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Bordetella bronchiseptica is a pathogen causing respiratory infections in mammals. With the improving understanding of companion animals' welfare, addressing the side effects of bordetella vaccine gains importance in dogs. Studies on diverse subunit vaccines are actively pursued in humans to safely and effectively control bordetellosis. Therefore, our objective was to develop a canine bordetella vaccine inspired by human vaccine development. We evaluated the immunogenicity of the two bacterial components: the outer membrane proteins (OMPs) and the dermonecrotic toxin (DNT) from a canine isolate of B. bronchiseptica. In-silico analysis identified eight domains of DNT, and Domain 3 was selected as the most promising antigen candidate. Additionally, the OMPs were extracted and examined using SDS-PAGE and Western blot analysis. The distinct immunological characteristic of OMPs and DNT-3 were examined individually and in combination. Gene expression and cytokine production were also evaluated in DH82 cells after stimulation with those antigens. Treatment with OMPs resulted in higher level of Th1 related cytokines, while DNT-3 induced a predominant response associated with Th17 and Th2 in the cytokine production. Synergistic effects were observed exclusively on IL-23, indicating increase of a potential risk of side effects when OMPs and DNT act together. These findings provide valuable insights into the reactogenicity of conventional Bordetella vaccines. Further, the presented preclinical data in this study offer an alternative method of the development for an optimal next-generation Bordetella vaccine for companion animals and humans, replacing the acellular vaccines containing both toxin and protein components.
Assuntos
Proteínas da Membrana Bacteriana Externa , Infecções por Bordetella , Bordetella bronchiseptica , Doenças do Cão , Bordetella bronchiseptica/imunologia , Animais , Cães , Proteínas da Membrana Bacteriana Externa/imunologia , Infecções por Bordetella/imunologia , Infecções por Bordetella/veterinária , Infecções por Bordetella/microbiologia , Infecções por Bordetella/prevenção & controle , Doenças do Cão/imunologia , Doenças do Cão/microbiologia , Vacinas Bacterianas/imunologia , Citocinas/imunologia , Fatores de Virulência de Bordetella/imunologia , TransglutaminasesRESUMO
Urea reacts with chlorine to form chlorinated ureas (chloroureas), and fully chlorinated urea (tetrachlorourea) is further hydrolyzed into CO2 and chloramines. This study found that the oxidative degradation of urea by chlorination was enhanced by the pH swing, wherein the reaction proceeded under an acidic pH (e.g., pH = 3) in the first stage, and the solution pH was subsequently increased to a neutral or alkaline value (e.g., pH > 7) in the second-stage reaction. The degradation rate of urea by pH-swing chlorination increased with increasing chlorine dose and pH during the second-stage reaction. The pH-swing chlorination was based on the opposite pH dependence of sub-processes comprising urea chlorination. The formation of monochlorourea was favored under acidic pH conditions; however, the subsequent conversion into di- and trichloroureas was favored under neutral or alkaline pH conditions. The deprotonation of monochlorourea (pKa = 9.7 ± 1.1) and dichlorourea (pKa = 5.1 ± 1.4) was suggested to be responsible for the accelerated reaction in the second stage under increased pH conditions. pH-swing chlorination was also effective in degrading urea at low concentrations (micromolar levels). In addition, the total nitrogen concentration significantly decreased during the degradation of urea because of the volatilization of chloramines and the release of other gaseous nitrogen compounds.
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Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic emaciating disease of ruminants that causes enormous economic losses to the bovine industry, globally. However, there are still remaining clues to be solved in the pathogenesis and diagnosis of the disease. Therefore, an in vivo murine experimental model was tried to understand responses in early stage of MAP infection by oral and intraperitoneal (IP) routes. In the MAP infection size, and weight of spleen and liver were increased in the IP group compared with oral groups. Severe histopathological changes were also observed in the spleen and liver of IP infected mice at 12 weeks post-infection (PI). Acid-fast bacterial burden in the organs was closely related to histopathological lesions. In the cytokine production from splenocytes of MAP-infected mice, higher amounts of in TNF-α, IL-10, and IFN-γ were produced at early stage of IP-infected mice while IL-17 production was different at time and infected groups. This phenomenon may indicate the immune shift from Th1 to Th17 through the time course of MAP infection. Systemic and local responses in the MAP-infection were analyzed by using transcriptomic analysis in the spleens and mesenteric lymph nodes (MLN). Based on the analysis of biological processes at 6 weeks PI in spleen and MLN in each infection group, canonical pathways were analyzed with ingenuity pathway analysis in the immune responses and metabolism especially lipid metabolism. Infected host cells with MAP increased in the production of proinflammatory cytokines and reduced the availability of glucose at early stage of infection (p < 0.05). Also, host cells secreted cholesterol through cholesterol efflux to disturb energy source of MAP. These results reveal immunopathological and metabolic responses in the early stage of MAP infection through the development of a murine model.
Assuntos
Doenças dos Bovinos , Mycobacterium avium subsp. paratuberculosis , Paratuberculose , Animais , Bovinos , Camundongos , Modelos Animais de Doenças , Paratuberculose/microbiologia , Citocinas , Colesterol , Doenças dos Bovinos/microbiologiaRESUMO
Carbapenems are broad-spectrum antibiotics widely used for the treatment of human infections caused by multidrug-resistant (MDR) Gram-negative bacteria. However, emerging carbapenemase-producing Enterobacterales (CPE) are rising as a public threat to human and animal health. We screened clinical bacterial isolates from 241 dogs and 18 cats hospitalized at Veterinary Medical Teaching Hospital, Seoul National University, from 2018 to 2020 for carbapenemase production. In our study, 5 strains of metallo-ß-lactamase NDM-5-producing Escherichia coli and Klebsiella pneumoniae were isolated from 4 different dogs. Multilocus sequence typing (MLST) results showed that all E. coli strains were ST410 and all K. pneumoniae strains were ST378. Whole genome analysis of the plasmid showed that blaNDM-5 is carried on a IncX3 plasmid, showing a high concordance rate with plasmids detected worldwide in human and animal isolates. The blaNDM gene was associated with the bleMBL gene and the ISAba125 element, truncated with the IS5 element. The results of this study show that CPE has already become as a threat to both animals and humans in our society, posing the necessity to solve it in terms of "One Health". Therefore, preventive strategies should be developed to prevent the spread of CPE in animal and human societies.
Assuntos
Escherichia coliRESUMO
Research has been undertaken to understand the host immune response to Brucella canis infection because of the importance of the disease in the public health field and the clinical field. However, the previous mechanisms governing this infection have not been elucidated. Therefore, in vitro models, which mimic the in vivo infection route using a canine epithelial cell line, D17, and a canine macrophage, DH82, were established to determine these mechanisms by performing an analysis of the transcriptomes in the cells. In this study, a coculture model was constructed by using the D17 cell line and DH82 cell line in a transwell plate. Also, a single cell line culture system using DH82 was performed. After the stimulation of the cells in the two different systems infected with B. canis, the gene expression in the macrophages of the two different systems was analyzed by using RNA-sequencing (RNA-seq), and a transcriptomic analysis was performed by using the Ingenuity Pathway Analysis (IPA). Gene expression patterns were analyzed in the DH82 cell line at 2, 12, and 24 h after the stimulation with B. canis. Changes in the upregulated or downregulated genes showing 2-fold or higher were identified at each time point by comparing with the non-stimulated group. Differentially expressed genes (DEGs) between the two culture models were identified by using the IPA program. Generally, the number of genes expressed in the single cell line culture was higher than the number of genes expressed in the coculture model for all-time points. The expression levels of those genes were higher in the single cell line culture (p < 0.05). This analysis indicated that the immune response-related pathways, especially, the dendritic cell maturation, Triggering receptor expression on myeloid cells 1 (TREM1) signaling, and Toll-like receptor (TLR) signaling pathway, were significantly induced in both the culture systems with higher p-values and z-scores. An increase in the expression level of genes related to the pathways was observed over time. All pathways are commonly associated with a manifestation of pro-inflammatory cytokines and early immune responses. However, the Peroxisome proliferator-activation receptor (PPAR) signaling and Liver X Receptor/Retinoid X Receptor (LXR/RXR) signaling associated with lipid metabolism were reduced. These results indicate that early immune responses might be highly activated in B. canis infection. Therefore, these results might suggest clues to reveal the early immune response of the canine to B. canis infection, particularly TLR signaling.
RESUMO
Johne's disease (JD) caused by Mycobacterium avium subsp. paratuberculosis (MAP) is a chronic, wasting infectious disease in ruminants that causes enormous economic losses to the dairy and beef cattle industries. Understanding the mechanism of persistency of MAP is key to produce novel ideas for the development of new diagnostic methods or prevention techniques. We sought interactions between the host and MAP using epithelial passage model, which mimic initial stage of infection. From the transcriptomic analysis of bovine immune cells (PBMCs), it was suggested that infection through the epithelial cells elicited prolonged Th17-derived immune response, as indicated by upregulation of IL-17A, IL-17F and RORC until 120 h p.i., compared to directly infected PBMCs. Global downregulation of gene expression was observed after 72 h p.i., especially for genes encoding cell surface receptors of phagocytic cells, such as Toll-like receptors and MHC class II molecules. In addition, the cholesterol efflux transporters ABCA1, ABCG1, and APOE, which are regulated by the LXR/RXR pathway, were downregulated. In summary, it would be suggested that the host initiate immune response to activate Th17-derived cytokines, and MAP survives persistently by altering the host adaptive immune response by suppressing surface receptors and manipulating lipid metabolism in phagocytic cells.
Assuntos
Leucócitos Mononucleares/imunologia , Paratuberculose/imunologia , Fagócitos/citologia , Células Th17/imunologia , Animais , Bovinos , Diferenciação Celular , Células Epiteliais/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Leucócitos Mononucleares/citologia , Mycobacterium avium subsp. paratuberculosis/patogenicidade , Paratuberculose/microbiologia , Fagócitos/imunologia , Receptores Toll-Like/metabolismo , TranscriptomaRESUMO
Mucosal delivery of antigens can induce both humoral and cellular immune responses. Particularly, the nasal cavity is a strongly inductive site for mucosal immunity among several administration routes, as it is generally the first point of contact for inhaled antigens. However, the delivery of antigens to the nasal cavity has some disadvantages such as rapid clearance and disposition of inhaled materials. For these reasons, remarkable efforts have been made to develop antigen delivery systems which suit the nasal route. The use of nanoparticles as delivery vehicles enables protection of the antigen from degradation and sustains the release of the loaded antigen, eventually resulting in improved vaccine and/or drug efficacy. Chitosan, which exhibits low toxicity, biodegradability, good cost performance, and strong mucoadhesive properties, is a useful material for nanoparticles. The present review provides an overview of the mucosal immune response induced by nanoparticles, recent advances in the use of nanoparticles, and nasal delivery systems with chitosan nanoparticles.
Assuntos
Antígenos/administração & dosagem , Quitosana/química , Portadores de Fármacos , Nanopartículas , Vacinas/administração & dosagem , Adesividade , Administração Intranasal , Animais , Antígenos/química , Preparações de Ação Retardada , Composição de Medicamentos , Estabilidade de Medicamentos , Humanos , Imunidade nas Mucosas/efeitos dos fármacos , Nanomedicina , Mucosa Nasal/efeitos dos fármacos , Mucosa Nasal/imunologia , Mucosa Nasal/metabolismo , Vacinas/químicaRESUMO
Non-tuberculous mycobacteria (NTM) are ubiquitous microorganisms that have the potential to cause disease in both humans and animals. Recently, NTM infections have rapidly increased in South Korea, especially in urbanized areas. However, the distribution of species and the antibiotic resistance profile of NTM in environmental sources have not yet been investigated. Therefore, we analyzed the distribution of species and the antibiotic resistance profile of NTM in soil within urban areas of South Korea. A total of 132 isolates of NTM were isolated from soil samples from 1 municipal animal shelter and 4 urban area parks. Among the 132 isolates, 105 isolates were identified as slowly growing mycobacteria (SGM) and 27 isolates as rapidly growing mycobacteria (RGM) based on the sequences of the rpoB and hsp65 genes. The antibiotic resistance patterns of NTM isolates differed from species to species. Additionally, a mutation in the rrs gene found in this study was not associated with aminoglycoside resistance. In conclusion, our results showed that NTM isolates from South Korean soil exhibit multidrug resistance to streptomycin, amikacin, azithromycin, ethambutol, isoniazid, and imipenem. These results suggest that NTM may pose a public threat.
RESUMO
Infection with Brucella abortus causes contagious zoonosis, brucellosis, and leads to abortion in animals and chronic illness in humans. Chitosan nanoparticles (CNs), biocompatible and nontoxic polymers, acts as a mucosal adjuvant. In our previous study, B. abortus malate dehydrogenase (Mdh) was loaded in CNs, and it induced high production of pro-inflammatory cytokines in THP-1 cells and systemic IgA in BALB/C mice. In this study, the time-series gene expression analysis of nasal-associated lymphoid tissue (NALT) was performed to identify the mechanism by which Mdh affect the target site of nasal immunization. We showed that intranasal immunization of CNs-Mdh reduced cell viability of epithelial cells and muscle cells at first 1 h, then induced cellular movement of immune cells such as granulocytes, neutrophils and lymphocytes at 6h, and activated IL-6 signaling pathway at 12h within NALT. These activation of immune cells also promoted signaling pathway for high-mobility group box 1 protein (HMGB1), followed by the maturation of DCs required for mucosal immunity. The CNs also triggered the response to other organism and inflammatory response, showing it is immune-enhancing adjuvant. The ELISA showed that significant production of specific IgA was detected in the fecal excretions and genital secretions from the CNs-Mdh-immunized group after 2 weeks-post immunization. Collectively, these results suggest that B. abortus Mdh-loaded CNs triggers activation of HMGB1, IL-6 and DCs maturation signaling within NALT and induce production of systemic IgG and IgA.
Assuntos
Formação de Anticorpos/fisiologia , Brucella abortus/imunologia , Brucelose/prevenção & controle , Imunização/métodos , Tecido Linfoide/imunologia , Malato Desidrogenase/imunologia , Administração Intranasal , Animais , Formação de Anticorpos/efeitos dos fármacos , Brucella abortus/metabolismo , Brucelose/imunologia , Quitosana/administração & dosagem , Quitosana/química , Quitosana/imunologia , Quitosana/farmacologia , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Feminino , Imunidade nas Mucosas/efeitos dos fármacos , Imunogenicidade da Vacina , Tecido Linfoide/efeitos dos fármacos , Malato Desidrogenase/administração & dosagem , Malato Desidrogenase/metabolismo , Malato Desidrogenase/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Mucosa Nasal/efeitos dos fármacos , Mucosa Nasal/imunologiaRESUMO
Drug delivery by the nasal or oral route is considered the preferred route of administration because it can induce systemic mucosal immunity. However, few studies have examined the immunogenicity and transport of antigen at the level of the microfold (M) cell, the epithelial cell that specializes in antigen sampling at mucosal surfaces. In our previous study, Brucella abortus malate dehydrogenase (Mdh) was loaded in chitosan nanoparticles (CNs), and it induced high production of proinflammatory cytokines in THP-1 cells and systemic IgA in BALB/C mice. In the present study, an in vitro M cell model was used in which Caco-2 cells and Raji B cells were co-cultured to investigate the impact of the uptake and immunogenicity of B. abortus Mdh on nanoparticle transport in human M cells. Our results showed that loaded CNs induced enhanced transport of Mdh in the M cell model. ELISAs showed significantly higher production of IL-1ß and IL-6 in the CN-Mdh stimulation group than that seen in the Mdh stimulation group. The observed increase of gene expression of TLR2, MyD88, TRAF6, IRF4 and CD14 implied that MyD88-dependent TLR2 signaling was activated by stimulation with CNs-Mdh. These results suggest that Mdh and CNs may function synergistically to enhance Th2-related responses triggered by the MyD88-dependent TLR2 signaling pathway and could induce an inflammatory response in M cells as an M cell-targeted delivery system. This study will contribute to the development of not only effective antigens for intracellular bacteria, including B. abortus, but also vaccine delivery systems that target M cells.
RESUMO
Brucella spp. is the causative agent of brucellosis, one of the worldwide diseases. The pathogen infects humans and animals mainly through the digestive or respiratory tract. Therefore, induction of mucosal immunity is required as the first line of defense. In this study, three Brucella abortus recombinant proteins, malate dehydrogenase (rMdh), outer membrane proteins (rOmp) 10 and 19 were loaded in mucoadhesive chitosan nanoparticles (CNs) and induction of mucosal and systemic immunity were investigated after intranasal immunization of BALB/c mice. These antigens were also coimmunized as cocktail (rCocktail) to evaluate multiple antigen specific vaccine candidates. At 6-weeks post-immunization (wpi), antigen specific total IgG was increased in all of the immunized groups, predominantly IgG1. In addition, spleenocyte from rMdh-, rOmp19-, and rCocktail-immunized groups significantly produced IFN-γ and IL-4 suggesting the induction of a mixed Th1-Th2 response. For mucosal immunity, anti-Mdh IgA from nasal washes and fecal excretions, and anti-Omps IgA from sera, nasal washes, genital secretions and fecal excretions were significantly increased in single antigen immunized groups. In the rCocktail-immunized group, anti-Mdh IgA were significantly increased while anti-Omps IgA was not. Collectively, this study indicates that comprise of B. abortus antigen-loaded CNs elicited the antigen-specific IgA with a Th2-polarized immune responses and combination of the highly immunogenic antigens elicited IgG specific to each type of antigen.
Assuntos
Anticorpos Antibacterianos/sangue , Proteínas da Membrana Bacteriana Externa/imunologia , Vacina contra Brucelose/imunologia , Malato Desidrogenase/imunologia , Nanopartículas/administração & dosagem , Células Th1/imunologia , Células Th2/imunologia , Animais , Proteínas da Membrana Bacteriana Externa/administração & dosagem , Vacina contra Brucelose/administração & dosagem , Brucella abortus/imunologia , Brucelose/prevenção & controle , Quitosana/administração & dosagem , Citocinas/imunologia , Feminino , Imunização , Imunogenicidade da Vacina , Imunoglobulina G/sangue , Interferon gama/sangue , Malato Desidrogenase/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Proteínas Recombinantes/imunologiaRESUMO
Thermal conditions are an important environmental factor in maintaining healthy pigs because they affect feed intake, growth efficiency, reproduction and immune responses in pigs. RAVI, a regenerative far-infrared heating system, can effect pig production by emitting an optimal far-infrared wavelength. Far-infrared radiation has been reported to increase microvascular dilation and vascular flow volume. The purpose of this study was to evaluate the immunobiological differences between pigs raised with the RAVI system and the gasoline heater system. Twenty-six-week-old weaned pigs were raised in two rooms that were equipped with a RAVI system or a gasoline heater for 8 weeks. A porcine atrophic rhinitis vaccine was administered after two weeks and transcriptome analysis in whole blood were analyzed at 2-week intervals. Signaling pathway analyses of the RAVI group at 8 weeks showed the activation of pathways related to nitric oxide (NO) production. This suggests that the application of RAVI might induce the production of NO and iNOS, which are important for increasing the immune activity. Similar to the result of microarray, phenotypic changes were also observed at a later period of the experiment. The increase in body weight in the RAVI group was significantly higher than the gasoline heater group at 8 weeks. The antibody titer against the vaccine in the RAVI group was also higher than that the gasoline heater group at 4 weeks and 8 weeks. This evaluation of the use of a far-infrared heating system with pigs will be helpful for applications in the pig farm industry and pig welfare.
Assuntos
Criação de Animais Domésticos/métodos , Calefação , Rinite Atrófica/veterinária , Sus scrofa/imunologia , Animais , Perfilação da Expressão Gênica , Abrigo para Animais , Distribuição Aleatória , Rinite Atrófica/imunologia , Vacinas/administração & dosagemRESUMO
Among various vaccines against Actinobacillus pleuropneumoniae, subunit vaccines using recombinant proteins of ApxI, ApxII, and ApxIII as vaccine antigens have shown good efficacy in terms of safety and protection. Therefore, subunit vaccines are being applied worldwide and the development of new subunit vaccines is actively being conducted. To evaluate the efficacy of the subunit vaccines, it is important to measure immune responses to each Apx toxin separately. However, the cross-reactivity of antibodies makes it difficult to measure specific immune reactivity to each toxin. In the present study, specific antigen regions among the toxins were identified and cloned to solve this problem. The antigenicity of each recombinant protein was demonstrated by Western blot. Using the recombinant proteins, we developed enzyme-linked immunosorbent assay (ELISA) methods that can detect specific immune responses to each Apx toxin in laboratory guinea pigs. We suggest that the ELISA method developed in this study can be an important tool in the evaluation of vaccine efficiency and vaccine development.
Assuntos
Infecções por Actinobacillus/prevenção & controle , Actinobacillus pleuropneumoniae/imunologia , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas Hemolisinas/imunologia , Infecções por Actinobacillus/imunologia , Infecções por Actinobacillus/microbiologia , Actinobacillus pleuropneumoniae/genética , Animais , Proteínas de Bactérias/genética , Western Blotting , Clonagem Molecular , Reações Cruzadas/imunologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática/veterinária , Cobaias , Proteínas Hemolisinas/genética , Alinhamento de Sequência , Vacinas SintéticasRESUMO
The aim of this study was to investigate the induction of mucosal immune responses by an important Brucella abortus antigen, malate dehydrogenase (Mdh), loaded in mucoadhesive chitosan nanoparticles (CNs) and immunized intranasally in a BALB/c mouse model. The production of cytokines was investigated in human leukemic monocyte cells (THP-1 cells) after stimulation with the nanoparticles. Mdh-loaded CNs (CNs-Mdh) induced higher interleukin (IL)-6 production than unloaded antigens and TF loaded CNs (CNs-TF). Using ELISpot to quantify cytokines and antibody-secreting cells in the intranasally immunized mice, IL-4 and IgG-secreting cells were found to be significantly increased at 4â¯weeks and 6â¯weeks post-immunization in the CNs-Mdh immunized group, respectively. Increases in Mdh-specific IgG, IgG1, and IgG2a antibodies were confirmed at 6â¯weeks after immunization, indicating a predominant IgG1 response. Analysis of the mucosal immune response in the intranasally immunized mice revealed, Mdh-specific IgA and total IgA in the nasal washes, genital secretions, fecal extracts and sera that were remarkably increased in the CNs-Mdh-immunized group compared to the CNs-TF-immunized group except total IgA of nasal wash. Therefore, the results indicated that the intranasal immunization of CNs-loaded B. abortus Mdh antigen effectively induced antigen-specific mucosal immune responses through the elicitation of Th2-related immune responses.
Assuntos
Brucella abortus/imunologia , Brucelose/prevenção & controle , Quitosana , Imunoglobulina A/imunologia , Malato Desidrogenase/imunologia , Nanopartículas , Células Th2/imunologia , Administração Intranasal , Animais , Vacina contra Brucelose/administração & dosagem , Vacina contra Brucelose/imunologia , Brucella abortus/enzimologia , Linhagem Celular , Quitosana/química , Citocinas/biossíntese , ELISPOT , Humanos , Imunidade nas Mucosas , Imunização , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Malato Desidrogenase/administração & dosagem , Malato Desidrogenase/química , Camundongos , Nanopartículas/química , Proteínas Recombinantes , Células Th2/metabolismoRESUMO
Brucella abortus can survive and replicate within host macrophages, and great efforts have been made to demonstrate the genes involved in pathogenicity, such as internalization, in Brucella research. Here, intracellular responses were compared between THP-1 macrophage cells stimulated with B. abortus wild-type and four mutants (C1, C10, C27, and C32) using microarray to demonstrate the role of genes related to intracellular survival and replication. These mutants were generated by deleting genes encoding BAB_RS13225 (4-hydrobenzoate 3-monooxygenase, PHBH), BAB_RS00455 (heme exporter protein cytochrome C, CcmC), BAB_RS03675 (exopolyphosphatase, PPX), and BAB_RS13225 (peptidase M24). The results showed that mutants C1 and C10 induced significant suppression of survival levels and cytokine expression relative to wild-type in the THP-1 macrophage cells. These findings suggest that the BAB_RS13225 and BAB_RS00455 genes play important roles in survival within human macrophages. Conversely, mutants C27 and C32 induced significantly higher survival level than wild-type in the cells inhibiting cellular signal transduction. It is assumed that the BAB_RS03675 and BAB_RS13225 genes play a role in cellular resistance to B. abortus. Therefore, the disrupted genes are involved in B. abortus intracellular growth, and especially in its survival, and they could be effective targets for understanding the intracellular bacterium, B. abortus.
Assuntos
Brucella abortus/fisiologia , Interações entre Hospedeiro e Microrganismos , Macrófagos/microbiologia , Brucella abortus/genética , Brucella abortus/crescimento & desenvolvimento , Citocinas/análise , Citoplasma/microbiologia , Deleção de Genes , Regulação da Expressão Gênica , Redes Reguladoras de Genes/genética , Genes Bacterianos/genética , Humanos , Análise em Microsséries , Viabilidade Microbiana , Mutagênese Insercional , Células THP-1RESUMO
BACKGROUND: Since recognizing the interaction between Brucella and host cells is crucial to the elucidation of the infectious process, Brucella researches have prioritized the investigation of genes related to pathogenicity. To demonstrate the roles of Brucella genes, RAW 264.7 cells were infected with the Brucella abortus wild-type and mutant strains (generated using transposon mutagenesis), after which the different transcriptional responses of the infected cells were determined using microarray. RESULTS: Following infection, enhanced strategies for intracellular survival, such as down-regulation of genes associated with cytokine responses and apoptosis, were observed in RAW 264.7 cells infected with C3 mutant strain when compared to the transcriptional responses of wild-type infected cells. Using sequence analysis, we determined the mutation site of a C3 mutant strain as the ATP-binding cassette transporter permease (BruAb2_1031). These results were evidenced by an increased level of intracellular survival of the C3 mutant strain. CONCLUSIONS: Characteristics of each mutant strain including bacterial growth rate, abilities to induce cytokine production in macrophages after infection, internalization, and levels of intracellular survival and replication, were investigated by performing RAW 264.7 cell infection experiments. Our results indicate that the BruAb2_1031 gene might be closely related with intracellular survival of B. abortus in RAW 264.7 cells.
Assuntos
Proteínas de Bactérias/genética , Brucella abortus/genética , Brucelose/microbiologia , Citoplasma/microbiologia , Mutação , Fagócitos/microbiologia , Transcriptoma , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Apoptose , Brucella abortus/crescimento & desenvolvimento , Citocinas/metabolismo , Elementos de DNA Transponíveis , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas de Membrana Transportadoras/genética , Camundongos , Mutagênese , Células RAW 264.7/microbiologia , Análise de SequênciaRESUMO
A zoonotic pathogen, Brucella spp. is the causative agent of brucellosis, which results in abortion and loss in milk production in domestic animals, and undulant fever, osteoarticular pain and splenomegaly in humans. Due to the capability of the bacteria to modulate the host cell functions and survive in macrophages, early detection and eradication of the intracellular bacteria has received significant attention. Moreover, understanding the immunological alterations in Brucella infection is crucial to help develop control measures. Cytokines and toll-like receptors (TLRs) are some of the major compounds that play important roles in modulating the innate immunity and acquired immunity in host after infection. In this study, therefore, human leukemic monocyte cells (THP-1â¯cells) were stimulated with five Brucella abortus cellular components: outer membrane protein 10 (OMP10), outer membrane protein 19 (OMP19), thiamine transporter substrate-binding protein (TbpA), arginase (RocF), and malate dehydrogenase (Mdh). Post stimulation, the cytokine productions and TLR expressions in the cells were evaluated at different time points (12â¯h and 24â¯h), and analyzed using ELISA and real time RT-PCR, respectively. In the production of cytokines, it was observed that the production of TNF-α and IL-6 was highly induced in THP-1â¯cells stimulated with five recombinant protein antigens. Also, TLR8 was induced in a time-dependent manner after stimulation with two recombinant proteins, rOMP19 and rMdh, until 24â¯h. These results suggest that the two B. abortus antigens, rOMP19 and rMdh, might be involved in TLR8 signaling pathway in THP-1â¯cells in a time-dependent manner. These two proteins are therefore potentially effective antigen candidates which would help to provide better understandings of the immune responses after Brucella infection.
Assuntos
Antígenos de Bactérias/imunologia , Brucella abortus/imunologia , Interleucina-6/metabolismo , Monócitos/imunologia , Receptor 8 Toll-Like/biossíntese , Fator de Necrose Tumoral alfa/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Células THP-1RESUMO
Brucella abortus is a bacterium that causes brucellosis and is the causative agent of worldwide zoonoses. Pathogenesis of the B. abortus infection is complicated, and several researchers have attempted to elucidate the infection mechanism of B. abortus. While several proteins have been revealed as pathogenic factors by previous researchers, the underlying mechanism of B. abortus infection is unresolved. In this study, we identified proteins showing different expression levels in B. abortus mutants with different biological characteristics that were generated by random insertion of a transposon. Five mutants were selected based on biological characteristics, in particular, their growth features. Total proteins of mutant and wild-type B. abortus were purified and subjected to two-dimensional gel electrophoresis. Thirty protein spots of each mutant with expression increases or decreases were selected; those with a change of more than 2-fold were compared with the wild-type. Selected spots underwent liquid chromatography tandem mass spectrometry for peptide analysis. DnaK and ClpB, involved in protein aggregation, increased. SecA and GAPDH, associated with energy metabolism, decreased in some mutants with a growth rate slower than that of the wild-type. Mutants with slower growth showed a decrease in energy metabolism-related proteins, while mutants with faster growth showed an increase in pathogenicity-related proteins.
Assuntos
Brucella abortus/metabolismo , Proteínas de Bactérias/biossíntese , Brucella abortus/genética , Brucella abortus/crescimento & desenvolvimento , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Mutagênese Insercional , Espectrometria de Massas em TandemRESUMO
Brucellosis is an important zoonotic disease caused by Brucella species. The disease is difficult to control due to the intracellular survival of the bacterium and the lack of precise understanding of pathogenesis. Despite of continuous researches on the pathogenesis of Brucella spp. infection, there is still question on the pathogenesis, especially earlier immune response in the bacterial infection. Malate dehydrogenase (MDH), elongation factor (Tsf), and arginase (RocF), which showed serological reactivity, were purified after gene cloning, and their immune modulating activities were then analyzed in a murine model. Cytokine production profiles were investigated by stimulating RAW 264.7 cells and naïve splenocytes with the three recombinant proteins. Also, immune responses were analyzed by ELISA and an ELIspot assay after immunizing mice with the three proteins. Only TNF-α was produced in stimulated RAW 264.7 cells, whereas Th1-related cytokines, IFN-γ and IL-2, were induced in naïve splenocytes. In contrast, Th2-type immune response was more strongly induced in antigen-secreting cells in the splenocytes obtained 28 days after immunizing mice with the three proteins, as were IgM and IgG. The induction of Th2-related antibody, IgG1, was higher than the Th1-related antibody, IgG2a, in immunized mice. These results suggest that the three proteins strongly induce Th2-type immune response in vivo, even though Th1-related cytokines were produced in vitro.
Assuntos
Antígenos de Bactérias/imunologia , Arginase/imunologia , Brucella abortus/imunologia , Brucelose/imunologia , Imunidade Celular , Malato Desidrogenase/imunologia , Fatores de Alongamento de Peptídeos/imunologia , Células Th2/microbiologia , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Arginase/genética , Linfócitos B , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Brucella abortus/química , Brucella abortus/genética , Brucelose/microbiologia , Citocinas/imunologia , Citocinas/metabolismo , DNA Bacteriano , Ensaio de Imunoadsorção Enzimática , Feminino , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Vetores Genéticos , Imunização , Imunoglobulina G , Imunoglobulina M , Interferon gama/metabolismo , Interleucina-2/metabolismo , Malato Desidrogenase/genética , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico/metabolismo , Fatores de Alongamento de Peptídeos/genética , Células RAW 264.7/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Células Th1/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The oscillational motion of bacteria and non-biological particles on a positively polarized substrate surface were investigated in this study using several bacterial species (Staphylococcus epidermidis ATCC12228 and Pseudomonas aeruginosa PA14) and polystyrene particles (modified with sulfate or carboxylate) that have different cell/particle size, surface potential, surface ionizable functional group, and surface appendage with respect to the mean square displacement (MSD) and motion trajectory. The attractive/repulsive interactions between the bacteria/particle and a positively polarized substrate surface are further discussed with the results of the motion analysis based on the extended Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. As our major findings, all the bacterial species and particles showed oscillational motion, a kind of sub-diffusive motion that is more limited than the Brownian motion of the suspended bacteria/particles, on a positively polarized substrate surface. However, the motion properties among the bacteria/particles were found to differ in motion radius and MSD. As the size and negative surface potential of the bacteria/particle got smaller, the oscillational motion became more active, which may result from a decrease in attractive interactions such as van der Waals interaction and electrostatic attractive interaction. In the case in which some surface functional group (e.g., sulfate group) contributed to the formation of a strong Lewis acid-base interaction, the oscillational motion was significantly reduced regardless of the surface potential of the particle. The bacterial surface appendages were found to have no influence in explaining motion differences between the bacteria and non-biological particle.