RESUMEN
BACKGROUND: Deinococcus radiodurans is a robust bacterium that can withstand harsh environments that cause oxidative stress to macromolecules due to its cellular structure and physiological functions. Cells release extracellular vesicles for intercellular communication and the transfer of biological information; their payload reflects the status of the source cells. Yet, the biological role and mechanism of Deinococcus radiodurans-derived extracellular vesicles remain unclear. AIM: This study investigated the protective effects of membrane vesicles derived from D. radiodurans (R1-MVs) against H2O2-induced oxidative stress in HaCaT cells. RESULTS: R1-MVs were identified as 322 nm spherical molecules. Pretreatment with R1-MVs inhibited H2O2-mediated apoptosis in HaCaT cells by suppressing the loss of mitochondrial membrane potential and reactive oxygen species (ROS) production. R1-MVs increased the superoxide dismutase (SOD) and catalase (CAT) activities, restored glutathione (GSH) homeostasis, and reduced malondialdehyde (MDA) production in H2O2-exposed HaCaT cells. Moreover, the protective effect of R1-MVs against H2O2-induced oxidative stress in HaCaT cells was dependent on the downregulation of mitogen-activated protein kinase (MAPK) phosphorylation and the upregulation of the nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway. Furthermore, the weaker protective capabilities of R1-MVs derived from ΔDR2577 mutant than that of the wild-type R1-MVs confirmed our inferences and indicated that SlpA protein plays a crucial role in R1-MVs against H2O2-induced oxidative stress. CONCLUSION: Taken together, R1-MVs exert significant protective effects against H2O2-induced oxidative stress in keratinocytes and have the potential to be applied in radiation-induced oxidative stress models.
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Foot-and-mouth disease virus (FMDV) is highly infectious and causes a major plague in animal farming. Unfolded protein response is one of the major cellular responses to pathogenic infections, which performs a crucial role in cell survival, apoptosis, and antiviral innate immune response. In this study, we showed that FMDV infection activated two unfolded protein response branches (PERK-eIF2α and ATF6 signaling) in both baby hamster kidney cells (BHK-21) and porcine kidney (PK-15) cells, whereas it suppressed the IRE1α-XBP1 signaling by decreasing IRE1α level. Further study revealed IRE1α signaling as an important antiviral innate immune mechanism against FMDV. Sec62, the transport protein, was greatly decreased at the late stages of FMDV infection. By overexpression and knockdown study, we also found that the expression of Sec62 was positively involved in the levels of IRE1α and RIG-I and subsequent activation of downstream antiviral signaling pathways in FMDV-infected PK-15 cells. Taken together, our study demonstrates that Sec62 is an important antiviral factor that upregulates IRE1α-RIG-I-dependent antiviral innate immune responses, and FMDV evades antiviral host defense mechanism by downregulating Sec62-IRE1α/RIG-I.
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Antivirales/inmunología , Proliferación Celular/fisiología , Virus de la Fiebre Aftosa/inmunología , Transducción de Señal/inmunología , Proteínas Virales/inmunología , Replicación Viral/inmunología , Animales , Línea Celular , Cricetinae , Endorribonucleasas , Inmunidad Innata/inmunología , Proteínas de Transporte de Membrana/inmunología , Proteínas Serina-Treonina Quinasas/inmunología , Receptores de Superficie Celular/inmunología , Porcinos , Respuesta de Proteína Desplegada/inmunologíaRESUMEN
Extracellular vesicles (EVs) have recently been isolated from different plants. Plant-derived EVs have been proposed as potent therapeutics and drug-delivery nanoplatforms for delivering biomolecules, including proteins, RNAs, DNAs, and lipids. Herein, Petasites japonicus-derived EVs (PJ-EVs) were isolated through a series of centrifugation steps and characterized using dynamic light scattering and transmission electron microscopy. Immunomodulatory effects of PJ-EVs were assessed using dendritic cells (DCs). PJ-EVs exhibited a spherical morphology with an average size of 122.6 nm. They induced the maturation of DCs via an increase in the expression of surface molecules (CD80, CD86, MHC-I, and MHC-II), production of Th1-polarizing cytokines (TNF-α and IL-12p70), and antigen-presenting ability; however, they reduced the antigen-uptake ability. Furthermore, maturation of DCs induced by PJ-EVs was dependent on the activation and phosphorylation of MAPK and NF-κB signal pathways. Notably, PJ-EV-treated DCs strongly induced the proliferation and differentiation of naïve T cells toward Th1-type T cells and cytotoxic CD8+ T cells along with robust secretion of IFN-γ and IL-2. In conclusion, our study indicates that PJ-EVs can be potent immunostimulatory candidates with an ability of strongly inducing the maturation of DCs.
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Adyuvantes Inmunológicos/farmacología , Diferenciación Celular/efectos de los fármacos , Células Dendríticas/inmunología , Vesículas Extracelulares/inmunología , Petasites/citología , Plantas Comestibles/citología , Animales , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Femenino , Activación de Linfocitos/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Células TH1/inmunologíaRESUMEN
Although cisplatin is one of most effective chemotherapeutic drugs that is widely used to treat various types of cancer, it can cause undesirable damage in immune cells and normal tissue because of its strong cytotoxicity and non-selectivity. This study was conducted to investigate the cytoprotective effects of Cudrania tricuspidata fruit-derived polysaccharides (CTPS) against cisplatin-induced cytotoxicity in macrophages, lung cancer cell lines, and a mouse model, and to explore the possibility of application of CTPS as a supplement for anticancer therapy. Both cisplatin alone and cisplatin with CTPS induced a significant cytotoxicity in A549 and H460 lung cancer cells, whereas cytotoxicity was suppressed by CTPS in cisplatin-treated RAW264.7 cells. CTPS significantly attenuated the apoptotic and necrotic population, as well as cell penetration in cisplatin-treated RAW264.7 cells, which ultimately inhibited the upregulation of Bcl-2-associated X protein (Bax), cytosolic cytochrome c, poly (adenosine diphosphateribose) polymerase (PARP) cleavage, and caspases-3, -8, and -9, and the downregulation of B cell lymphoma-2 (Bcl-2). The CTPS-induced cytoprotective action was mediated with a reduction in reactive oxygen species production and mitochondrial transmembrane potential loss in cisplatin-treated RAW264.7 cells. In agreement with the results obtained above, CTPS induced the attenuation of cell damage in cisplatin-treated bone marrow-derived macrophages (primary cells). In in vivo studies, CTPS significantly inhibited metastatic colonies and bodyweight loss as well as immunotoxicity in splenic T cells compared to the cisplatin-treated group in lung metastasis-induced mice. Furthermore, CTPS decreased the level of CRE and BUN in serum. In summation, these results suggest that CTPS-induced cytoprotective action may play a role in alleviating the side effects induced by chemotherapeutic drugs.
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Cisplatino/toxicidad , Frutas/química , Macrófagos/efectos de los fármacos , Melanoma Experimental/tratamiento farmacológico , Moraceae/química , Extractos Vegetales/farmacología , Polisacáridos/farmacología , Animales , Antineoplásicos/toxicidad , Apoptosis , Proliferación Celular , Femenino , Humanos , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Macrófagos/patología , Melanoma Experimental/inducido químicamente , Melanoma Experimental/patología , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Sustancias Protectoras/farmacología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Bombyx batryticatus, a protein-rich edible insect, is widely used as a traditional medicine in China. Several pharmacological studies have reported the anticancer activity of B. batryticatus extracts; however, the capacity of B. batryticatus extracts as immune potentiators for increasing the efficacy of cancer immunotherapy is still unverified. In the present study, we investigated the immunomodulatory role of B. batryticatus protein-rich extract (BBPE) in bone marrow-derived dendritic cells (BMDCs) and DC vaccine-immunized mice. BBPE-treated BMDCs displayed characteristics of mature immune status, including high expression of surface molecules (CD80, CD86, major histocompatibility complex (MHC)-I, and MHC-II), increased production of proinflammatory cytokines (tumor necrosis factor-α and interleukin-12p70), enhanced antigen-presenting ability, and reduced endocytosis. BBPE-treated BMDCs promoted naive CD4+ and CD8+ T-cell proliferation and activation. Furthermore, BBPE/ovalbumin (OVA)-pulsed DC-immunized mice showed a stronger OVA-specific multifunctional T-cell response in CD4+ and CD8+ T cells and a stronger Th1 antibody response than mice receiving differently treated DCs, which showed the enhanced protective effect against tumor growth in E.G7 tumor-bearing mice. Our data demonstrate that BBPE can be a novel immune potentiator for a DC-based vaccine in anticancer therapy.
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Adyuvantes Inmunológicos , Presentación de Antígeno/inmunología , Vacunas contra el Cáncer/inmunología , Células Dendríticas/fisiología , Proteínas de Insectos/metabolismo , Células TH1/inmunología , Extractos de Tejidos/farmacología , Animales , Bombyx , Proliferación Celular , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Femenino , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
Although our previous study revealed that gamma-irradiated chrysin enhanced anti-inflammatory activity compared to intact chrysin, it remains unclear whether the chrysin derivative, CM1, produced by gamma irradiation, negatively regulates toll-like receptor (TLR) signaling. In this study, we investigated the molecular basis for the downregulation of TLR4 signal transduction by CM1 in macrophages. We initially determined the appropriate concentration of CM1 and found no cellular toxicity below 2 µg/mL. Upon stimulation with lipopolysaccharide (LPS), CM1 modulated LPS-stimulated inflammatory action by suppressing the release of proinflammatory mediators (cytokines TNF-α and IL-6) and nitric oxide (NO) and downregulated the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. Furthermore, CM1 markedly elevated the expression of the TLR negative regulator toll-interacting protein (Tollip) in dose- and time-dependent manners. LPS-induced expression of cell surface molecules (CD80, CD86, and MHC class I/II), proinflammatory cytokines (TNF-α and IL-6), COX-2, and iNOS-mediated NO were inhibited by CM1; these effects were prevented by the knockdown of Tollip expression. Additionally, CM1 did not affect the downregulation of LPS-induced expression of MAPKs and NF-κB signaling in Tollip-downregulated cells. These findings provide insight into effective therapeutic intervention of inflammatory disease by increasing the understanding of the negative regulation of TLR signaling induced by CM1.
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Flavonoides/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/metabolismo , Animales , Antiinflamatorios/farmacología , Flavonoides/metabolismo , Flavonoides/efectos de la radiación , Inflamación/tratamiento farmacológico , Interleucina-6 , Lipopolisacáridos/efectos adversos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Óxido Nítrico , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Receptores Toll-Like/metabolismo , Factor de Necrosis Tumoral alfaRESUMEN
An aerobic, Gram-stain-negative, non-motile, non-spore-forming, rod-shaped and pink-colored bacterial strain, designated BRD72T, was isolated from a crater lake (Baengnokdam) at the top of Mt. Hallasan in the Republic of Korea. Cells were catalase-positive and oxidase-negative. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that the isolate was a member of the genus Hymenobacter and most closely related to Hymenobacter marinus KJ035T (96.2% similarity). The isolate was found to produce carotenoid pigment, but not flexirubin-type pigment. The predominant fatty acids of strain BRD72T were summed feature 3 (C16:1 ω7c and/or C16:1 ω6c, 21.6%), iso-C15:0 (17.9%), anteiso-C15:0 (13.3%) and summed feature 4 (iso-C17:1 I and/or anteiso-C17:1 B, 11.3%). The major polar lipids were phosphatidylethanolamine, an unidentified amino lipid, and two unidentified aminophospholipids. The main respiratory quinone was menaquinone-7 (MK-7), and the main polyamine was homospermidine. The DNA G+C content was 59.8 mol%. Based on the phylogenetic, physiological, and chemotaxonomic characteristics, strain BRD72T represents a novel species, for which the name Hymenobacter baengnokdamensis sp. nov. is proposed. The type strain is BRD72T (= KCTC 72649T = JCM 33837T).
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Lagos , Suelo , Técnicas de Tipificación Bacteriana , Bacteroidetes , ADN Bacteriano/genética , Ácidos Grasos/análisis , Filogenia , ARN Ribosómico 16S/genética , República de Corea , Análisis de Secuencia de ADN , Vitamina K 2RESUMEN
Deinococcus radiodurans is an extremophilic bacterium well-known for its extraordinary resistance to ionizing radiation and other DNA damage- and oxidative stress-generating agents. In addition to its efficient DNA damage repair and oxidative stress resistance mechanisms, protein family expansions and stress-induced genes/proteins are also regarded as important components that add to the robustness of this bacterium. D. radiodurans encodes specific expansions of 13 DinB/YfiT homologs, which is a relatively large number when compared to those found in Gram-positive bacteria. In this study, we investigated the expression profiles of 13 dinB genes after γ-irradiation, mitomycin C and H2O2 treatment. dr0053 had the highest expression levels after DNA-damage inducing γ-irradiation and MMC treatment, increasing â¼200-fold and â¼16-fold, respectively. We also determined the crystal structure of DR0053 at 2.07â¯Å resolution. DR0053 adopted a typical four-helix bundle structure that is characteristic of DinB/YfiT proteins. A putative metal binding site was occupied by zinc even though the highly conserved His triad of DinB/YfiT proteins was replaced by Glu-Asn-His.
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Proteínas Bacterianas/química , Deinococcus/química , Alquilantes/farmacología , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Deinococcus/efectos de los fármacos , Deinococcus/genética , Deinococcus/efectos de la radiación , Rayos gamma , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de la radiación , Mitomicina/farmacología , Modelos Moleculares , Conformación ProteicaRESUMEN
We recently identified a previously unidentified sphingosine-1-phosphate (S1P) signaling mechanism that stimulates production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), in mammalian cells exposed to external perturbations, such as UVB irradiation and other oxidative stressors that provoke subapoptotic levels of endoplasmic reticulum (ER) stress, independent of the well-known vitamin D receptor-dependent mechanism. ER stress increases cellular ceramide and one of its distal metabolites, S1P, which activates NF-κB followed by C/EBPα activation, leading to CAMP production, but in a S1P receptor-independent fashion. We now show that S1P activates NF-κB through formation of a previously unidentified signaling complex, consisting of S1P, TRAF2, and RIP1 that further associates with three stress-responsive proteins; i.e., heat shock proteins (GRP94 and HSP90α) and IRE1α. S1P specifically interacts with the N-terminal domain of heat shock proteins. Because this ER stress-initiated mechanism is operative in both epithelial cells and macrophages, it appears to be a universal, highly conserved response, broadly protective against diverse external perturbations that lead to increased ER stress. Finally, these studies further illuminate how ER stress and S1P orchestrate critical stress-specific signals that regulate production of one protective response by stimulating production of the key innate immune element, CAMP.
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Péptidos Catiónicos Antimicrobianos/metabolismo , Estrés del Retículo Endoplásmico , Lisofosfolípidos/metabolismo , Transducción de Señal , Esfingosina/análogos & derivados , Animales , Péptidos Catiónicos Antimicrobianos/genética , Western Blotting , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Proteínas de Choque Térmico/metabolismo , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones Noqueados , Microscopía Fluorescente , FN-kappa B/metabolismo , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Unión Proteica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esfingosina/metabolismo , Factor 2 Asociado a Receptor de TNF/genética , Factor 2 Asociado a Receptor de TNF/metabolismo , CatelicidinasRESUMEN
Streptococcus pneumoniae is a major respiratory pathogen that can cause pneumonia, meningitis, and otitis media. Although capsular polysaccharide-based vaccines are commercially available, there is a need for broad-spectrum, serotype-independent, and cost-effective vaccines. Recently, an intranasal vaccine formulated with gamma-irradiated nonencapsulated S. pneumoniae whole cells has been developed and its immunogenicity is under investigation. Since innate immunity influences the subsequent adaptive immunity, in the present study, we investigated the immunostimulatory activity of gamma-irradiated S. pneumoniae (r-SP) in the human bronchial epithelial cell-line, BEAS-2B, by comparing with heat-inactivated S. pneumoniae (h-SP) and formalin-inactivated S. pneumoniae (f-SP). r-SP potently induced interleukin (IL)-6 and IL-8 at both mRNA and protein levels in a dose- and time-dependent manner, whereas h-SP and f-SP poorly induced them. Of note, the mRNA levels of IL-6 and IL-8 were approximately two-fold higher when cells were stimulated with 3â¯×â¯107â¯CFU/ml of r-SP for 3â¯h, while the protein levels of IL-6 and IL-8 were approximately five-fold higher after stimulation with 3â¯×â¯107â¯CFU/ml of r-SP for 24â¯h. Furthermore, r-SP exhibited potent activation of Toll-like receptor 2 compared with h-SP or f-SP. The expression of IL-6 and IL-8 induced by r-SP was mediated through the activation of mitogen-activated protein kinases. Remarkably, when r-SP was further treated with heat or formalin, there was a decrease in the aforementioned activities. Taken together, we suggest that r-SP stimulates the human respiratory epithelial cells to produce the cytokines IL-6 and IL-8, which might influence the induction of adaptive immune responses.
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Células Epiteliales/inmunología , Células Epiteliales/microbiología , Rayos gamma , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Streptococcus pneumoniae/inmunología , Streptococcus pneumoniae/efectos de la radiación , Vacunas Bacterianas/inmunología , Línea Celular , Formaldehído , Perfilación de la Expresión Génica , Calor , Humanos , Streptococcus pneumoniae/efectos de los fármacos , Vacunas de Productos Inactivados/inmunologíaRESUMEN
Streptococcus agalactiae (group B streptococcus [GBS]) is a leading cause of invasive diseases in neonates and severe infections in elderly individuals. GBS serine-rich repeat glycoprotein 1 (Srr1) acts as a critical virulence factor by facilitating GBS invasion into the central nervous system through interaction with the fibrinogen Aα chain. This study revealed that srr1 is highly conserved, with 86.7% of GBS clinical isolates expressing the protein. Vaccination of mice with different Srr1 truncated peptides revealed that only Srr1 truncates containing the latch domain protected against GBS meningitis. Furthermore, the latch peptide alone was immunogenic and elicited protective antibodies, which efficiently enhanced antibody-mediated opsonophagocytic killing of GBS by HL60 cells and provided heterogeneous protection against 4 different GBS serogroups. Taken together, these findings indicated that the latch domain of Srr1 may constitute an effective peptide vaccine candidate for GBS.
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Protección Cruzada , Inmunidad Heteróloga , Meningitis Bacterianas/prevención & control , Infecciones Estreptocócicas/prevención & control , Vacunas Estreptocócicas/inmunología , Streptococcus agalactiae/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Actividad Bactericida de la Sangre , Modelos Animales de Enfermedad , Masculino , Meningitis Bacterianas/inmunología , Meningitis Bacterianas/microbiología , Ratones , Proteínas Opsoninas/sangre , Fagocitosis , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , Vacunas Estreptocócicas/administración & dosificación , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
Group B streptococcus (GBS) infection is a leading cause of sepsis and meningitis among infants, and is associated with high rates of morbidity and mortality in many countries. Protection against GBS typically involves antibody-mediated opsonization by phagocytes and complement components. The present study evaluated serotype-specific functional antibodies to GBS among Korean infants and in intravenous immunoglobulin (IVIG) products. An opsonophagocytic killing assay (OPA) was used to calculate the opsonization indices (OIs) of functional antibodies to serotypes Ia, Ib, and III in 19 IVIG products from 5 international manufacturers and among 98 Korean infants (age: 0-11 months). The GBS Ia, Ib, and III serotypes were selected because they are included in a trivalent GBS vaccine formulation that is being developed. The OI values for the IVIG products were 635-5,706 (serotype Ia), 488-1,421 (serotype Ib), and 962-3,315 (serotype III), and none of the IVIG lots exhibited undetectable OI values (< 4). The geometric mean OI values were similar for all 3 serotypes when we compared the Korean manufacturers. The seropositive rate among infants was significantly lower for serotype Ia (18.4%), compared to serotype Ib and serotype III (both, 38.8%). Infant age of ≥ 3 months was positively correlated with the seropositive rates for each serotype. Therefore, only a limited proportion of infants exhibited protective immunity against serotype Ia, Ib, and III GBS infections. IVIG products that exhibit high antibody titers may be a useful therapeutic or preventive measure for infants. Further studies are needed to evaluate additional serotypes and age groups.
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Inmunoglobulinas Intravenosas/uso terapéutico , Infecciones Estreptocócicas/tratamiento farmacológico , Anticuerpos Antibacterianos/inmunología , Pueblo Asiatico , Humanos , Inmunoglobulinas Intravenosas/farmacología , Lactante , Recién Nacido , Proteínas Opsoninas/inmunología , Proteínas Opsoninas/metabolismo , República de Corea , Serogrupo , Infecciones Estreptocócicas/microbiología , Streptococcus agalactiae/efectos de los fármacos , Streptococcus agalactiae/aislamiento & purificación , Streptococcus agalactiae/metabolismoRESUMEN
Deinococcus radiodurans, which is extremely resistant to ionizing radiation and oxidative stress, is known to have three catalases (DR1998, DRA0146, and DRA0259). In this study, to investigate the role of each catalase, we constructed catalase mutants (Δdr1998, ΔdrA0146, and ΔdrA0259) of D. radiodurans. Of the three mutants, Δdr1998 exhibited the greatest decrease in hydrogen peroxide (H2O2) resistance and the highest increase in intracellular reactive oxygen species (ROS) levels following H2O2 treatments, whereas ΔdrA0146 showed no change in its H2O2 resistance or ROS level. Catalase activity was not attenuated in ΔdrA0146, and none of the three bands detected in an in-gel catalase activity assay disappeared in ΔdrA0146. The purified His-tagged recombinant DRA0146 did not show catalase activity. In addition, the phylogenetic analysis of the deinococcal catalases revealed that the DR1998-type catalase is common in the genus Deinococcus, but the DRA0146-type catalase was found in only 4 of 23 Deinococcus species. Taken together, these results indicate that DR1998 plays a critical role in the anti-oxidative system of D. radiodurans by detoxifying H2O2, but DRA0146 does not have catalase activity and is not involved in the resistance to H2O2 stress.
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Catalasa/metabolismo , Deinococcus/enzimología , Peróxido de Hidrógeno/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Catalasa/clasificación , Activación Enzimática , Especificidad por SustratoRESUMEN
Cellular robustness is an important trait for industrial microbes, because the microbial strains are exposed to a multitude of different stresses during industrial processes, such as fermentation. Thus, engineering robustness in an organism in order to push the strains toward maximizing yield has become a significant topic of research. We introduced the deinococcal response regulator DR1558 into Escherichia coli (strain Ec-1558), thereby conferring tolerance to hydrogen peroxide (H2O2). The reactive oxygen species (ROS) level in strain Ec-1558 was reduced due to the increased KatE catalase activity. Among four regulators of the oxidative-stress response, OxyR, RpoS, SoxS, and Fur, we found that the expression of rpoS increased in Ec-1558, and we confirmed this increase by Western blot analysis. Electrophoretic mobility shift assays showed that DR1558 bound to the rpoS promoter. Because the alternative sigma factor RpoS regulates various stress resistance-related genes, we performed stress survival analysis using an rpoS mutant strain. Ec-1558 was able to tolerate a low pH, a high temperature, and high NaCl concentrations in addition to H2O2, and the multistress tolerance phenotype disappeared in the absence of rpoS. Microarray analysis clearly showed that a variety of stress-responsive genes that are directly or indirectly controlled by RpoS were upregulated in strain Ec-1558. These findings, taken together, indicate that the multistress tolerance conferred by DR1558 is likely routed through RpoS. In the present study, we propose a novel strategy of employing an exogenous response regulator from polyextremophiles for strain improvement.
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Deinococcus/genética , Escherichia coli/fisiología , Regulación Bacteriana de la Expresión Génica , Ingeniería Genética , Peróxido de Hidrógeno/metabolismo , Estrés Oxidativo , Estrés Fisiológico , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Perfilación de la Expresión GénicaRESUMEN
Group B streptococcus (GBS) can cause severe disease in susceptible hosts, including newborns, pregnant women, and the elderly. GBS serine-rich repeat (Srr) surface glycoproteins are important adhesins/invasins in multiple host tissues, including the vagina. However, exact molecular mechanisms contributing to their importance in colonization are unknown. We have recently determined that Srr proteins contain a fibrinogen-binding region (BR) and hypothesize that Srr-mediated fibrinogen binding may contribute to GBS cervicovaginal colonization. In this study, we observed that fibrinogen enhanced wild-type GBS attachment to cervical and vaginal epithelium, and that this was dependent on Srr1. Moreover, purified Srr1-BR peptide bound directly to host cells, and peptide administration in vivo reduced GBS recovery from the vaginal tract. Furthermore, a GBS mutant strain lacking only the Srr1 "latching" domain exhibited decreased adherence in vitro and decreased persistence in a mouse model of GBS vaginal colonization, suggesting the importance of Srr-fibrinogen interactions in the female reproductive tract.
Asunto(s)
Adhesinas Bacterianas/fisiología , Fibrinógeno/metabolismo , Glicoproteínas de Membrana/fisiología , Infecciones Estreptocócicas/microbiología , Streptococcus agalactiae/patogenicidad , Vagina/microbiología , Animales , Adhesión Bacteriana , Línea Celular , Modelos Animales de Enfermedad , Epitelio/microbiología , Femenino , Ratones , Serina , Streptococcus agalactiae/fisiología , Enfermedades Vaginales/microbiologíaRESUMEN
The serine-rich repeat glycoproteins of Gram-positive bacteria comprise a large family of cell wall proteins. Streptococcus agalactiae (group B streptococcus, GBS) expresses either Srr1 or Srr2 on its surface, depending on the strain. Srr1 has recently been shown to bind fibrinogen, and this interaction contributes to the pathogenesis of GBS meningitis. Although strains expressing Srr2 appear to be hypervirulent, no ligand for this adhesin has been described. We now demonstrate that Srr2 also binds human fibrinogen and that this interaction promotes GBS attachment to endothelial cells. Recombinant Srr1 and Srr2 bound fibrinogen in vitro, with affinities of KD = 2.1 × 10(-5) and 3.7 × 10(-6) M, respectively, as measured by surface plasmon resonance spectroscopy. The binding site for Srr1 and Srr2 was localized to tandem repeats 6-8 of the fibrinogen Aα chain. The structures of both the Srr1 and Srr2 binding regions were determined and, in combination with mutagenesis studies, suggest that both Srr1 and Srr2 interact with a segment of these repeats via a "dock, lock, and latch" mechanism. Moreover, properties of the latch region may account for the increased affinity between Srr2 and fibrinogen. Together, these studies identify how greater affinity of Srr2 for fibrinogen may contribute to the increased virulence associated with Srr2-expressing strains.
Asunto(s)
Proteínas Bacterianas/metabolismo , Fibrinógeno/metabolismo , Glicoproteínas/metabolismo , Streptococcus agalactiae/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Modelos Moleculares , Mutagénesis , Unión Proteica , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
The serine-rich repeat glycoprotein Srr1 of Streptococcus agalactiae (GBS) is thought to be an important adhesin for the pathogenesis of meningitis. Although expression of Srr1 is associated with increased binding to human brain microvascular endothelial cells (hBMEC), the molecular basis for this interaction is not well defined. We now demonstrate that Srr1 contributes to GBS attachment to hBMEC via the direct interaction of its binding region (BR) with human fibrinogen. When assessed by Far Western blotting, Srr1 was the only protein in GBS extracts that bound fibrinogen. Studies using recombinant Srr1-BR and purified fibrinogen in vitro confirmed a direct protein-protein interaction. Srr1-BR binding was localized to amino acids 283-410 of the fibrinogen Aα chain. Structural predictions indicated that the conformation of Srr1-BR is likely to resemble that of SdrG and other related staphylococcal proteins that bind to fibrinogen through a "dock, lock, and latch" mechanism (DLL). Deletion of the predicted latch domain of Srr1-BR abolished the interaction of the BR with fibrinogen. In addition, a mutant GBS strain lacking the latch domain exhibited reduced binding to hBMEC, and was significantly attenuated in an in vivo model of meningitis. These results indicate that Srr1 can bind fibrinogen directly likely through a DLL mechanism, which has not been described for other streptococcal adhesins. This interaction was important for the pathogenesis of GBS central nervous system invasion and subsequent disease progression.
Asunto(s)
Proteínas Bacterianas/metabolismo , Encéfalo/metabolismo , Endotelio/metabolismo , Fibrinógeno/metabolismo , Glicoproteínas/metabolismo , Meningitis Bacterianas/microbiología , Infecciones Estreptocócicas/microbiología , Streptococcus agalactiae/metabolismo , Streptococcus agalactiae/patogenicidad , Adhesinas Bacterianas/metabolismo , Animales , Adhesión Bacteriana , Sitios de Unión , Encéfalo/microbiología , Células Endoteliales/metabolismo , Células Endoteliales/microbiología , Endotelio/microbiología , Humanos , Meningitis Bacterianas/metabolismo , Ratones , Unión Proteica , Conformación Proteica , Análisis de Secuencia de Proteína , Infecciones Estreptocócicas/metabolismoRESUMEN
Streptococcus pneumoniae (pneumococcus) is an opportunistic pathogen that can cause severe infectious diseases such as pneumonia, meningitis, and otitis media. Despite the availability of antibiotics and pneumococcal vaccines against some invasive serotypes, pneumococcal infection remains a tremendous clinical challenge due to the increasing frequency of infection by antimicrobial resistant, nonencapsulated, and/or non-vaccine serotype strains. Short-chain fatty acids (SCFAs), which are produced at various mucosal sites in the body, have potent antimicrobial activity, including inhibition of pathogen growth and/or bacterial biofilm formation. In this study, we investigated the antimicrobial activity of SCFAs (acetate, propionate, and butyrate) against various serotypes pneumococci. Propionate generally inhibited the growth of S. pneumoniae serotypes included in the pneumococcal conjugate vaccine (PCV) 13, except for serotypes 3 and 7F, though butyrate and acetate showed no or low inhibition, depending on the serotypes. Of note, butyrate showed strong inhibition against serotype 3, the most prevalent invasive strain since the introduction of the PCV. No SCFAs showed inhibitory effects against serotype 7F. Remarkably, the nonencapsulated pneumococcal strain had more sensitivity to SCFAs than encapsulated parental strains. Taken together, these results suggest that propionate showing the most potent inhibition of pneumococcal growth may be used as an alternative treatment for pneumococcal infection, and that butyrate could be used against serotype 3, which is becoming a serious threat.
Asunto(s)
Infecciones Neumocócicas , Streptococcus pneumoniae , Humanos , Lactante , Serogrupo , Propionatos/farmacología , Infecciones Neumocócicas/microbiología , Infecciones Neumocócicas/prevención & control , Antibacterianos/farmacología , Vacunas Neumococicas/farmacología , Ácidos Grasos Volátiles , Butiratos/farmacología , Vacunas Conjugadas , Acetatos/farmacología , SerotipificaciónRESUMEN
Recently, respiratory syncytial virus (RSV) vaccines based on the prefusion F (pre-F) antigen were approved in the United States. We aimed to develop an enzyme-linked immunosorbent assay (ELISA)-based protocol for the practical and large-scale evaluation of RSV vaccines. Two modified pre-F proteins (DS-Cav1 and SC-TM) were produced by genetic recombination and replication using an adenoviral vector. The protocol was established by optimizing the concentrations of the coating antigen (pre-F proteins), secondary antibodies, and blocking buffer. To validate the protocol, we examined its accuracy, precision, and specificity using serum samples from 150 participants across various age groups and the standard serum provided by the National Institute of Health. In the linear correlation analysis, coating concentrations of 5 and 2.5 µg/mL of DS-Cav1 and SC-TM showed high coefficients of determination (r > 0.90), respectively. Concentrations of secondary antibodies (alkaline phosphatase-conjugated anti-human immunoglobulin G, diluted 1:2000) and blocking reagents (5% skim milk/PBS-T) were optimized to minimize non-specific reactions. High accuracy was observed for DS-Cav1 (r = 0.90) and SC-TM (r = 0.86). Further, both antigens showed high precision (coefficient of variation < 15%). Inhibition ELISA revealed cross-reactivity of antibodies against DS-Cav1 and SC-TM, but not with the attachment (G) protein.
Asunto(s)
Anticuerpos Antivirales , Ensayo de Inmunoadsorción Enzimática , Infecciones por Virus Sincitial Respiratorio , Vacunas contra Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/normas , Humanos , Infecciones por Virus Sincitial Respiratorio/prevención & control , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Vacunas contra Virus Sincitial Respiratorio/inmunología , Virus Sincitial Respiratorio Humano/inmunología , Lactante , Preescolar , Adulto , Niño , Adolescente , Persona de Mediana Edad , Adulto Joven , Femenino , Sensibilidad y Especificidad , Antígenos Virales/inmunología , Masculino , Proteínas Virales de Fusión/inmunología , AncianoRESUMEN
This is a cross-sectional serosurveillance study for RSV. Between June and September of 2021, a total of 150 sera were collected from 30 individuals in each age group (<5, 5-18, 19-49, 50-64, and ≥65 years). Seroprevalence was estimated using enzyme-linked immunosorbent assays targeting two stabilized prefusion F (preF; DS-Cav1 and SC-TM) and G proteins. The overall seroprevalence was low in young children and older adults, despite them having a higher risk of severe RSV infection. There was a remarkable difference in age-stratified seroprevalence rates between anti-preF and anti-G protein antibodies. Given the high disease burden and low seroprevalence in both infants and old adults, RSV vaccination would be crucial for pregnant women and people aged over 60 years.