RESUMO
Fusobacterium nucleatum (Fn) is abundant in the human oral cavity and has been associated with periodontal disease, which in-turn has been linked to respiratory disease development. Tight junctions (TJs) line the airway and alveoli surfaces serving as a first line of defense against multiple pathogens. Fn has already been linked to respiratory diseases, however, how Fn affects the alveolar TJ was not fully elucidated. Here, we designed and analyzed a TJ network, grew Fn cells and inoculated it in vitro (16HBE and primary cells) and in vivo (mice lung), measured transepithelial electrical resistance, performed RT-PCR, checked for in vitro cell and mice lung permeability, and determined air space size through morphometric measurements. We found that Fn can potentially affect TJs proteins that are directly exposed to the alveolar surface. Additionally, Fn could possibly cause neutrophil accumulation and an increase in alveolar space. Moreover, Fn putatively may cause an increase in paracellular permeability in the alveoli.
Assuntos
Células Epiteliais Alveolares , Junções Íntimas , Camundongos , Animais , Humanos , Junções Íntimas/metabolismo , Fusobacterium nucleatum , Pulmão , Permeabilidade , Células Epiteliais/metabolismoRESUMO
SARS-CoV-2 (SARS2) regularly mutates resulting to variants of concern (VOC) which have higher virulence and transmissibility rates while concurrently evading available therapeutic strategies. This highlights the importance of amino acid mutations occurring in the SARS2 spike protein structure since it may affect virus biology. However, this was never fully elucidated. Here, network analysis was performed based on the COVID-19 genomic epidemiology network between December 2019-July 2021. Representative SARS2 VOC spike protein models were generated and quality checked, protein model superimposition was done, and common contact based on contact mapping was established. Throughout this study, we found that: (1) certain individual variant-specific amino acid mutations can affect the spike protein structural pattern; (2) certain individual variant-specific amino acid mutations had no affect on the spike protein structural pattern; and (3) certain combination of variant-specific amino acids are putatively epistatic mutations that can potentially influence the VOC spike protein structural pattern. This manuscript was submitted as part of a theme issue on "Modelling COVID-19 and Preparedness for Future Pandemics".
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Mutação , Aminoácidos/genéticaRESUMO
Porphyromonas gingivalis (Pg) a major periodontal pathogen involved in periodontal disease development and progression. Moreover, Pg has two fimbriae surface proteins (FimA and Mfa1) that are genetically distinct and make-up the fimbrial shaft which in-turn form crucial attachment to oral bacteria and multiple host cells. However, unlike FimA, Mfa1 attachment to non-periodontal cells has not been fully elucidated. Considering Pg-associated periodontal disease contributes to pulmonary disease development, we investigated whether Mfa1 can functionally interact with human bronchial epithelial cells and, likewise, trigger a functional response. Initially, we simulated molecular docking and performed both luciferase and neutralization assays to confirm Mfa1-related functional interaction. Subsequently, we treated BEAS-2B cells with purified Mfa1 and performed cytokine quantification through real time-PCR and ELISA to establish Mfa1-related functional response. We found that both Mfa1-TLR2 and Mfa1-TLR4 docking is possible, however, only Mfa1-TLR2 showed a functional interaction. Additionally, we observed that both IL-8 and IL-6 gene expression and protein levels were induced confirming Mfa1-related functional response. Taken together, we propose that BEAS-2B human bronchial epithelial cells are able to recognize Pg Mfa1 and induce both IL-8 and IL-6 inflammatory responses.
Assuntos
Proteínas de Bactérias/metabolismo , Brônquios/patologia , Células Epiteliais/metabolismo , Proteínas de Fímbrias/metabolismo , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Porphyromonas gingivalis/fisiologia , Receptor 2 Toll-Like/metabolismo , Linhagem Celular , Fímbrias Bacterianas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Porphyromonas gingivalis/química , Ligação Proteica , Mapeamento de Interação de Proteínas , Receptor 4 Toll-Like/química , Receptor 4 Toll-Like/metabolismoRESUMO
Fusobacterium nucleatum (Fn) is generally an opportunistic oral pathogen that adheres to mammalian mucosal sites, triggering a host inflammatory response. In general, Fn is normally found within the human oral cavity; however, it was previously reported that Fn is a risk factor for certain respiratory diseases. Surprisingly, this was never fully elucidated. Here, we investigated the virulence potential of heat-killed Fn on primary human tracheal, bronchial, and alveolar epithelial cells. In this study, we measured the secretion of inflammatory- (IL-8 and IL-6), stress- (total heme and hydrogen peroxide), and cell death-related (caspase-1 and caspase-3) signals. We established that the inflammatory response mechanism varies in each epithelial cell type: (1) along tracheal cells, possible Fn adherence would trigger increased heme secretion and regulated inflammatory response; (2) along bronchial cells, potential Fn adherence would simultaneously initiate an increase in secreted H2O2 and inflammatory response (ascribable to decreased secreted heme amounts); and (3) along alveolar cells, putative Fn adherence would instigate the increased secretion of inflammatory responses attributable to a decrease in secreted heme levels. Moreover, regardless of the epithelial cell-specific inflammatory mechanism, we believe these are putative, not harmful. Taken together, we propose that any potential Fn-driven inflammation along the respiratory tract would be initiated by differing epithelial cell-specific inflammatory mechanisms that are collectively dependent on secreted heme.
Assuntos
Células Epiteliais Alveolares/metabolismo , Fusobacterium nucleatum/química , Heme/metabolismo , Temperatura Alta , Células Epiteliais Alveolares/patologia , Caspase 1/metabolismo , Caspase 3/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismoRESUMO
Staphylococcus aureus is associated with several clinically significant infections among humans and infections associated with antibiotic-resistant strains are growing in frequency. Antivirulence strategies shift the target of drugs from bacterial growth to the infection process resulting to milder evolutionary pressure for the development of bacterial resistant strains. Staphyloxanthin (STX) is a yellowish-orange carotenoid pigment synthesized by S. aureus and this carotenoid functions as an important virulence factor for the bacteria. In this study, we elucidated whether network analytics can be used as a viable tool to identify significant components in the STX biosynthetic network which in-turn could serve as possible antivirulence drug targets. For confirmation, we correlated our results to known drugs that were able to inhibit STX biosynthesis. Throughout this study, we established that crtN(1) activity and 4,4'-diaponeurosporene amounts are significant components in the STX biosynthetic network and, moreover, network analytics can aid in identifying antivirulence drug targets within the STX biosynthetic network. Similarly, we found that network analytics is capable of identifying multiple potential targets simultaneously. Taken together, we propose that an effective antivirulence drug against S. aureus STX biosynthesis would involve targeting crtN(1) activity, 4,4'-diaponeurosporene levels, or both components.
Assuntos
Antibacterianos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Xantofilas/biossíntese , Carotenoides/biossíntese , Carotenoides/farmacologia , Terapia de Alvo Molecular , Staphylococcus aureus/enzimologia , Staphylococcus aureus/patogenicidade , Triterpenos/farmacologia , Virulência/efeitos dos fármacosRESUMO
Neuropathic pain is absent from the early stages of periodontal disease possibly due to neurite retraction. Butyric acid (BA) is a periodontopathic metabolite that activates several stress-related signals and, likewise, induce neurite retraction. Neuronal cell death is associated to neurite retraction which would suggest that BA-induced neurite retraction is ascribable to neuronal cell death. However, the underlying mechanism of BA-related cell death signaling remains unknown. In this study, we exposed NGF-treated PC12 cells to varying BA concentrations [0 (control), 0.5, 1.0, 5.0 mM] and determined selected stress-related (H2O2, glutathione reductase, calcium (Ca(2+)), plasma membrane Ca(2+) ATPase (PMCA), and GADD153/CHOPS) and cell death-associated (extrinsic: FasL, TNF-α, TWEAK, and TRAIL; intrinsic: cytochrome C (CytC), NF-kB, CASP8, CASP9, CASP10, and CASP3) signals. Similarly, we confirmed cell death execution by chromatin condensation. Our results showed that low (0.5 mM) and high (1.0 and 5.0 mM) BA levels differ in stress and cell death signaling. Moreover, at periodontal disease-level BA concentration (5 mM), we observed that only FasL amounts were affected and occurred concurrently with chromatin condensation insinuating that cells have fully committed to neurodegeneration. Thus, we believe that both stress and cell death signaling in NGF-treated PC12 cells are affected differently depending on BA concentration. In a periodontal disease scenario, we hypothesize that during the early stages, low BA amounts accumulate resulting to both stress- and cell death-related signals that favor neurite non-proliferation, whereas, during the later stages, high BA amounts accumulate resulting to both stress- and cell death-related signals that favor neurodegeneration. More importantly, we propose that neuropathic pain absence at any stage of periodontal disease progression is ascribable to BA accumulation regardless of amount.
Assuntos
Apoptose , Ácido Butírico/metabolismo , Neuralgia/patologia , Estresse Oxidativo , Doenças Periodontais/patologia , Animais , Progressão da Doença , Fator de Crescimento Neural/metabolismo , Neuralgia/metabolismo , Neuritos/metabolismo , Células PC12 , Doenças Periodontais/metabolismo , Ratos , Transdução de Sinais , Fator de Transcrição CHOP/metabolismoRESUMO
The oral microbiome is composed of detrimental and beneficial microbial communities producing several microbial factors that could contribute to the development of the oral microbiome and, likewise, may lead to the development of host diseases. Metabolites, like short-chain fatty acids, are commonly produced by the oral microbiome and serve various functions. Among the periodontal short-chain fatty acids, butyric acid is mainly produced by periodontopathic bacteria and, attributable to the butyrate paradox, is postulated to exhibit a dual function depending on butyric acid concentration. A better understanding of the interconnecting networks that would influence butyric acid function in the oral cavity may shed a new light on the current existing knowledge and view regarding butyric acid.
Assuntos
Bactérias/metabolismo , Ácido Butírico/metabolismo , Boca/microbiologia , Doenças Periodontais/microbiologia , Bactérias/efeitos dos fármacos , Humanos , MicrobiotaRESUMO
Periodontal diseases have long been postulated to contribute to systemic diseases and, likewise, it has been proposed that periodontal disease treatment may ameliorate certain systemic diseases. Short-chain fatty acids (SCFA) are major secondary metabolites produced by oral anaerobic bacteria and, among the SCFAs, butyric acid (BA) in high amounts contribute to periodontal disease development. Periodontal disease level-butyric acid (PDL-BA) is found among patients suffering from periodontal disease and has previously shown to induce oxidative stress, whereas, oxidative stress is correlated to endoplasmic reticulum (ER) stress. This would imply that PDL-BA may likewise stimulate ER stress, however, this was never elucidated. A better understanding of the correlation between PDL-BA and systemic ER stress stimulation could shed light on the possible systemic effects of PDL-BA-related periodontal diseases. Here, PDL-BA was injected into the gingival mucosa and the systemic blood obtained from the rat jugular was collected at 0, 15, 60, and 180 min post-injection. Collected blood samples were purified and only the blood cytosol was used throughout this study. Subsequently, we measured blood cytosolic GADD153, Ca(2+), representative apoptotic and inflammatory caspases, and NF-κB amounts. We found that PDL-BA presence increased blood cytosolic GADD153 and Ca(2+) amounts. Moreover, we observed that blood cytosolic caspases and NF-κB were activated only at 60 and 180 min post-injection in the rat gingival mucosa. This suggests that PDL-BA administered through the gingival mucosa may influence the systemic blood via ER stress stimulation and, moreover, prolonged PDL-BA retention in the gingival mucosa may play a significant role in ER stress-related caspase and NF-κB activation. In a periodontal disease scenario, we propose that PDL-BA-related ER stress stimulation leading to the simultaneous activation of apoptosis and inflammation may contribute to periodontal disease pathogenesis.
Assuntos
Ácido Butírico/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Doenças Periodontais/sangue , Animais , Apoptose/efeitos dos fármacos , Cálcio/sangue , Caspases/sangue , Citosol/metabolismo , Gengiva/metabolismo , Gengiva/microbiologia , Masculino , NF-kappa B/sangue , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Fator de Transcrição CHOP/sangue , Fator de Transcrição CHOP/metabolismoRESUMO
Porphyromonas gingivalis requires heme to grow, however, heme availability and concentration in the periodontal pockets vary. Fluctuations in heme concentration may affect each P. gingivalis strain differently, however, this was never fully demonstrated. Here, we elucidated the effects of varying hemin concentrations in representative P. gingivalis strains. Throughout this study, representative P. gingivalis strains [FDC381 (type I), MPWIb-01 (type Ib), TDC60 (type II), ATCC49417 (type III), W83 (type IV), and HNA99 (type V)] were used and grown for 24 h in growth media under varying hemin concentrations (5 × , 1 × , 0.5 × , 0.1 × ). Samples were lysed and protein standardized. Arg-gingipain (Rgp), H2O2, and superoxide dismutase (SOD) levels were subsequently measured. We focused our study on 24 h-grown strains which excluded MPWIb-01 and HNA99. Rgp activity among the 4 remaining strains varied with Rgp peaking at: 1 × for FDC381, 5 × for TDC60, 0.5 × for ATCC49417, 5 × and 0.5 × for W83. With regards to H2O2 and SOD amounts: FDC381 had similar H2O2 amounts in all hemin concentrations while SOD levels varied; TDC60 had the lowest H2O2 amount at 1 × while SOD levels became higher in relation to hemin concentration; ATCC49417 also had similar H2O2 amounts in all hemin concentrations while SOD levels were higher at 1 × and 0.5 × ; and W83 had statistically similar H2O2 and SOD amounts regardless of hemin concentration. Our results show that variations in hemin concentration affect each P. gingivalis strain differently.
Assuntos
Hemina/administração & dosagem , Porphyromonas gingivalis/efeitos dos fármacos , Adesinas Bacterianas/metabolismo , Meios de Cultura , Cisteína Endopeptidases/metabolismo , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Genótipo , Cisteína Endopeptidases Gingipaínas , Peróxido de Hidrogênio/metabolismo , Doenças Periodontais/microbiologia , Bolsa Periodontal/metabolismo , Bolsa Periodontal/microbiologia , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/crescimento & desenvolvimento , Porphyromonas gingivalis/metabolismo , Especificidade da Espécie , Superóxido Dismutase/metabolismoRESUMO
Influenza is a serious respiratory disease among immunocompromised individuals, such as the elderly, and its prevention is an urgent social issue. Influenza viruses rely on neuraminidase (NA) activity to release progeny viruses from infected cells and spreading the infection. NA is, therefore, an important target of anti-influenza drugs. A causal relationship between bacteria and influenza virus infection has not yet been established, however, a positive correlation between them has been reported. Thus, in this study, we examined the biological effects of oral mitis group streptococci, which are predominant constituents of human oral florae, on the release of influenza viruses. Among them, Streptococcus oralis ATCC 10557 and Streptococcus mitis ATCC 6249 were found to exhibit NA activity and their culture supernatants promoted the release of influenza virus and cell-to-cell spread of the infection. In addition, culture supernatants of these NA-producing oral bacteria increased viral M1 protein expression levels and cellular ERK activation. These effects were not observed with culture supernatants of Streptococcus sanguinis ATCC 10556 which lacks the ability to produce NA. Although the NA inhibitor zanamivir suppressed the release of progeny viruses from the infected cells, the viral release was restored upon the addition of culture supernatants of NA-producing S. oralis ATCC 10557 or S. mitis ATCC 6249. These findings suggest that an increase in the number of NA-producing oral bacteria could elevate the risk of and exacerbate the influenza infection, hampering the efficacy of viral NA inhibitor drugs.
Assuntos
Antivirais/farmacologia , Influenza Humana/tratamento farmacológico , Influenza Humana/microbiologia , Neuraminidase/metabolismo , Streptococcus mitis/enzimologia , Streptococcus oralis/enzimologia , Zanamivir/farmacologia , Análise de Variância , Western Blotting , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Zanamivir/antagonistas & inibidoresRESUMO
Porphyromonas gingivalis requires optimal hemin to grow while non-optimal hemin hampers growth. Hemin induces H2O2 production while H2O2 has a dual function. In P. gingivalis ATCC 33277, we found similar physiological effects under hemin-excess and hemin-limited concentrations which we propose is related to two different functions of the H2O2 molecule.
Assuntos
Hemina/metabolismo , Peróxido de Hidrogênio/metabolismo , Porphyromonas gingivalis/fisiologia , Estresse Fisiológico , Adesinas Bacterianas/metabolismo , Butiratos/metabolismo , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases Gingipaínas , Porphyromonas gingivalis/crescimento & desenvolvimento , Porphyromonas gingivalis/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Autophagy is an important cellular process that triggers a coordinated action involving multiple individual proteins and protein complexes while SARS-CoV-2 (SARS2) was found to both hinder autophagy to evade host defense and utilize autophagy for viral replication. Interestingly, the possible significant stages of the autophagy biochemical network in relation to the corresponding autophagy-targeted SARS2 proteins from the different variants of concern (VOC) were never established. In this study, we performed the following: autophagy biochemical network design and centrality analyses; generated autophagy-targeted SARS2 protein models; and superimposed protein models for structural comparison. We identified 2 significant biochemical pathways (one starts from the ULK complex and the other starts from the PI3P complex) within the autophagy biochemical network. Similarly, we determined that the autophagy-targeted SARS2 proteins (Nsp15, M, ORF7a, ORF3a, and E) are structurally conserved throughout the different SARS2 VOC suggesting that the function of each protein is preserved during SARS2 evolution. Interestingly, among the autophagy-targeted SARS2 proteins, the M protein coincides with the 2 significant biochemical pathways we identified within the autophagy biochemical network. In this regard, we propose that the SARS2 M protein is the main determinant that would influence autophagy outcome in regard to SARS2 infection.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Autofagia , Replicação ViralRESUMO
The cAMP-dependent protein kinase (PKA) signaling pathway plays a crucial role in the pathogenesis of many NF-kappaB-related diseases. However, there have been controversial reports with regard to the PKA actions in the regulation of NF-kappaB activity. In this study, we have demonstrated the effect of PKA on NF-kappaB activity in view of AKIP1 action; and in 293 and HeLa cells, where the endogenous AKIP1 expression is minimal, PKA-activating agents inhibited the NF-kappaB-dependent reporter gene expression, blocked the interaction of PKAc and p65 subunit of NF-kappaB, and attenuated PKA-dependent phosphorylation of p65 on Ser-276. This inhibitory function of PKAc in NF-kappaB signaling was reversed by overexpression of AKIP1 in 293 cells. In the breast cancer cell line, MDA-MB231 cells and MCF7 cells, where the endogenous AKIP1 is abundant, the PKA signal was found to be synergized with NF-kappaB activation; PKA-activating agents enhanced NF-kappaB-dependent transcriptional activity and the interaction between p65 and PKAc and augmented the phosphorylation of p65 on Ser-276. After RNAi knockdown of AKIP1 in these breast cancer cells, we observed that PKA-activating agents antagonized NF-kappaB-dependent activation. Meanwhile, PKA inhibitor suppressed NF-kappaB-induced breast cancer cell proliferation and multiple NF-kappaB-dependent anti-apoptotic gene expression. It is likely that expression of AKIP1 determines the relationship between these two signal transduction pathways. These findings explained controversial results from various independent groups regarding the action of PKA signaling on the NF-kappaB activation cascade and suggested a possible therapeutic potential of PKA inhibitor in developing anti-cancer strategies.
Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Apoptose/genética , Neoplasias da Mama/patologia , Proteína de Ligação a CREB/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , AMP Cíclico/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Serina/metabolismo , Fator de Transcrição RelA/química , Transcrição Gênica/efeitos dos fármacosRESUMO
Coronavirus disease 2019 (COVID-19) pandemic has been attributed to SARS-CoV-2 (SARS2) and, consequently, SARS2 has evolved into multiple SARS2 variants driving subsequent waves of infections. In particular, variants of concern (VOC) were identified to have both increased transmissibility and virulence ascribable to mutational changes occurring within the spike protein resulting to modifications in the protein structural orientation which in-turn may affect viral pathogenesis. However, this was never fully elucidated. Here, we generated spike models of endemic HCoVs (HCoV 229E, HCoV OC43, HCoV NL63, HCoV HKU1, SARS CoV, MERS CoV), original SARS2, and VOC (alpha, beta, gamma, delta). Model quality check, structural superimposition, and structural comparison based on RMSD values, TM scores, and contact mapping were all performed. We found that: 1) structural comparison between the original SARS2 and VOC whole spike protein model have minor structural differences (TM > 0.98); 2) the whole VOC spike models putatively have higher structural similarity (TM > 0.70) to spike models from endemic HCoVs coming from the same phylogenetic cluster; 3) original SARS2 S1-CTD and S1-NTD models are structurally comparable to VOC S1-CTD (TM = 1.0) and S1-NTD (TM > 0.96); and 4) endemic HCoV S1-CTD and S1-NTD models are structurally comparable to VOC S1-CTD (TM > 0.70) and S1-NTD (TM > 0.70) models belonging to the same phylogenetic cluster. Overall, we propose that structural similarities (possibly ascribable to similar conformational epitopes) may help determine immune cross-reactivity, whereas, structural differences (possibly associated with varying conformational epitopes) may lead to viral infection (either reinfection or breakthrough infection).
RESUMO
Controlling the oral microbial flora is putatively thought to prevent not only oral diseases, but also systemic diseases caused by oral diseases. This study establishes the antibacterial effect of the novel bioactive substance "S-PRG filler" on oral bacteria. We examined the state of oxidative stress caused by the six types of ions released in eluate from the S-PRG filler in oral bacterial cells. Moreover, we investigated the effects of these ions on the growth and pathogenicity of Gram-positive and Gram-negative bacteria. We found that the released ions affected SOD amount and hydrogen peroxide in bacterial cells insinuating oxidative stress occurrence. In bacterial culture, growth inhibition was observed depending on the ion concentration in the medium. Additionally, released ions suppressed Streptococcus mutans adhesion to hydroxyapatite, S. oralis neuraminidase activity, and Porphyromonas gingivalis hemagglutination and gingipain activity in a concentration-dependent manner. From these results, it was suggested that the ions released from the S-PRG filler may suppress the growth and pathogenicity of the oral bacterial flora. This bioactive material is potentially useful to prevent the onset of diseases inside and outside of the oral cavity, which in turn may have possible applications for oral care and QOL improvement.
RESUMO
The SARS-CoV-2 (SARS2) is the cause of the coronavirus disease 2019 (COVID-19) pandemic. One unique structural feature of the SARS2 spike protein is the presence of a furin-like cleavage site (FLC) which is associated with both viral pathogenesis and host tropism. Specifically, SARS2 spike protein binds to the host ACE-2 receptor which in-turn is cleaved by furin proteases at the FLC site, suggesting that SARS2 FLC structural variations may have an impact on viral infectivity. However, this has not yet been fully elucidated. This study designed and analyzed a COVID-19 genomic epidemiology network for December 2019 to July 2020, and subsequently generated and analyzed representative SARS2 spike protein models from significant node clusters within the network. To distinguish possible structural variations, a model quality assessment was performed before further protein model analyses and superimposition of the protein models, particularly in both the receptor-binding domain (RBD) and FLC. Mutant spike models were generated with the unique 681PRRA684 amino acid sequence found within the deleted FLC. We found 9 SARS2 FLC structural patterns that could potentially correspond to nine node clusters encompassing various countries found within the COVID-19 genomic epidemiology network. Similarly, we associated this with the rapid evolution of the SARS2 genome. Furthermore, we observed that either in the presence or absence of the unique 681PRRA684 amino acid sequence no structural changes occurred within the SARS2 RBD, which we believe would mean that the SARS2 FLC has no structural influence on SARS2 RBD and may explain why host tropism was maintained.
RESUMO
Porphyromonas gingivalis (Pg) is a periodontopathic pathogen that may affect MUC5AC-related mucus hypersecretion along airway epithelial cells. Here, we attempted to establish whether Pg virulence factors (lipopolysaccharide, FimA fimbriae, gingipains) affect MUC5AC in immortalized and primary bronchial cells. We report that MUC5AC gene expression and protein levels are affected by Pg culture supernatant, but not by lipopolysaccharide or FimA fimbriae. Cells treated with either Pg single (Kgp or Rgp) or double (Kgp/Rgp) mutants had altered levels of MUC5AC gene expression and protein levels, and MUC5AC staining of double mutant-treated mouse lung cells showed that MUC5AC protein levels were unaffected. Taken together, we propose that Pg gingipains may be the primary virulence factor that influences both MUC5AC gene expression and protein levels.
Assuntos
Mucina-5AC/metabolismo , Doenças Periodontais/complicações , Porphyromonas gingivalis/imunologia , Infecções Respiratórias/imunologia , Animais , Brônquios/imunologia , Brônquios/metabolismo , Brônquios/patologia , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Cisteína Endopeptidases Gingipaínas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Masculino , Camundongos , Mucina-5AC/análise , Doenças Periodontais/imunologia , Doenças Periodontais/microbiologia , Porphyromonas gingivalis/metabolismo , Cultura Primária de Células , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Infecções Respiratórias/microbiologia , Infecções Respiratórias/patologia , Organismos Livres de Patógenos Específicos , Fatores de Virulência/metabolismoRESUMO
Previous attempts to express the human immunodeficiency virus 1 (HIV-1) Tat (trans-activator of transcription) protein in plants resulted in a number of physiological abnormalities, such as stunted growth and absence of seed formation, that could not be explained. In the study reported here, we expressed Tat in tomato and observed phenotypic abnormalities, including stunted growth, absence of root formation, chlorosis, and plant death, as a result of reduced cytokinin levels. These reduced levels were ascribed to a differentially expressed CKO35 in Tat-bombarded tomato. Of the two CKO isoforms that are naturally expressed in tomato, CKO43 and CKO37, only the expression of CKO37 was affected by Tat. Our analysis of the Tat confirmed that the Arg-rich and RGD motifs of Tat have functional relevance in tomato and that independent mutations at these motifs caused inhibition of the differentially expressed CKO isoform and the extracellular secretion of the Tat protein, respectively, in our Tat-bombarded tomato samples.
Assuntos
HIV-1/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Solanum lycopersicum/crescimento & desenvolvimento , Produtos do Gene tat do Vírus da Imunodeficiência Humana/toxicidade , Motivos de Aminoácidos , Cloroplastos/metabolismo , Citocininas/biossíntese , Indução Enzimática , Solanum lycopersicum/genética , Modelos Biológicos , Oligopeptídeos/toxicidade , Oxirredutases/biossíntese , Oxirredutases/genética , Fenótipo , Doenças das Plantas/genética , Raízes de Plantas/ultraestrutura , Brotos de Planta/ultraestrutura , Plantas Geneticamente Modificadas/genética , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Proteínas Recombinantes de Fusão/toxicidade , Relação Estrutura-Atividade , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
HIV-1 Tat plays a major role in viral replication and is essential for AIDS development making it an ideal vaccine target providing that both humoral and cellular immune responses are induced. Plant-based antigen production, due to its cheaper cost, appears ideal for vaccine production. In this study, we created a plant-optimized tat and mutant (Cys30Ala/Lys41Ala) tat (mtat) gene and ligated each into a pBI121 expression vector with a stop codon and a gusA gene positioned immediately downstream. The vector construct was bombarded into tomato leaf calli and allowed to develop. We thus generated recombinant tomato plants preferentially expressing a Tat-GUS fusion protein over a Tat-only protein. In addition, plants bombarded with either tat or mtat genes showed no phenotypic difference and produced 2-4 microg Tat-GUS fusion protein per milligram soluble plant protein. Furthermore, tomato extracts intradermally inoculated into mice were found to induce a humoral and, most importantly, cellular immunity.
Assuntos
Vacinas contra a AIDS/imunologia , HIV-1/genética , Plantas Geneticamente Modificadas , Solanum lycopersicum/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/imunologia , Animais , Sequência de Bases , Genes Reporter , Genes tat , Vetores Genéticos/genética , Anticorpos Anti-HIV/biossíntese , Imunoglobulina G/biossíntese , Solanum lycopersicum/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fenótipo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Mutação Puntual , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinação , Vacinas Sintéticas/imunologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/biossíntese , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
Coronaviruses (CoV) are enveloped positive-stranded RNA viruses and, historically, there are seven known human-infecting CoVs with varying degrees of virulence. CoV attachment to the host is the first step of viral pathogenesis and mainly relies on the spike glycoprotein located on the viral surface. Among the human-infecting CoVs, only the infection of SARS CoV 2 (SARS2) among humans resulted to a pandemic which would suggest that the protein structural conformation of SARS2 spike protein is distinct as compared to other human-infecting CoVs. Surprisingly, the possible differences and similarities in the protein structural conformation between the various human-infecting CoV spike proteins have not been fully elucidated. In this study, we utilized a computational approach to generate models and analyze the seven human-infecting CoV spike proteins, namely: HCoV 229E, HCoV OC43, HCoV NL63, HCoV HKU1, SARS CoV, MERS CoV, and SARS2. Model quality assessment of all CoV models generated, structural superimposition of the whole protein model and selected S1 domains (S1-CTD and S1-NTD), and structural comparison based on RMSD values, Tm scores, and contact mapping were all performed. We found that the structural orientation of S1-CTD is a potential structural feature associated to both the CoV phylogenetic cluster and lineage. Moreover, we observed that spike models in the same phylogenetic cluster or lineage could potentially have similar protein structure. Additionally, we established that there are potentially three distinct S1-CTD orientation (Pattern I, Pattern II, Pattern III) among the human-infecting CoVs. Furthermore, we postulate that human-infecting CoVs in the same phylogenetic cluster may have similar S1-CTD and S1-NTD structural orientation. Taken together, we propose that the SARS2 spike S1-CTD follows a Pattern III orientation which has a higher degree of similarity with SARS1 and some degree of similarity with both OC43 and HKU1 which coincidentally are in the same phylogenetic cluster and lineage, whereas, the SARS2 spike S1-NTD has some degree of similarity among human-infecting CoVs that are either in the same phylogenetic cluster or lineage.