-treated MVs or ethylenediaminetetraacetic acid-treated MVs compared to those after treatment with intact MVs. Our results suggest that salt stress can affect the production of L. monocytogenes MVs, thus causing different pro-inflammatory responses in host cells.


Assuntos
Proteínas de Bactérias/imunologia , Células CACO-2/imunologia , Células Epiteliais/imunologia , Listeria monocytogenes/metabolismo , Estresse Salino/fisiologia , Proteínas de Bactérias/genética , Sobrevivência Celular , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/genética , Citocinas/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Fator sigma/genética , Fator sigma/imunologia , Estresse Fisiológico/fisiologia
9.
Res Vet Sci ; 124: 387-392, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31077966

RESUMO

Streptococcus equi subspecies zooepidemicus (SEZ) is a zoonotic pathogen with adhesive and invasive properties. Due to the shortcomings of antibiotics and traditional inactivated vaccine, identifying protective antigens against SEZ would be helpful to the development of novel vaccines. MAP has been identified as a membrane anchored protein with a typical LPXTG-like cell wall-anchored motif. In present study, the objective was to evaluate the effects of MAP as a subunit vaccine with mouse model. The Western blot analysis shown that the purified recombinant MAP presented good immunoreactive to convalescent porcine sera against SEZ. The protein could elicit a remarkable humoral antibody response and protect 80% of mice against lethal dose challenge of SEZ in mouse model. Moreover, the hyperimmune sera against MAP could efficiently kill the bacteria in whole blood killing assay and conferred significant protection against SEZ in passive immunization experiments. This study suggests with good reasons that MAP could be a novel and effective vaccine candidate for SEZ.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus equi/imunologia , Animais , Antígenos de Bactérias/genética , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Proteínas de Bactérias/genética , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Infecções Estreptocócicas/microbiologia , Streptococcus equi/genética
10.
Microbiol Immunol ; 63(7): 280-284, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31087695

RESUMO

In 2018, a patient was diagnosed with Shimokoshi type scrub typhus in Yamagata Prefecture, Japan. The causative pathogen was likely a variant type because 43 (8.3%) of 521 deduced amino acid sequences of the 56-kDa type-specific antigen (TSA) were different from those of the Shimokoshi prototype strain. The patient's paired sera showed low antibody titers against the Shimokoshi prototype strain. Two cases of scrub typhus reported in the Tohoku region during 2011-2012 also involved the same 56-kDa TSA gene sequence. These findings suggest the presence of diversity in Shimokoshi type Orientia tsutsugamushi, which may impede the laboratory diagnosis of scrub typhus.


Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Orientia tsutsugamushi/genética , Orientia tsutsugamushi/patogenicidade , Tifo por Ácaros/imunologia , Tifo por Ácaros/microbiologia , Sequência de Aminoácidos , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Sequência de Bases , Genes Bacterianos/genética , Humanos , Japão , Proteínas de Membrana/imunologia , Peso Molecular , Tifo por Ácaros/diagnóstico
11.
Infect Immun ; 87(7)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31036601

RESUMO

Haemophilus ducreyi causes chancroid and is a major cause of cutaneous ulcers in children. Due to environmental reservoirs, both class I and class II H. ducreyi strains persist in cutaneous ulcer regions of endemicity following mass drug administration of azithromycin, suggesting the need for a vaccine. The hemoglobin receptor (HgbA) is a leading vaccine candidate, but its efficacy in animal models is class specific. Controlled human infection models can be used to evaluate vaccines, but only a class I strain (35000HP) has been characterized in this model. As a prelude to evaluating HgbA vaccines in the human model, we tested here whether a derivative of 35000HP containing a class II hgbA allele (FX548) is as virulent as 35000HP in humans. In eight volunteers infected at three sites with each strain, the papule formation rate was 95.8% for 35000HP versus 62.5% for FX548 (P = 0.021). Excluding doses of FX548 that were ≥2-fold higher than those of 35000HP, the pustule formation rate was 25% for 35000HP versus 11.7% for FX548 (P = 0.0053). By Western blot analysis, FX548 and 35000HP expressed equivalent amounts of HgbA in whole-cell lysates and outer membranes. The growth of FX548 and 35000HP was similar in media containing hemoglobin or hemin. By whole-genome sequencing and single-nucleotide polymorphism analysis, FX548 contained no mutations in open reading frames other than hgbA We conclude that by an unknown mechanism, FX548 is partially attenuated in humans and is not a suitable strain for HgbA vaccine efficacy trials in the model.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Cancroide/prevenção & controle , Vacinas Anti-Haemophilus/imunologia , Haemophilus ducreyi/imunologia , Adulto , Alelos , Proteínas de Bactérias/administração & dosagem , Proteínas de Transporte/administração & dosagem , Cancroide/imunologia , Cancroide/microbiologia , Feminino , Vacinas Anti-Haemophilus/administração & dosagem , Vacinas Anti-Haemophilus/genética , Haemophilus ducreyi/genética , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
12.
Mol Immunol ; 111: 182-197, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31078054

RESUMO

The type VI secretion system (T6SS) has recently emerged as a new pattern of protein secretions in Campylobacter jejuni (C. jejuni). Within the T6SS cluster, hemolysin co-regulated protein (hcp) is considered as a hallmark of functional T6SS and holds key role in bacterial virulence. As poultry is the primary reservoir of C. jejuni and the major sources for human infection, we evaluated the capacity of recombinant hcp (rhcp) immunization in blocking C. jejuni colonization in chickens with an aim to control bacterial transmission to humans via poultry food chain. Considering the mucosal route is the primary portal for C. jejuni entry and gut mucosa offers the apposite site for C. jejuni adherence, we investigated the immune-protective potential of intra-gastric administration of rhcp using chitosan-based nanoparticles. To achieve this goal, full length coding sequence of hcp gene from C. jejuni was cloned and expressed in E. coli. Purified rhcp was entrapped in chitosan-Sodium tripolyphosphate nanoparticles (CS-TPP NPs) and orally gavaged in chickens. Our results suggest that intra-gastric immunization of CS-TPP-rhcp induces consistent and steady increase in intestinal (sIgA) and systemic antibody (IgY) response against rhcp with significant reduction in cecal load of C. jejuni. The protection afforded by rhcp associated cellular responses with Th1 and Th17 profile in terms of increased expression of NFkB, IL-1ß, IL-8, IL-6, IFN-γ and IL-17 A genes. Though systemic immunization of rhcp with IFA resulting in a robust systemic (IgY) and local (sIgA) antibody response, mucosal administration of rhcp loaded CS-TPP NPs was found to be superior in terms of bacterial clearance. Altogether, present study suggests that chitosan based intra-gastric delivery of rhcp have several advantages over the injectable composition and could be a promising vaccine approach to effectively control C. jejuni colonization in chickens.


Assuntos
Formação de Anticorpos/imunologia , Campylobacter jejuni/imunologia , Galinhas/imunologia , Mucosa Gástrica/imunologia , Proteínas com Ferro-Enxofre/imunologia , Proteínas Recombinantes/imunologia , Sistemas de Secreção Tipo VI/imunologia , Animais , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Ceco/imunologia , Ceco/microbiologia , Galinhas/microbiologia , Escherichia coli/imunologia , Mucosa Gástrica/microbiologia , Proteínas Hemolisinas/imunologia , Imunização/métodos , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/microbiologia , Células Th1/imunologia , Células Th17/imunologia
13.
Mol Immunol ; 112: 115-122, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31082645

RESUMO

Mycobacterium tuberculosis (M. tuberculosis) persistent infection might cause the dysfunction of hematopoiesis. To investigate whether M. tuberculosis persistent antigen stimulation impairs the proliferation and differentiation of hematopoietic stem and progenitor cells characterized as lineage- c-Kit+ (LK cells), C57BL/6 mice were primed with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and boosted with a cocktail of M. tuberculosis antigens ESAT6, CFP10 and Mtb10.4-HspX (MH) along with adjuvant N, N'-dimethyl-N, N'-dioctadecylammonium bromide (DDA) plus polyinosinic-polycytidylic acid (Poly I:C) weekly for 12 or 22 weeks. The cytokine production by splenic T cells, proliferation of LK cells and transcriptional events during differentiation of bone marrow (BM) c-Kit+ cells were investigated. Meanwhile, the mice were treated with interleukin 2 (IL-2) and the therapeutic effects were analyzed. We found that antigen specific interferon-γ (IFN-γ) production by splenic CD4+ T cells increased following antigen stimulation for 12 weeks, but it declined after continuous stimulation for 22 weeks. The long-term exposure of mice to M. tuberculosis antigen compromised the proliferation of LK cells. Moreover, the expression of transcription factors in the c-Kit+ cells was adjusted, with up-regulation of IRF8 and Batf2 involved in myeloid differentiation and down-regulation of NOTCH1 and GATA2 participated in T-cell lineage commitment. The concentrations of IFN-γ in BM of the persistent antigen group were higher than that in sham control at the 12th week, while the concentrations of IL-2 in BM of the persistent antigen group were lower compared with the transient antigen stimulation control. Following IL-2 treatment, the concentrations of IL-2 in BM increased while IFN-γ got declined. IL-2 treatment could restore the expression levels of those transcription factors and the proliferating activity of LK cells impaired by persistent antigen stimulation. Our results indicate that M. tuberculosis antigen persistent stimulation decreases the proliferating activity of LK cells, promotes myelopoietic differentiation, and represses lymphopoietic differentiation as a consequence of elevated IFN-γ production. IL-2 supplementation contributes to maintaining the homeostasis of hemopoiesis.


Assuntos
Antígenos de Bactérias/imunologia , Medula Óssea/imunologia , Proliferação de Células/fisiologia , Mycobacterium tuberculosis/imunologia , Transcrição Genética/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/imunologia , Medula Óssea/microbiologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Feminino , Transplante de Células-Tronco Hematopoéticas/métodos , Interferon gama/imunologia , Interleucina-2/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium bovis/imunologia , Fatores de Transcrição/imunologia
14.
BMC Infect Dis ; 19(1): 366, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31039752

RESUMO

BACKGROUND: Independent of HIV infection, extrapulmonary TB (EPTB) risk is increased in women, persons of black race or foreign birth, and by genetic variants in vitamin D receptor (VDR), interleukin-1 beta (IL-1ß), and toll-like receptor (TLR)-2; functional correlates are unclear. We evaluated macrophage expression of VDR, TLR2, cathelicidin, and TNF-α, and production of IL-1ß in HIV-seronegative persons with previous EPTB, previous pulmonary TB, latent M. tuberculosis infection, and uninfected TB contacts. Persons with previous pleural TB were excluded due to enhanced immune responses at the site of disease. METHODS: Macrophages were stimulated with TLR-2 agonist M. tuberculosis lipoprotein (LpqH), live and gamma-irradiated M. tuberculosis. RESULTS: M. tuberculosis - infected macrophages from persons with previous EPTB had increased VDR expression (29.17 relative value unit increase in median expression vs. uninfected contacts, after adjusting for foreign-born status; P = 0.02). Macrophages from persons with previous EPTB had a 38.88 µg/mL increase in median IL-1ß production after stimulation with LpqH compared to uninfected contacts (P = 0.01); the effect was similar (44.99 µg/mL) but not statistically significant after controlling for foreign-born status. Median 25-hydroxyvitamin D levels were low but not significantly different between groups. CONCLUSIONS: There was increased macrophage expression of VDR after stimulation with live M. tuberculosis in persons with previous extrapulmonary TB. If post-treatment VDR expression reflects expression prior to disease, it may identify persons at risk for extrapulmonary TB.


Assuntos
Macrófagos/metabolismo , Mycobacterium tuberculosis/fisiologia , Receptores de Calcitriol/metabolismo , Tuberculose/patologia , Adulto , Idoso , Proteínas de Bactérias/imunologia , Estudos de Casos e Controles , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Feminino , Raios gama , Expressão Gênica , Humanos , Interleucina-1beta/análise , Macrófagos/citologia , Macrófagos/microbiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efeitos da radiação , Receptores de Calcitriol/genética , Receptor 2 Toll-Like/agonistas , Tuberculose/imunologia , Vitamina D/análogos & derivados , Vitamina D/sangue
15.
Nat Commun ; 10(1): 2260, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31113958

RESUMO

ABC exporters harness the energy of ATP to pump substrates across membranes. Extracellular gate opening and closure are key steps of the transport cycle, but the underlying mechanism is poorly understood. Here, we generated a synthetic single domain antibody (sybody) that recognizes the heterodimeric ABC exporter TM287/288 exclusively in the presence of ATP, which was essential to solve a 3.2 Å crystal structure of the outward-facing transporter. The sybody binds to an extracellular wing and strongly inhibits ATPase activity by shifting the transporter's conformational equilibrium towards the outward-facing state, as shown by double electron-electron resonance (DEER). Mutations that facilitate extracellular gate opening result in a comparable equilibrium shift and strongly reduce ATPase activity and drug transport. Using the sybody as conformational probe, we demonstrate that efficient extracellular gate closure is required to dissociate the NBD dimer after ATP hydrolysis to reset the transporter back to its inward-facing state.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Simulação de Dinâmica Molecular , Domínio AAA/genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Espectroscopia de Ressonância de Spin Eletrônica , Mutação , Multimerização Proteica , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Thermotoga maritima
16.
Res Microbiol ; 170(4-5): 182-191, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30953690

RESUMO

This paper presents the effects of the composition of different media (i.e., Tryptic soy broth (TSB), Brain heart infusion (BHI), Listeria enrichment broth (LEB), Fraser broth (FB) and University of Vermont medium (UVM)) on the detection of a short peptide fragment PepD specific to the p60 protein (p60) of L. monocytogenes by a monoclonal antibody (anti-PepD mAb). Expression of the p60 obtained was demonstrated to be proportional to the cellular growth of Listeria monocytogenes regardless of the tested growth medium. However, the early growth of L. monocytogenes and the expression of the p60 were negatively affected by the presence of selective agents present in LEB, FB and UVM. Among those three selective enrichment media commonly used for L. monocytogenes, LEB allowed a better expression of L. monocytogenes p60 after an incubation period of 18 h. Optimization of the LEB revealed that the dextrose concentration was the critical factor for improving the expression of p60 and promotes the early expression of p60. Moreover, an optimal dextrose concentration of 0.5% (w/v) in LEB, coupled with anti-PepD mAb immobilized to solid support, reduced the detection of p60 from 18 h to 9 h for an initial concentration of L. monocytogenes of 108 CFU/ml.


Assuntos
Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/imunologia , Meios de Cultura/química , Lipoproteínas/biossíntese , Lipoproteínas/imunologia , Listeria monocytogenes/crescimento & desenvolvimento , Técnicas Bacteriológicas , Ensaio de Imunoadsorção Enzimática , Microbiologia de Alimentos , Listeria monocytogenes/isolamento & purificação
17.
Microb Pathog ; 132: 87-99, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31029716

RESUMO

Treponema is a diverse bacterial genus, the species of which can be pathogenic, symbiotic, or free living. These treponemes can cause various diseases in humans and other animals, such as periodontal disease, bovine digital dermatitis and animal skin lesions. However, the most important and well-studied disease of treponemes that affects humans is 'syphilis'. This disease is caused by Treponema pallidum subspecie pallidum with 11-12 million new cases around the globe on an annual basis. In this study we analyze the transportome of ten Treponema species, with emphasis on the types of encoded transport proteins and their substrates. Of the ten species examined, two (T. primitia and T. azonutricium) reside as symbionts in the guts of termites; six (T. pallidum, T. paraluiscuniculi, T. pedis, T. denticola, T. putidum and T. brennaborense) are pathogens of either humans or animals, and T. caldarium and T. succinifaciens are avirulent species, the former being thermophilic. All ten species have a repertoire of transport proteins that assists them in residing in their respective ecological niches. For instance, oral pathogens use transport proteins that take up nutrients uniquely present in their ecosystem; they also encode multiple multidrug/macromolecule exporters that protect against antimicrobials and aid in biofilm formation. Proteins of termite gut symbionts convert cellulose into other sugars that can be metabolized by the host. As often observed for pathogens and symbionts, several of these treponemes have reduced genome sizes, and their small genomes correlate with their dependencies on the host. Overall, the transportomes of T. pallidum and other pathogens have a conglomerate of parasitic lifestyle-assisting proteins. For example, a T. pallidum repeat protein (TprK) mediates immune evasion; outer membrane proteins (OMPs) allow nutrient uptake and end product export, and several ABC transporters catalyze sugar uptake, considered pivotal to parasitic lifestyles. Taken together, the results of this study yield new information that may help open new avenues of treponeme research.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Genômica/métodos , Treponema/classificação , Treponema/genética , Treponema/fisiologia , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Proteínas de Transporte/classificação , Farmacorresistência Bacteriana/genética , Microbioma Gastrointestinal , Tamanho do Genoma , Interações Hospedeiro-Patógeno , Humanos , Evasão da Resposta Imune , Porinas/genética , Porinas/imunologia , Proteoma , Especificidade da Espécie , Especificidade por Substrato , Simbiose , Sífilis/microbiologia , Treponema/patogenicidade , Treponema pallidum/genética
18.
Biomed Res ; 40(2): 87-95, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30982804

RESUMO

Helicobacter pylori (H. pylori) urease is a key protein for persistent infection of the bacteria in the stomach. Although H. pylori generally induce anti-H. pylori-specific antibodies (Abs), these Abs do not usually work for eradication or prevention of the H. pylori infection. In our previous study, we identified a linear epitope composed of 19-mer peptides termed UB-33, CHHLDKSIKEDVQFADSRI, within the large subunit of H. pylori urease. Anti-UB-33-specific Abs neutralized the enzymatic activity of H. pylori urease in vitro. In the present study, we evaluated the effect of immunization of BALB/c mice with H. pylori UB-33 peptide. After confirming the production of anti-UB-33-specific Abs, mice were challenged orally with H. pylori Sydney Strain-1 (SS-1). Mice producing anti-UB-33-specific Abs were not infected with SS-1, and the amount of SS-1 isolate in their stomach was significantly reduced. Also, the urease-negative mutant of H. pylori, HPP1801, did not colonize in the stomach, indicating that H. pylori urease was a critical element for infection of H. pylori in the gastric mucosa. Moreover, mice producing UB-33-specific Abs apparently suppressed H. pylori infection in the stomach where anti-UB-33 Abs were secreted in the gastric juice, indicating that H. pylori colonization was inhibited in the presence of anti-UB-33 Abs. In addition, the neutralization activity of sera from mice immunized with purified urease was less potent than that in the sera from mice immunized with UB-33. Furthermore, the recognition of epitope UB-33 was mediated through Toll-like receptor 2 (TLR2) on the B-1 cells using TLR2-knockout BALB/c mice in vivo. These results indicate that liner peptide UB-33 should be used for immunization to induce neutralizing Abs instead of purified H. pylori urease to prevent H. pylori infection and their colonization in the stomach.


Assuntos
Anticorpos Antibacterianos/biossíntese , Anticorpos Neutralizantes/biossíntese , Proteínas de Bactérias/imunologia , Infecções por Helicobacter/prevenção & controle , Imunização/métodos , Peptídeos/imunologia , Urease/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Epitopos/química , Epitopos/imunologia , Feminino , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/imunologia , Mucosa Gástrica/microbiologia , Deleção de Genes , Expressão Gênica , Infecções por Helicobacter/genética , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/crescimento & desenvolvimento , Helicobacter pylori/imunologia , Soros Imunes/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/administração & dosagem , Peptídeos/síntese química , Estômago/efeitos dos fármacos , Estômago/imunologia , Estômago/microbiologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Urease/deficiência , Urease/genética
19.
Microb Pathog ; 131: 9-14, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30930220

RESUMO

Acinetobacter baumannii is considered as a major cause of nosocomial infection worldwide. Various vaccine formulations have been mostly studied based on secreted or surface-exposed proteins of A. baumannii in murine models. Serum resistance proteins are critical virulence factors in bloodstream infections. AbOmpA and PKF are two major factors involved in serum resistance and could be considered as promising vaccine targets. In this study IgG1, IgG2c, Total-IgG concentrations, survival rates and spleen bacterial loads were studied in C57/BL mice model according to PKF, AbOmpA and AbOmpA + PKF vaccine formulations. The findings showed significant raises of IgG2c and Total-IgG in all three vaccinated groups in comparison with the control group. Whereas, there were low concentrations of IgG1 in all immunization plans. Colony counts of mice spleen showed the bacterial load of PKF plan had the most decrease of bacterial load (DBL = 5 log10 CFU/g). Taken together, this evaluation indicated that PKF vaccination plan induced a polarized Th1 response and rendered an effective protection against bloodstream infection caused by A. baumannii.


Assuntos
Infecções por Acinetobacter/imunologia , Infecções por Acinetobacter/prevenção & controle , Acinetobacter baumannii/patogenicidade , Formação de Anticorpos/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Fatores R/sangue , Sepse/microbiologia , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/genética , Animais , Carga Bacteriana , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Clonagem Molecular , Modelos Animais de Doenças , Genes Bacterianos/genética , Imunização , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos C57BL , Fatores R/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Baço/imunologia , Taxa de Sobrevida , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Fatores de Virulência/genética , Fatores de Virulência/imunologia
20.
Fish Shellfish Immunol ; 89: 420-427, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30974221

RESUMO

As an important pathogen in aquaculture, Pseudomonas plecoglossicida has caused heavy losses. It was determined with RNA-seq that the expression of a LysR-type transcriptional regulator gene (L321_20267) of P. plecoglossicida at 18 °C was significantly higher than that at 28 °C, which was verified by quantitative real-time PCR (qRT-PCR). RNAi significantly reduced the content of L321_20267 mRNA in P. plecoglossicida, with a maximal decrease of 90.63%. Compared with the wild-type strain, infection with the L321_20267-RNAi strain resulted in a 50% reduction in mortality and an onset time delay of Epinephelus coioides, as well as alleviation of the symptoms in E. coioides spleens. Compared with the wild-type strain of P. plecoglossicida, the L321_20267-RNAi strain resulted in a significant change in the spleen transcriptome of infected E. coioides. The results of GO and KEGG analysis showed that genes of serine hydrolase activity, the antigen processing and presentation pathway, the B cell receptor signalling pathway and the chemokine signalling pathway were most affected by the L321_20267 gene of P. plecoglossicida. Meanwhile, the immune genes were related to different numbers of miRNAs and lncRNAs, and some miRNAs were related to more than one gene. The results indicated that 1. L321_20267 is a virulence gene of P. plecoglossicida; 2. the upregulation of the immune pathways facilitated E. coioides to remove the L321_20267-RNAi strain compared with the wild-type strain of P. plecoglossicida; and 3. the immune genes were regulated by miRNA and lncRNA in a complex manner.


Assuntos
Proteínas de Bactérias/imunologia , Bass/imunologia , Imunidade Inata , Pseudomonas/fisiologia , Fatores de Transcrição/imunologia , Animais , Genes Bacterianos/imunologia , Pseudomonas/genética
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