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1.
Nature ; 611(7935): 312-319, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36261521

RESUMO

Infectious diseases are among the strongest selective pressures driving human evolution1,2. This includes the single greatest mortality event in recorded history, the first outbreak of the second pandemic of plague, commonly called the Black Death, which was caused by the bacterium Yersinia pestis3. This pandemic devastated Afro-Eurasia, killing up to 30-50% of the population4. To identify loci that may have been under selection during the Black Death, we characterized genetic variation around immune-related genes from 206 ancient DNA extracts, stemming from two different European populations before, during and after the Black Death. Immune loci are strongly enriched for highly differentiated sites relative to a set of non-immune loci, suggesting positive selection. We identify 245 variants that are highly differentiated within the London dataset, four of which were replicated in an independent cohort from Denmark, and represent the strongest candidates for positive selection. The selected allele for one of these variants, rs2549794, is associated with the production of a full-length (versus truncated) ERAP2 transcript, variation in cytokine response to Y. pestis and increased ability to control intracellular Y. pestis in macrophages. Finally, we show that protective variants overlap with alleles that are today associated with increased susceptibility to autoimmune diseases, providing empirical evidence for the role played by past pandemics in shaping present-day susceptibility to disease.


Assuntos
DNA Antigo , Predisposição Genética para Doença , Imunidade , Peste , Seleção Genética , Yersinia pestis , Humanos , Aminopeptidases/genética , Aminopeptidases/imunologia , Peste/genética , Peste/imunologia , Peste/microbiologia , Peste/mortalidade , Yersinia pestis/imunologia , Yersinia pestis/patogenicidade , Seleção Genética/imunologia , Europa (Continente)/epidemiologia , Europa (Continente)/etnologia , Imunidade/genética , Conjuntos de Dados como Assunto , Londres/epidemiologia , Dinamarca/epidemiologia
2.
Antimicrob Agents Chemother ; 66(11): e0078722, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36200773

RESUMO

OXA-48 is the most common carbapenemase in Enterobacterales in Germany and many other European countries. Depending on the genomic location of blaOXA-48, OXA-48-producing isolates vary in phenotype and intra- and interspecies transferability of blaOXA-48. In most bacterial isolates, blaOXA-48 is located on one of seven variants of Tn1999 (Tn1999.1 to Tn1999.6 and invTn1999.2). Here, a novel Tn1999 variant, Tn1999.7, is described, which was identified in 11 clinical isolates from 2016 to 2020. Tn1999.7 differs from Tn1999.1 by the insertion of the 8,349-bp Tn3 family transposon Tn7442 between the lysR gene and blaOXA-48 open reading frame. Tn7442 carries genes coding for a restriction endonuclease and a DNA methyltransferase as cargo, forming a type III restriction modification system. Tn1999.7 was carried on an ~71-kb IncL plasmid in 9/11 isolates. In one isolate, Tn1999.7 was situated on an ~76-kb plasmid, harboring an additional insertion sequence in the plasmid backbone. In one isolate, the plasmid size is only ~63 kb due to a deletion adjacent to Tn7442 that extends into the plasmid backbone. Mean conjugation rates of the Tn1999.7-harboring plasmids in J53 ranged from 4.47 × 10-5 to 2.03 × 10-2, similar to conjugation rates of other pOXA-48-type IncL plasmids. The stability of plasmids with Tn1999.7 was significantly higher than that of a Tn1999.2-harboring plasmid in vitro. This increase in stability could be related to the insertion of a restriction-modification system, which can promote postsegregational killing. The increased plasmid stability associated with Tn1999.7 could contribute to the further spread of OXA-48.


Assuntos
Proteínas de Bactérias , Elementos de DNA Transponíveis , Plasmídeos , beta-Lactamases , Proteínas de Bactérias/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo , Elementos de DNA Transponíveis/genética , Europa (Continente) , Alemanha , Plasmídeos/genética , Enterobacteriaceae/genética , Enterobacteriaceae/patogenicidade , Variação Genética
3.
Harmful Algae ; 118: 102288, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36195431

RESUMO

Chytrid parasites are increasingly recognized as ubiquitous and potent control agents of phytoplankton, including bloom-forming toxigenic cyanobacteria. In order to explore the fate of the cyanobacterial toxin microcystins (MCs) and assess potential upregulation of their production under parasite attack, a laboratory experiment was conducted to evaluate short- and long-term variation in extracellular and intracellular MC in the cyanobacteria Planktothrix agardhii and P. rubescens, both under chytrid infection and in the presence of lysates of previously infected cyanobacteria. MCs release under parasite infection was limited and not different to uninfected cyanobacteria, with extracellular toxin shares never exceeding 10%, substantially below those caused by mechanical lysis induced by a cold-shock. Intracellular MC contents in P. rubescens under infection were not significantly different from uninfected controls, whereas infected P. agardhii showed a 1.5-fold increase in intracellular MC concentrations, but this was detected within the first 48 hours after parasite inoculation and not later, indicating no substantial MC upregulation in cells being infected. The presence of lysates of previously infected cyanobacteria did not elicit higher intracellular MC contents in exposed cyanobacteria, speaking against a putative upregulation of toxin production induced via quorum sensing in response to parasite attack. These results indicate that chytrid epidemics can constitute a bloom decay mechanism that is not accompanied by massive release of toxins into the medium.


Assuntos
Quitridiomicetos , Cianobactérias , Quitridiomicetos/patogenicidade , Toxinas de Cianobactérias , Microcistinas , Fitoplâncton/microbiologia
4.
Nature ; 610(7931): 381-388, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36198800

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and caused the devastating global pandemic of coronavirus disease 2019 (COVID-19), in part because of its ability to effectively suppress host cell responses1-3. In rare cases, viral proteins dampen antiviral responses by mimicking critical regions of human histone proteins4-8, particularly those containing post-translational modifications required for transcriptional regulation9-11. Recent work has demonstrated that SARS-CoV-2 markedly disrupts host cell epigenetic regulation12-14. However, how SARS-CoV-2 controls the host cell epigenome and whether it uses histone mimicry to do so remain unclear. Here we show that the SARS-CoV-2 protein encoded by ORF8 (ORF8) functions as a histone mimic of the ARKS motifs in histone H3 to disrupt host cell epigenetic regulation. ORF8 is associated with chromatin, disrupts regulation of critical histone post-translational modifications and promotes chromatin compaction. Deletion of either the ORF8 gene or the histone mimic site attenuates the ability of SARS-CoV-2 to disrupt host cell chromatin, affects the transcriptional response to infection and attenuates viral genome copy number. These findings demonstrate a new function of ORF8 and a mechanism through which SARS-CoV-2 disrupts host cell epigenetic regulation. Further, this work provides a molecular basis for the finding that SARS-CoV-2 lacking ORF8 is associated with decreased severity of COVID-19.


Assuntos
COVID-19 , Epigênese Genética , Histonas , Interações entre Hospedeiro e Microrganismos , Mimetismo Molecular , SARS-CoV-2 , Proteínas Virais , COVID-19/genética , COVID-19/metabolismo , COVID-19/virologia , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Epigenoma/genética , Histonas/química , Histonas/metabolismo , Humanos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
5.
FASEB J ; 36(11): e22599, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36250902

RESUMO

Emerging evidence suggest that C3aR plays important roles in homeostasis, host defense and disease. Although it is known that C3aR is protective in several models of acute bacterial infections, the role for C3aR in chronic infection is largely unknown. Here we show that C3aR is protective in experimental chronic pyelonephritis. Global C3aR deficient (C3ar-/- ) mice had higher renal bacterial load, more pronounced renal histological lesions, increased renal apoptotic cell accumulation, tissue inflammation and extracellular matrix deposition following renal infection with uropathogenic E. coli (UPEC) strain IH11128, compared to WT control mice. Myeloid C3aR deficient (Lyz2-C3ar-/- ) mice exhibited a similar disease phenotype to global C3ar-/- mice. Pharmacological treatment with a C3aR agonist reduced disease severity in experimental chronic pyelonephritis. Furthermore, macrophages of C3ar-/- mice exhibited impaired ability to phagocytose UPEC. Our data clearly demonstrate a protective role for C3aR against experimental chronic pyelonephritis, macrophage C3aR plays a major role in the protection, and C3aR is necessary for phagocytosis of UPEC by macrophages. Our observation that C3aR agonist curtailed the pathology suggests a therapeutic potential for activation of C3aR in chronic infection.


Assuntos
Infecções por Escherichia coli , Pielonefrite , Receptores de Complemento , Animais , Camundongos , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/patologia , Inflamação/imunologia , Inflamação/microbiologia , Inflamação/patologia , Rim/microbiologia , Rim/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Pielonefrite/imunologia , Pielonefrite/microbiologia , Pielonefrite/patologia , Pielonefrite/prevenção & controle , Escherichia coli Uropatogênica/patogenicidade , Receptores de Complemento/agonistas , Receptores de Complemento/deficiência , Receptores de Complemento/genética , Receptores de Complemento/imunologia , Matriz Extracelular/metabolismo
6.
Science ; 378(6617): 242-245, 2022 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-36264794
7.
Environ Pollut ; 314: 120294, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36181932

RESUMO

Per- and Poly-fluoroalkyl substances (PFAS) are major persistent environmental contaminants. Epidemiological studies have linked PFAS exposures to altered immunity and increased occurrence of infections in children. However, the mechanisms leading to immune susceptibility to bacterial infections remains unclear. To elucidate the mechanism, transcriptional alteration in the Caenorhabditis elegans model caused by a PFAS contaminated environmental water and two reconstituted PFAS solutions were evaluated using RNA-sequencing. PFAS affected the expression of several genes involved in C. elegans immune surveillance to Gram-positive bacteria (cpr-2, tag-38, spp-1, spp-5, clec-7, clec-172). The combined exposure to PFAS and Staphylococcus aureus significantly reduced C. elegans survival and increased intestinal membrane permeability. Furthermore, the growth of S. aureus in the presence of PFAS increased the expression of virulence genes, specifically, the virulence gene regulator saeR and α-hemolysin, hla, which resulted in increased hemolytic activity. The present study demonstrated that PFAS exposure not only increased C. elegans susceptibility to pathogens by reducing host immunity and increasing intestinal membrane permeability, but also increased bacteria virulence. This presents a broader implication for humans and other animals, where environmental contaminants simultaneously reduce host resilience, while, increasing microbial pathogenicity.


Assuntos
Caenorhabditis elegans , Fluorcarbonetos , Staphylococcus aureus , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/imunologia , Caenorhabditis elegans/microbiologia , Fluorcarbonetos/toxicidade , Proteínas Hemolisinas , Imunidade , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Virulência/genética , Poluentes Ambientais/toxicidade
8.
Commun Biol ; 5(1): 910, 2022 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-36065015

RESUMO

Phenol-soluble modulin α (PSMα) is identified as potent virulence factors in Staphylococcus aureus (S. aureus) infections. Very little is known about the role of PSMß which belongs to the same toxin family. Here we compared the role of PSMs in S. aureus-induced septic arthritis in a murine model using three isogenic S. aureus strains differing in the expression of PSMs (Newman, Δpsmα, and Δpsmß). The effects of PSMs on neutrophil NADPH-oxidase activity were determined in vitro. We show that the PSMα activates neutrophils via the formyl peptide receptor (FPR) 2 and reduces their NADPH-oxidase activity in response to the phorbol ester PMA. Despite being a poor neutrophil activator, PSMß has the ability to reduce the neutrophil activating effect of PSMα and to partly reverse the effect of PSMα on the neutrophil response to PMA. Mice infected with S. aureus lacking PSMα had better weight development and lower bacterial burden in the kidneys compared to mice infected with the parental strain, whereas mice infected with bacteria lacking PSMß strain developed more severe septic arthritis accompanied with higher IL-6 and KC. We conclude that PSMα and PSMß play distinct roles in septic arthritis: PSMα aggravates systemic infection, whereas PSMß protects arthritis development.


Assuntos
Artrite Infecciosa , Toxinas Bacterianas , Infecções Estafilocócicas , Staphylococcus aureus , Animais , Artrite Infecciosa/metabolismo , Toxinas Bacterianas/metabolismo , Camundongos , NADP/metabolismo , Oxirredutases/metabolismo , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade
9.
Nature ; 611(7935): 346-351, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36130725

RESUMO

Clinical outcomes of severe acute respiratory syndrome 2 (SARS-CoV-2) infection are highly heterogeneous, ranging from asymptomatic infection to lethal coronavirus disease 2019 (COVID-19). The factors underlying this heterogeneity remain insufficiently understood. Genetic association studies have suggested that genetic variants contribute to the heterogeneity of COVID-19 outcomes, but the underlying potential causal mechanisms are insufficiently understood. Here we show that common variants of the apolipoprotein E (APOE) gene, homozygous in approximately 3% of the world's population1 and associated with Alzheimer's disease, atherosclerosis and anti-tumour immunity2-5, affect COVID-19 outcome in a mouse model that recapitulates increased susceptibility conferred by male sex and advanced age. Mice bearing the APOE2 or APOE4 variant exhibited rapid disease progression and poor survival outcomes relative to mice bearing the most prevalent APOE3 allele. APOE2 and APOE4 mice exhibited increased viral loads as well as suppressed adaptive immune responses early after infection. In vitro assays demonstrated increased infection in the presence of APOE2 and APOE4 relative to APOE3, indicating that differential outcomes are mediated by differential effects of APOE variants on both viral infection and antiviral immunity. Consistent with these in vivo findings in mice, our results also show that APOE genotype is associated with survival in patients infected with SARS-CoV-2 in the UK Biobank (candidate variant analysis, P = 2.6 × 10-7). Our findings suggest APOE genotype to partially explain the heterogeneity of COVID-19 outcomes and warrant prospective studies to assess APOE genotyping as a means of identifying patients at high risk for adverse outcomes.


Assuntos
Apolipoproteínas E , COVID-19 , Genética Humana , Camundongos Transgênicos , SARS-CoV-2 , Animais , Humanos , Masculino , Camundongos , Apolipoproteína E2/genética , Apolipoproteína E3/genética , Apolipoproteína E4/genética , Apolipoproteínas E/genética , COVID-19/genética , COVID-19/mortalidade , COVID-19/virologia , Camundongos Transgênicos/genética , Camundongos Transgênicos/virologia , Estudos Prospectivos , SARS-CoV-2/patogenicidade , Modelos Animais de Doenças
10.
J Biol Chem ; 298(10): 102441, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36055404

RESUMO

Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging ß-barrel pore-forming toxin. Upon binding to the target membranes, VCC monomers first assemble into oligomeric prepore intermediates and subsequently transform into transmembrane ß-barrel pores. VCC harbors a designated pore-forming motif, which, during oligomeric pore formation, inserts into the membrane and generates a transmembrane ß-barrel scaffold. It remains an enigma how the molecular architecture of the pore-forming motif regulates the VCC pore-formation mechanism. Here, we show that a specific pore-forming motif residue, E289, plays crucial regulatory roles in the pore-formation mechanism of VCC. We find that the mutation of E289A drastically compromises pore-forming activity, without affecting the structural integrity and membrane-binding potential of the toxin monomers. Although our single-particle cryo-EM analysis reveals WT-like oligomeric ß-barrel pore formation by E289A-VCC in the membrane, we demonstrate that the mutant shows severely delayed kinetics in terms of pore-forming ability that can be rescued with elevated temperature conditions. We find that the pore-formation efficacy of E289A-VCC appears to be more profoundly dependent on temperature than that of the WT toxin. Our results suggest that the E289A mutation traps membrane-bound toxin molecules in the prepore-like intermediate state that is hindered from converting into the functional ß-barrel pores by a large energy barrier, thus highlighting the importance of this residue for the pore-formation mechanism of VCC.


Assuntos
Proteínas de Bactérias , Citotoxinas , Proteínas Citotóxicas Formadoras de Poros , Vibrio cholerae , Fatores de Virulência , Membrana Celular/metabolismo , Citotoxinas/química , Citotoxinas/genética , Vibrio cholerae/patogenicidade , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Fatores de Virulência/química , Fatores de Virulência/genética , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Motivos de Aminoácidos , Mutação , Ácido Glutâmico/química , Ácido Glutâmico/genética
11.
BMC Microbiol ; 22(1): 219, 2022 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-36115948

RESUMO

BACKGROUND: The prevalence of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin (PVL) gene is higher in Africa (≈50%) compared to Europe (< 5%). The study aimed to measure anti-PVL-antibodies in Africans and Germans in a multi-center study and to test whether detected antibodies can neutralize the cytotoxic effect of PVL on polymorphonuclear leukocytes (PMNs). METHODS: Sera from asymptomatic Africans (n = 22, Nigeria, Gabon) and Caucasians (n = 22, Germany) were used to quantify antibody titers against PVL and α-hemolysin (in arbitrary units [AU]) by ELISA. PMNs from one African and German donor were exposed to 5 nM recombinant PVL to measure the neutralizing effect of serial dilutions of pooled sera from African and Caucasian participants, or donor sera at 0.625 and 2.5% (v/v). RESULTS: Anti-PVL-antibodies were significantly higher in Africans than in Germans (1.9 vs. 0.7 AU, p < 0.0001). The pooled sera from the study participants neutralized the cytotoxic effect of PVL on African and German PMNs in a dose dependent manner. Also, neutralization of PVL on PMNs from the African and German donors had a stronger effect with African sera (half-maximal inhibitory concentration (IC50) = 0.27 and 0.47%, respectively) compared to Caucasian sera (IC50 = 3.51 and 3.59% respectively). CONCLUSION: Africans have higher levels of neutralizing anti-PVL-antibodies. It remains unclear if or at what level these antibodies protect against PVL-related diseases.


Assuntos
Anticorpos Neutralizantes/sangue , Leucocidinas , Neutrófilos , Infecções Estafilocócicas , Staphylococcus aureus , Anticorpos Neutralizantes/imunologia , Toxinas Bacterianas/sangue , Toxinas Bacterianas/imunologia , Exotoxinas/sangue , Exotoxinas/imunologia , Alemanha/epidemiologia , Proteínas Hemolisinas , Humanos , Leucocidinas/sangue , Leucocidinas/imunologia , Neutrófilos/imunologia , Nigéria/epidemiologia , Infecções Estafilocócicas/sangue , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Staphylococcus aureus/patogenicidade
12.
Sheng Wu Gong Cheng Xue Bao ; 38(9): 3390-3405, 2022 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-36151808

RESUMO

Influenza B virus (IBV) is more likely to cause complications than influenza A virus (IAV) and even causes higher disease burden than IAV in a certain season, but IBV has received less attention. In order to analyze the genetic evolution characteristics of the clinical strain IBV (B/Guangxi-Jiangzhou/1352/2018), we constructed genetic evolution trees and analyzed the homology and different amino acids of hemagglutinin and neuraminidase referring to the vaccine strains recommended by World Health Organization (WHO). We found that strain B/Guangxi-Jiangzhou/1352/2018 was free of interlineage reassortment and poorly matched with the vaccine strain B/Colorado/06/2017 of the same year. We also determined the median lethal dose (LD50) and the pathogenicity of strain B/Guangxi-Jiangzhou/1352/2018 in mice. The results showed that the LD50 was 105.9 TCID50 (median tissue culture infective dose), the IBV titer in the lungs reached peak 1 d post infection and the mRNA level of the most of inflammatory cytokines in the lungs reached peak 12 h post infection. The alveoli in the lungs were severely damaged and a large number of inflammatory cells were infiltrated post infection. The study demonstrated that the clinical strain IBV (B/Guangxi-Jiangzhou/1352/2018) could infect mice and induce typical lung inflammation. This will facilitate the research on the pathogenesis and transmission mechanism of IBV, and provide an ideal animal model for evaluation of new vaccines, antiviral and anti-inflammatory drug.


Assuntos
Vírus da Influenza B , Influenza Humana , Aminoácidos/genética , Animais , Antivirais/farmacologia , China , Citocinas/metabolismo , Hemaglutininas/metabolismo , Humanos , Vírus da Influenza B/genética , Vírus da Influenza B/patogenicidade , Influenza Humana/imunologia , Influenza Humana/virologia , Camundongos , Neuraminidase/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Filogenia , RNA Mensageiro/metabolismo , Virulência/genética
13.
Proc Natl Acad Sci U S A ; 119(40): e2206990119, 2022 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-36161913

RESUMO

Rapid detection of pathogenic bacteria within a few minutes is the key to control infectious disease. However, rapid detection of pathogenic bacteria in clinical samples is quite a challenging task due to the complex matrix, as well as the low abundance of bacteria in real samples. Herein, we employ a label-free single-particle imaging approach to address this challenge. By tracking the scattering intensity variation of single particles in free solution, the morphological heterogeneity can be well identified with particle size smaller than the diffraction limit, facilitating the morphological identification of single bacteria from a complex matrix in a label-free manner. Furthermore, the manipulation of convection in free solution enables the rapid screening of low-abundance bacteria in a small field of view, which significantly improves the sensitivity of single-particle detection. As a proof of concept demonstration, we are able to differentiate the group B streptococci (GBS)-positive samples within 10 min from vaginal swabs without using any biological reagents. This is the most rapid and low-cost method to the best of our knowledge. We believe that such a single-particle imaging approach will find wider applications in clinical diagnosis and disease control due to its high sensitivity, rapidity, simplicity, and low cost.


Assuntos
Bactérias , Doenças Transmissíveis , Análise de Célula Única , Bactérias/isolamento & purificação , Bactérias/patogenicidade , Doenças Transmissíveis/diagnóstico por imagem , Feminino , Humanos , Tamanho da Partícula , Análise de Célula Única/métodos , Esfregaço Vaginal
14.
J Virol ; 96(18): e0081022, 2022 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-36069552

RESUMO

Stress granules (SGs) are dynamic structures that store cytosolic messenger ribonucleoproteins. SGs have recently been shown to serve as a platform for activating antiviral innate immunity; however, several pathogenic viruses suppress SG formation to evade innate immunity. In this study, we investigated the relationship between rabies virus (RABV) virulence and SG formation, using viral strains with different levels of virulence. We found that the virulent Nishigahara strain did not induce SG formation, but its avirulent offshoot, the Ni-CE strain, strongly induced SG formation. Furthermore, we demonstrated that the amino acid at position 95 in the RABV matrix protein (M95), a pathogenic determinant for the Nishigahara strain, plays a key role in inhibiting SG formation, followed by protein kinase R (PKR)-dependent phosphorylation of the α subunit of eukaryotic initiation factor 2α (eIF2α). M95 was also implicated in the accumulation of RIG-I, a viral RNA sensor protein, in SGs and in the subsequent acceleration of interferon induction. Taken together, our findings strongly suggest that M95-related inhibition of SG formation contributes to the pathogenesis of RABV by allowing the virus to evade the innate immune responses of the host. IMPORTANCE Rabies virus (RABV) is a neglected zoonotic pathogen that causes lethal infections in almost all mammalian hosts, including humans. Recently, RABV has been reported to induce intracellular formation of stress granules (SGs), also known as platforms that activate innate immune responses. However, the relationship between SG formation capacity and pathogenicity of RABV has remained unclear. In this study, by comparing two RABV strains with completely different levels of virulence, we found that the amino acid mutation from valine to alanine at position 95 of matrix protein (M95), which is known to be one of the amino acid mutations that determine the difference in virulence between the strains, plays a major role in SG formation. Importantly, M95 was involved in the accumulation of RIG-I in SGs and in promoting interferon induction. These findings are the first report of the effect of a single amino acid substitution associated with SGs on viral virulence.


Assuntos
Vírus da Raiva , Grânulos de Estresse , Proteínas da Matriz Viral , Aminoácidos/metabolismo , Animais , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Interferons/imunologia , Proteínas Quinases/imunologia , RNA Viral/metabolismo , Vírus da Raiva/genética , Vírus da Raiva/patogenicidade , Ribonucleoproteínas/metabolismo , Grânulos de Estresse/genética , Grânulos de Estresse/imunologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologia , Proteínas Virais/genética , Proteínas Virais/metabolismo
15.
J Virol ; 96(19): e0066122, 2022 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-36106873

RESUMO

Members of the mosquito-borne flavivirus genus such as dengue (DENV), West Nile (WNV), and Zika (ZIKV) viruses cause distinct diseases and affect different tissues. We previously found that the secreted flaviviral nonstructural protein 1 (NS1) interacts with endothelial cells and disrupts endothelial barrier function in a tissue-specific manner consistent with the disease tropism of the respective viruses. However, the underlying molecular mechanism of this tissue-specific NS1-endothelial cell interaction is not well understood. To elucidate the distinct role(s) that the wing and ß-ladder domains of NS1 play in NS1 interactions with endothelial cells, we constructed flavivirus NS1 chimeras that exchanged the wing and ß-ladder domains in a pairwise manner between DENV, WNV, and ZIKV NS1. We found that both the NS1 wing and ß-ladder domains conferred NS1 tissue-specific endothelial dysfunction, with the wing conferring cell binding and the ß-ladder involved in inducing endothelial hyperpermeability as measured by transendothelial electrical resistance. To narrow down the amino acids dictating cell binding specificity, we utilized the DENV-WNV NS1 chimera and identified residues 91 to 93 (GDI) of DENV NS1 as a molecular motif determining binding specificity. Further, using an in vivo mouse model of localized leak, we found that the GDI motif of the wing domain was essential for triggering DENV NS1-induced vascular leak in mouse dermis. Taken together, we identify molecular determinants of flavivirus NS1 that confer NS1 binding and vascular leak and highlight the importance of the NS1 wing domain for flavivirus pathogenesis. IMPORTANCE Flavivirus NS1 is secreted into the bloodstream from infected cells during a viral infection. Dengue virus NS1 contributes to severe dengue pathology such as endothelial dysfunction and vascular leak independently of the virus. We have shown that multiple flavivirus NS1 proteins result in endothelial dysfunction in a tissue-specific manner consistent with their respective viral tropism. Here, we aimed to identify the molecular determinants that make some, but not other, flavivirus NS1 proteins bind to select endothelial cells in vitro and cause vascular leak in a mouse model. We identified the wing domain of NS1 as a primary determinant conferring differential endothelial dysfunction and vascular leak and narrowed the contributing amino acid residues to a three-residue motif within the wing domain. The insights from this study pave the way for future studies on the effects of flavivirus NS1 on viral dissemination and pathogenesis and offer potential new avenues for antiviral therapies.


Assuntos
Células Endoteliais , Flavivirus , Proteínas não Estruturais Virais , Tropismo Viral , Aminoácidos/metabolismo , Animais , Antivirais/metabolismo , Comunicação Celular , Vírus da Dengue/genética , Células Endoteliais/virologia , Flavivirus/metabolismo , Flavivirus/patogenicidade , Infecções por Flavivirus , Camundongos , Proteínas não Estruturais Virais/metabolismo , Vírus do Nilo Ocidental , Zika virus
16.
J Virol ; 96(19): e0130122, 2022 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-36121299

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remained genetically stable during the first 3 months of the pandemic, before acquiring a D614G spike mutation that rapidly spread worldwide and then generating successive waves of viral variants with increasingly high transmissibility. We set out to evaluate possible epistatic interactions between the early-occurring D614G mutation and the more recently emerged cleavage site mutations present in spike of the Alpha, Delta, and Omicron variants of concern. The P681H/R mutations at the S1/S2 cleavage site increased spike processing and fusogenicity but limited its incorporation into pseudoviruses. In addition, the higher cleavage rate led to higher shedding of the spike S1 subunit, resulting in a lower infectivity of the P681H/R-carrying pseudoviruses compared to those expressing the Wuhan wild-type spike. The D614G mutation increased spike expression at the cell surface and limited S1 shedding from pseudovirions. As a consequence, the D614G mutation preferentially increased the infectivity of P681H/R-carrying pseudoviruses. This enhancement was more marked in cells where the endosomal route predominated, suggesting that more stable spikes could better withstand the endosomal environment. Taken together, these findings suggest that the D614G mutation stabilized S1/S2 association and enabled the selection of mutations that increased S1/S2 cleavage, leading to the emergence of SARS-CoV-2 variants expressing highly fusogenic spikes. IMPORTANCE The first SARS-CoV-2 variant that spread worldwide in early 2020 carried a D614G mutation in the viral spike, making this protein more stable in its cleaved form at the surface of virions. The Alpha and Delta variants, which spread in late 2020 and early 2021, respectively, proved increasingly transmissible and pathogenic compared to the original strain. Interestingly, Alpha and Delta both carried the mutations P681H/R in a cleavage site that made the spike more cleaved and more efficient at mediating viral fusion. We show here that variants with increased spike cleavage due to P681H/R were even more dependent on the stabilizing effect of the D614G mutation, which limited the shedding of cleaved S1 subunits from viral particles. These findings suggest that the worldwide spread of the D614G mutation was a prerequisite for the emergence of more pathogenic SARS-CoV-2 variants with highly fusogenic spikes.


Assuntos
COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , COVID-19/virologia , Humanos , Mutação , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/genética
17.
J Virol ; 96(19): e0134422, 2022 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-36125302

RESUMO

Subtype H7 avian influenza A viruses (IAVs) are enzootic in wild aquatic birds and have caused sporadic spillovers into domestic poultry and humans. Here, we determined the distribution of fucosylated α2,3 sialoglycan (i.e., sialyl Lewis X [SLeX]) in chickens and five common dabbling duck species and the association between SLeX and cell/tissue/host tropisms of H7 IAVs. Receptor binding analyses showed that H7 IAVs bind to both α2,3-linked (SA2,3Gal) and α2,6-linked sialic acids (SA2,6Gal), but with a higher preference for SLeX; H7 IAVs replicated more efficiently in SLeX-overexpressed than SLeX-deficient MDCK cells. While chickens and all tested dabbling ducks expressed abundant SA2,3Gal and SA2,6Gal, SLeX was detected in both respiratory and gastrointestinal tissues of chickens and mallard ducks and in only the respiratory tissues of gadwall, green-wing teal, and northern shoveler but not in wood ducks. Viral-tissue binding assays showed that H7 IAVs bind to chicken colon crypt cells that express SLeX but fewer bind to mallard colon crypt cells, which do not express SLeX; H7 IAVs bind efficiently to epithelial cells of all tissues expressing SA2,3Gal. High viral replication was identified in both chickens and mallards infected with an H7 virus, regardless of SLeX expression, and viruses were detected in all cells to the same degree as viruses detected in the viral-tissue binding assays. In summary, this study suggests that SLeX facilitates infection of H7 viruses, but other types of SA2,3Gal glycan receptors shape the tissue/host tropisms of H7 IAVs. IMPORTANCE In addition to causing outbreaks in domestic poultry, subtype H7 IAVs can cause sporadic spillover infections in lower mammals and humans. In this study, we showed that SLeX expression varies among wild dabbling ducks. Although it facilitated virus binding and affected infection of H7 IAV in cells, SLeX expression is not the only determinant of viral replication at either the tissue or host level. This study suggested that access to heterologous SA2,3Gal glycan receptors, including fucosylated α2,3-linked sialoglycans, shape tissue and host tropism of H7 IAVs in aquatic wild birds.


Assuntos
Vírus da Influenza A , Influenza Aviária , Antígeno Sialil Lewis X , Tropismo Viral , Animais , Animais Selvagens/virologia , Galinhas/virologia , Cães , Patos/virologia , Vírus da Influenza A/patogenicidade , Vírus da Influenza A/fisiologia , Células Madin Darby de Rim Canino , Polissacarídeos , Ácidos Siálicos , Antígeno Sialil Lewis X/metabolismo
18.
J Virol ; 96(19): e0101522, 2022 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-36129261

RESUMO

Cutaneous beta genus human papillomaviruses (ß-HPVs) are suspected to promote the development of nonmelanoma skin cancer (NMSC) by destabilizing the host genome. Multiple studies have established the genome destabilizing capacities of ß-HPV proteins E6 and E7 as a cofactor with UV. However, the E6 protein from ß-HPV8 (HPV8 E6) induces tumors in mice without UV exposure. Here, we examined a UV-independent mechanism of HPV8 E6-induced genome destabilization. We showed that HPV8 E6 reduced the abundance of anaphase bridge resolving helicase, Bloom syndrome protein (BLM). The diminished BLM was associated with increased segregation errors and micronuclei. These HPV8 E6-induced micronuclei had disordered micronuclear envelopes but retained replication and transcription competence. HPV8 E6 decreased antiproliferative responses to micronuclei and time-lapse imaging revealed HPV8 E6 promoted cells with micronuclei to complete mitosis. Finally, whole-genome sequencing revealed that HPV8 E6 induced chromothripsis in nine chromosomes. These data provide insight into mechanisms by which HPV8 E6 induces genome instability independent of UV exposure. IMPORTANCE Some beta genus human papillomaviruses (ß-HPVs) may promote skin carcinogenesis by inducing mutations in the host genome. Supporting this, the E6 protein from ß-HPV8 (8 E6) promotes skin cancer in mice with or without UV exposure. Many mechanisms by which 8 E6 increases mutations caused by UV have been elucidated, but less is known about how 8 E6 induces mutations without UV. We address that knowledge gap by showing that 8 E6 causes mutations stemming from mitotic errors. Specifically, 8 E6 reduces the abundance of BLM, a helicase that resolves and prevents anaphase bridges. This hinders anaphase bridge resolution and increases their frequency. 8 E6 makes the micronuclei that can result from anaphase bridges more common. These micronuclei often have disrupted envelopes yet retain localization of nuclear-trafficked proteins. 8 E6 promotes the growth of cells with micronuclei and causes chromothripsis, a mutagenic process where hundreds to thousands of mutations occur in a chromosome.


Assuntos
Alphapapillomavirus , Cromotripsia , Proteínas Oncogênicas Virais , Neoplasias Cutâneas , Alphapapillomavirus/patogenicidade , Animais , Instabilidade Genômica , Camundongos , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , RecQ Helicases/metabolismo , Neoplasias Cutâneas/virologia
19.
Proc Natl Acad Sci U S A ; 119(40): e2201473119, 2022 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-36161886

RESUMO

Antimicrobial resistance (AMR) in soils represents a serious risk to human health through the food chain and human-nature contact. However, the active antibiotic-resistant bacteria (ARB) residing in soils that primarily drive AMR dissemination are poorly explored. Here, single-cell Raman-D2O coupled with targeted metagenomics is developed as a culture-independent approach to phenotypically and genotypically profiling active ARB against clinical antibiotics in a wide range of soils. This method quantifies the prevalence (contamination degree) and activity (spread potential) of soil ARB and reveals a clear elevation with increasing anthropogenic activities such as farming and the creation of pollution, thereby constituting a factor that is critical for the assessment of AMR risks. Further targeted sorting and metagenomic sequencing of the most active soil ARB uncover several uncultured genera and a pathogenic strain. Furthermore, the underlying resistance genes, virulence factor genes, and associated mobile genetic elements (including plasmids, insertion sequences, and prophages) are fully deciphered at the single-cell level. This study advances our understanding of the soil active AMR repertoire by linking the resistant phenome to the genome. It will aid in the risk assessment of environmental AMR and guide the combat under the One Health framework.


Assuntos
Antibacterianos , Bactérias , Farmacorresistência Bacteriana , Metagenômica , Microbiologia do Solo , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/patogenicidade , Elementos de DNA Transponíveis , Genes Bacterianos , Humanos , Análise de Célula Única , Solo , Fatores de Virulência/genética
20.
Fish Shellfish Immunol ; 130: 132-140, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36084889

RESUMO

Non-coding RNAs (ncRNAs) have been implicated in a variety of biological processes. However, most ncRNAs are of unknown function and are as-yet unannotated. The immune-related functions of ncRNAs in the pearl oyster Pinctada fucata martensii were explored based on transcriptomic differences in the expression levels of long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs) in the hemocytes of P.f. martensii after challenge by the pathogenic bacterium Vibrio parahaemolyticus. Across the challenged and control pearl oysters, 144 miRNAs and 14,571 lncRNAs were identified. In total, 13,375 ncRNAs were differentially expressed between the challenged and control pearl oysters; in the challenged pearl oysters as compared to the controls, 15 miRNAs and 5147 lncRNAs were upregulated, while 51 miRNAs and 8162 lncRNAs were downregulated. The sequencing results were validated using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. GO and KEGG pathway analysis showed that genes targeted by the differentially expressed ncRNAs were associated with the vascular endothelial growth factor (VEGF) signaling pathway and the nuclear factor kappa-B (NF-κB) signaling pathway. An lncRNA-mRNA-miRNA network that was developed based on the transcriptomic results of this study suggested that lncRNAs may compete with miRNAs for mRNA binding sites. This study may provide a useful framework for the detection of additional novel ncRNAs, as well as new insights into the pathogenic mechanisms underlying the response of P.f. martensii to V. parahaemolyticus.


Assuntos
MicroRNAs , Pinctada , RNA Longo não Codificante , RNA Mensageiro , Vibrio parahaemolyticus , Animais , Imunidade , MicroRNAs/genética , NF-kappa B/metabolismo , Pinctada/genética , Pinctada/imunologia , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Vibrio parahaemolyticus/patogenicidade
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