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1.
PLoS Biol ; 18(8): e3000814, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32797039

RESUMO

Plasmid-mediated horizontal gene transfer of antibiotic resistance and virulence in pathogenic bacteria underlies a major public health issue. Understanding how, in the absence of antibiotic-mediated selection, plasmid-bearing cells avoid being outnumbered by plasmid-free cells is key to developing counterstrategies. Here, we quantified the induction of the plasmidial sex pheromone pathway of Enterococcus faecalis to show that the integration of the stimulatory (mate-sensing) and inhibitory (self-sensing) signaling modules from the pCF10 conjugative plasmid provides a precise measure of the recipient-to-donor ratio, agnostic to variations in population size. Such ratiometric control of conjugation favors vertical plasmid transfer under low mating likelihood and allows activation of conjugation functions only under high mating likelihood. We further show that this strategy constitutes a cost-effective investment into mating effort because overstimulation produces unproductive self-aggregation and growth rate reduction. A mathematical model suggests that ratiometric control of conjugation increases plasmid fitness and predicts a robust long-term, stable coexistence of donors and recipients. Our results demonstrate how population-level parameters can control transfer of antibiotic resistance in bacteria, opening the door for biotic control strategies.


Assuntos
Proteínas de Bactérias/genética , Resistência Microbiana a Medicamentos/genética , Enterococcus faecalis/genética , Transferência Genética Horizontal , Feromônios/genética , Percepção de Quorum/efeitos dos fármacos , Antibacterianos/farmacologia , Carga Bacteriana , Proteínas de Bactérias/metabolismo , Conjugação Genética , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/metabolismo , Expressão Gênica , Aptidão Genética , Modelos Estatísticos , Feromônios/biossíntese , Plasmídeos/química , Plasmídeos/metabolismo , Percepção de Quorum/genética , Virulência
2.
Immunobiology ; 225(3): 151955, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32517882

RESUMO

SARS Coronavirus-2 (SARS-CoV-2) pandemic has become a global issue which has raised the concern of scientific community to design and discover a counter-measure against this deadly virus. So far, the pandemic has caused the death of hundreds of thousands of people upon infection and spreading. To date, no effective vaccine is available which can combat the infection caused by this virus. Therefore, this study was conducted to design possible epitope-based subunit vaccines against the SARS-CoV-2 virus using the approaches of reverse vaccinology and immunoinformatics. Upon continual computational experimentation, three possible vaccine constructs were designed and one vaccine construct was selected as the best vaccine based on molecular docking study which is supposed to effectively act against the SARS-CoV-2. Thereafter, the molecular dynamics simulation and in silico codon adaptation experiments were carried out in order to check biological stability and find effective mass production strategy of the selected vaccine. This study should contribute to uphold the present efforts of the researches to secure a definitive preventative measure against this lethal disease.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/tratamento farmacológico , Pandemias , Pneumonia Viral/tratamento farmacológico , Proteínas Virais/química , Vacinas Virais/biossíntese , Sequência de Aminoácidos , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/patogenicidade , Biologia Computacional/métodos , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Progressão da Doença , Epitopos/química , Epitopos/imunologia , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Antígenos HLA/genética , Antígenos HLA/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunogenicidade da Vacina , Simulação de Acoplamento Molecular , Plasmídeos/química , Plasmídeos/imunologia , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Conformação Proteica , Genética Reversa/métodos , Alinhamento de Sequência , Vacinas de Subunidades , Proteínas Virais/genética , Proteínas Virais/imunologia
3.
Virus Res ; 286: 198074, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32589897

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel human coronavirus causing the pandemic of severe pneumonia (Coronavirus Disease 2019, COVID-19). SARS-CoV-2 is highly pathogenic in human, having posed immeasurable public health challenges to the world. Innate immune response is critical for the host defense against viral infection and the dysregulation of the host innate immune responses probably aggravates SARS-CoV-2 infection, contributing to the high morbidity and lethality of COVID-19. It has been reported that some coronavirus proteins play an important role in modulating innate immunity of the host, but few studies have been conducted on SARS-CoV-2. In this study, we screened the viral proteins of SARS-CoV-2 and found that the viral ORF6, ORF8 and nucleocapsid proteins were potential inhibitors of type I interferon signaling pathway, a key component for antiviral response of host innate immune. All the three proteins showed strong inhibition on type I interferon (IFN-ß) and NF-κB-responsive promoter, further examination revealed that these proteins were able to inhibit the interferon-stimulated response element (ISRE) after infection with Sendai virus, while only ORF6 and ORF8 proteins were able to inhibit the ISRE after treatment with interferon beta. These findings would be helpful for the further study of the detailed signaling pathway and unveil the key molecular player that may be targeted.


Assuntos
Betacoronavirus/genética , Interações Hospedeiro-Patógeno/genética , Interferon beta/genética , NF-kappa B/genética , Proteínas do Nucleocapsídeo/genética , Proteínas Virais/genética , Betacoronavirus/imunologia , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Interferon beta/imunologia , Luciferases/genética , Luciferases/metabolismo , NF-kappa B/imunologia , Proteínas do Nucleocapsídeo/imunologia , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Elementos de Resposta , Vírus Sendai/genética , Vírus Sendai/imunologia , Transdução de Sinais , Transfecção/métodos , Proteínas Virais/imunologia
4.
Virus Res ; 286: 198074, 2020 09.
Artigo em Inglês | MEDLINE | ID: covidwho-611212

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel human coronavirus causing the pandemic of severe pneumonia (Coronavirus Disease 2019, COVID-19). SARS-CoV-2 is highly pathogenic in human, having posed immeasurable public health challenges to the world. Innate immune response is critical for the host defense against viral infection and the dysregulation of the host innate immune responses probably aggravates SARS-CoV-2 infection, contributing to the high morbidity and lethality of COVID-19. It has been reported that some coronavirus proteins play an important role in modulating innate immunity of the host, but few studies have been conducted on SARS-CoV-2. In this study, we screened the viral proteins of SARS-CoV-2 and found that the viral ORF6, ORF8 and nucleocapsid proteins were potential inhibitors of type I interferon signaling pathway, a key component for antiviral response of host innate immune. All the three proteins showed strong inhibition on type I interferon (IFN-ß) and NF-κB-responsive promoter, further examination revealed that these proteins were able to inhibit the interferon-stimulated response element (ISRE) after infection with Sendai virus, while only ORF6 and ORF8 proteins were able to inhibit the ISRE after treatment with interferon beta. These findings would be helpful for the further study of the detailed signaling pathway and unveil the key molecular player that may be targeted.


Assuntos
Betacoronavirus/genética , Interações Hospedeiro-Patógeno/genética , Interferon beta/genética , NF-kappa B/genética , Proteínas do Nucleocapsídeo/genética , Proteínas Virais/genética , Betacoronavirus/imunologia , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Interferon beta/imunologia , Luciferases/genética , Luciferases/metabolismo , NF-kappa B/imunologia , Proteínas do Nucleocapsídeo/imunologia , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Elementos de Resposta , Vírus Sendai/genética , Vírus Sendai/imunologia , Transdução de Sinais , Transfecção/métodos , Proteínas Virais/imunologia
5.
Immunobiology ; 225(3): 151955, 2020 05.
Artigo em Inglês | MEDLINE | ID: covidwho-309013

RESUMO

SARS Coronavirus-2 (SARS-CoV-2) pandemic has become a global issue which has raised the concern of scientific community to design and discover a counter-measure against this deadly virus. So far, the pandemic has caused the death of hundreds of thousands of people upon infection and spreading. To date, no effective vaccine is available which can combat the infection caused by this virus. Therefore, this study was conducted to design possible epitope-based subunit vaccines against the SARS-CoV-2 virus using the approaches of reverse vaccinology and immunoinformatics. Upon continual computational experimentation, three possible vaccine constructs were designed and one vaccine construct was selected as the best vaccine based on molecular docking study which is supposed to effectively act against the SARS-CoV-2. Thereafter, the molecular dynamics simulation and in silico codon adaptation experiments were carried out in order to check biological stability and find effective mass production strategy of the selected vaccine. This study should contribute to uphold the present efforts of the researches to secure a definitive preventative measure against this lethal disease.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/tratamento farmacológico , Pandemias , Pneumonia Viral/tratamento farmacológico , Proteínas Virais/química , Vacinas Virais/biossíntese , Sequência de Aminoácidos , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/patogenicidade , Biologia Computacional/métodos , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Progressão da Doença , Epitopos/química , Epitopos/imunologia , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Antígenos HLA/genética , Antígenos HLA/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunogenicidade da Vacina , Simulação de Acoplamento Molecular , Plasmídeos/química , Plasmídeos/imunologia , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Conformação Proteica , Genética Reversa/métodos , Alinhamento de Sequência , Vacinas de Subunidades , Proteínas Virais/genética , Proteínas Virais/imunologia
6.
PLoS One ; 15(5): e0233298, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469898

RESUMO

BACKGROUND: Evolutionary studies have been conducted that have investigated the chromosomal variance in the genus of Chlamydia. However, no all-encompassing genus-wide comparison has been performed on the plasmid. Therefore, there is a gap in the current knowledge on Chlamydia plasmid diversity. AIMS: This project is aimed to investigate and establish the nature and extent of diversity across the entire genus of Chlamydia, by comparing the sequences of all currently available plasmid carrying strains. METHODS: The PUBMED database was used to identify plasmid sequences from all available strains that met the set quality criteria for their inclusion in the study. Alignments were performed on the 51 strains that fulfilled the criteria using MEGA X software. Following that Maximum Likelihood estimation was used to construct 11 phylogenetic trees of the whole plasmid sequence, the individual 8 coding sequences, the iteron and a chromosomal gene ompA as a comparator. RESULTS: The genus-wide plasmid phylogeny produced three distinct lineages labelled as alpha, beta and gamma. Nineteen genotypes were found in the initial whole plasmid analysis. Their distribution was allocated as six C. pecorum, two C. pneumoniae, one C. gallinacea, one C. avium, one C. caviae, one C. felis, two C. psittaci, one C. trachomatis, one C. muridarum, and two C. suis. The chromosomal comparative gene ompA supported this distribution, with the same number of primary clades with the same species distribution. However, ompA sequence comparison resulted in fewer genotypes due to a reduced amount of available sequences (33 out of 51). All results were statistically significant. CONCLUSION: The results of this study indicate that the common bacterial ancestor of all the species had a plasmid, which has diverged over time. Moreover, it suggests that there is a strong evolutionary selection towards these species retaining their plasmids due to its high level of conservation across the genus, with the notable exception of C. pneumoniae. Furthermore, the evolutionary analysis showed that the plasmid and the chromosome have co-evolved.


Assuntos
Infecções por Chlamydia/microbiologia , Chlamydia/genética , Variação Genética , Plasmídeos/genética , Animais , Chlamydia/química , Genoma Bacteriano , Genótipo , Filogenia , Plasmídeos/química , Plasmídeos/classificação , Análise de Sequência de DNA
7.
Nat Chem Biol ; 16(7): 725-730, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32284602

RESUMO

Anti-CRISPR (Acr) proteins are powerful tools to control CRISPR-Cas technologies. However, the available Acr repertoire is limited to naturally occurring variants. Here, we applied structure-based design on AcrIIC1, a broad-spectrum CRISPR-Cas9 inhibitor, to improve its efficacy on different targets. We first show that inserting exogenous protein domains into a selected AcrIIC1 surface site dramatically enhances inhibition of Neisseria meningitidis (Nme)Cas9. Then, applying structure-guided design to the Cas9-binding surface, we converted AcrIIC1 into AcrIIC1X, a potent inhibitor of the Staphylococcus aureus (Sau)Cas9, an orthologue widely applied for in vivo genome editing. Finally, to demonstrate the utility of AcrIIC1X for genome engineering applications, we implemented a hepatocyte-specific SauCas9 ON-switch by placing AcrIIC1X expression under regulation of microRNA-122. Our work introduces designer Acrs as important biotechnological tools and provides an innovative strategy to safeguard CRISPR technologies.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , MicroRNAs/genética , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Genoma Humano , Células HEK293 , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , MicroRNAs/metabolismo , Modelos Moleculares , Mutagênese Insercional , Neisseria meningitidis/enzimologia , Neisseria meningitidis/genética , Plasmídeos/química , Plasmídeos/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , RNA Guia/genética , RNA Guia/metabolismo , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética
8.
Phys Chem Chem Phys ; 22(13): 6984-6992, 2020 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-32188961

RESUMO

Ectoine is a small zwitterionic osmolyte and compatible solute, which does not interfere with cell metabolism even at molar concentrations. Plasmid DNA (pUC19) was irradiated with ultraviolet radiation (UV-C at 266 nm) under quasi physiological conditions (PBS) and in pure water in the presence and absence of ectoine (THP(B)) and hydroxyectoine (THP(A)). Different types of UV induced DNA damage were analysed: DNA single-strand breaks (SSBs), abasic sites and cyclobutane pyrimidine dimers (CPDs). A complex interplay between these factors was observed with respect to the nature and occurrence of DNA damage with 266 nm photons. In PBS, the cosolutes showed efficient protection against base damage, whilst in pure water, a dramatic shift from SSB damage to base damage was observed when cosolutes were added. To test whether these effects are caused by ectoine binding to DNA, further experiments were conducted: small-angle X-ray scattering (SAXS), surface-plasmon resonance (SPR) measurements and Raman spectroscopy. The results show, for the first time, a close interaction between ectoine and DNA. This is in stark contrast to the assumption made by preferential exclusion models, which are often used to interpret the behaviour of compatible solutes within cells and with biomolecules. It is tentatively proposed that the alterations of UV damage to DNA are attributed to ectoine influence on nucleobases through the direct interaction between ectoine and DNA.


Assuntos
Diamino Aminoácidos/metabolismo , Dano ao DNA/genética , DNA/metabolismo , DNA/efeitos da radiação , Raios Ultravioleta , DNA/química , Plasmídeos/química , Plasmídeos/metabolismo , Plasmídeos/efeitos da radiação , Espalhamento a Baixo Ângulo , Ressonância de Plasmônio de Superfície , Difração de Raios X
9.
Nat Microbiol ; 5(4): 620-629, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32218510

RESUMO

CRISPR-Cas adaptive immune systems protect bacteria and archaea against their invading genetic parasites, including bacteriophages/viruses and plasmids. In response to this immunity, many phages have anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas targeting. To date, anti-CRISPR genes have primarily been discovered in phage or prophage genomes. Here, we uncovered acr loci on plasmids and other conjugative elements present in Firmicutes using the Listeria acrIIA1 gene as a marker. The four identified genes, found in Listeria, Enterococcus, Streptococcus and Staphylococcus genomes, can inhibit type II-A SpyCas9 or SauCas9, and are thus named acrIIA16-19. In Enterococcus faecalis, conjugation of a Cas9-targeted plasmid was enhanced by anti-CRISPRs derived from Enterococcus conjugative elements, highlighting a role for Acrs in the dissemination of plasmids. Reciprocal co-immunoprecipitation showed that each Acr protein interacts with Cas9, and Cas9-Acr complexes were unable to cleave DNA. Northern blotting suggests that these anti-CRISPRs manipulate single guide RNA length, loading or stability. Mirroring their activity in bacteria, AcrIIA16 and AcrIIA17 provide robust and highly potent broad-spectrum inhibition of distinct Cas9 proteins in human cells (for example, SpyCas9, SauCas9, SthCas9, NmeCas9 and CjeCas9). This work presents a focused analysis of non-phage Acr proteins, demonstrating a role in horizontal gene transfer bolstered by broad-spectrum CRISPR-Cas9 inhibition.


Assuntos
Proteína 9 Associada à CRISPR/antagonistas & inibidores , Sistemas CRISPR-Cas , Transferência Genética Horizontal , Plasmídeos/metabolismo , RNA Guia/antagonistas & inibidores , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Conjugação Genética , DNA/antagonistas & inibidores , DNA/genética , DNA/metabolismo , Enterococcus/genética , Enterococcus/virologia , Células HEK293 , Humanos , Listeria/genética , Listeria/virologia , Plasmídeos/química , Ligação Proteica , RNA Guia/genética , RNA Guia/metabolismo , Staphylococcus/genética , Staphylococcus/virologia , Streptococcus/genética , Streptococcus/virologia
10.
Biochem Biophys Res Commun ; 524(4): 825-831, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32037086

RESUMO

Chromatin organization starts from a "beads-on-a string" 10 nm fiber, a basic nucleosomal structure consisting of DNA and core histones. Given its regular nucleosome array on DNA backbone where N-terminal tails of each histone are exposed on the surface of chromatin fiber, we hypothesized that chromatin can be utilized as a heterologous peptide carrier to elicit a peptide-specific immune response. The plasmid DNA containing the Widom's clone 601 sequence and the recombinant chimeric histones containing the peptide derived from ras oncogene (G12V) were used to assemble the chromatin fiber in vitro. The immunogenicity of the assembled chromatin was tested in mice as a single vaccine component or formulated with adjuvants. G12V tagged-chromatin co-administered with adjuvants induced higher antibody responses against the G12V peptide than vaccination with adjuvant alone, while chimeric histones did not generate a significant antibody response. Interestingly, splenocytes from mice vaccinated with the G12V tagged-chromatin vaccine did not generate significant antigen-specific cytokine responses. Our studies suggest that chromatin can be utilized as an effective carrier of antigenic peptides for inducing specific antibody responses.


Assuntos
Vacinas Anticâncer/biossíntese , Genes ras/imunologia , Histonas/imunologia , Nanofibras/química , Biblioteca de Peptídeos , Peptídeos/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/química , Animais , Anticorpos/genética , Anticorpos/metabolismo , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Montagem e Desmontagem da Cromatina , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/prevenção & controle , Nucleossomos/química , Nucleossomos/imunologia , Nucleossomos/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Plasmídeos/química , Plasmídeos/imunologia , Plasmídeos/metabolismo , Vacinas de Subunidades , Xenopus laevis
11.
PLoS One ; 15(1): e0220484, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31990938

RESUMO

The growing occurrence of multidrug-resistant (MDR) Salmonella enterica in poultry has been reported with public health concern worldwide. We reported, recently, the occurrence of Escherichia coli and Salmonella enterica serovars carrying clinically relevant resistance genes in dairy cattle farms in the Wakiso District, Uganda, highlighting an urgent need to monitor food-producing animal environments. Here, we present the prevalence, antimicrobial resistance, and sequence type of 51 Salmonella isolates recovered from 379 environmental samples from chicken farms in Uganda. Among the Salmonella isolates, 32/51 (62.7%) were resistant to at least one antimicrobial, and 10/51 (19.6%) displayed multiple drug resistance. Through PCR, five replicon plasmids were identified among chicken Salmonella isolates including IncFIIS 17/51 (33.3%), IncI1α 12/51 (23.5%), IncP 8/51 (15.7%), IncX1 8/51 (15.7%), and IncX2 1/51 (2.0%). In addition, we identified two additional replicons through WGS (Whole Genome Sequencing; ColpVC and IncFIB). A significant seasonal difference between chicken sampling periods was observed (p = 0.0017). We conclude that MDR Salmonella highlights the risks posed to animals and humans. Implementing a robust, integrated surveillance system will aid in monitoring MDR zoonotic threats.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Genes Bacterianos , Plasmídeos/metabolismo , Doenças das Aves Domésticas/epidemiologia , Infecções por Salmonella/epidemiologia , Salmonella enterica/genética , Animais , Antibacterianos/classificação , Antibacterianos/farmacologia , Galinhas/microbiologia , Fazendas , Humanos , Vigilância Imunológica , Plasmídeos/química , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/transmissão , Prevalência , Replicon , Infecções por Salmonella/microbiologia , Infecções por Salmonella/transmissão , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/isolamento & purificação , Estações do Ano , Uganda/epidemiologia , Sequenciamento Completo do Genoma
12.
Nucleic Acids Res ; 48(5): e25, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31943080

RESUMO

Allele-specific protospacer adjacent motif (asPAM)-positioning SNPs and CRISPRs are valuable resources for gene therapy of dominant disorders. However, one technical hurdle is to identify the haplotype comprising the disease-causing allele and the distal asPAM SNPs. Here, we describe a novel CRISPR-based method (CRISPR-hapC) for haplotyping. Based on the generation (with a pair of CRISPRs) of extrachromosomal circular DNA in cells, the CRISPR-hapC can map haplotypes from a few hundred bases to over 200 Mb. To streamline and demonstrate the applicability of the CRISPR-hapC and asPAM CRISPR for allele-specific gene editing, we reanalyzed the 1000 human pan-genome and generated a high frequency asPAM SNP and CRISPR database (www.crispratlas.com/knockout) for four CRISPR systems (SaCas9, SpCas9, xCas9 and Cas12a). Using the huntingtin (HTT) CAG expansion and transthyretin (TTR) exon 2 mutation as examples, we showed that the asPAM CRISPRs can specifically discriminate active and dead PAMs for all 23 loci tested. Combination of the CRISPR-hapC and asPAM CRISPRs further demonstrated the capability for achieving highly accurate and haplotype-specific deletion of the HTT CAG expansion allele and TTR exon 2 mutation in human cells. Taken together, our study provides a new approach and an important resource for genome research and allele-specific (haplotype-specific) gene therapy.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Circular/genética , RNA Guia/genética , Alelos , Sequência de Bases , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , DNA Circular/metabolismo , Edição de Genes/métodos , Células HEK293 , Haplótipos , Células Hep G2 , Humanos , Plasmídeos/química , Plasmídeos/metabolismo , RNA Guia/metabolismo
13.
Nucleic Acids Res ; 48(5): e28, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31980824

RESUMO

We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for binding to overlapping target sites in a plasmid-borne lacI promoter variant. The assay utilizes two plasmids and an E. coli host strain, from which the gene of the Lac repressor (lacI) has been deleted. One of the plasmids carries the lacI gene with a unique NheI restriction site created in the lacI promoter. The potential recognition sequences of the tested protein are inserted into the NheI site. Introduction of the plasmids into the E. coliΔlacI host represses the constitutive ß-galactosidase synthesis of the host bacterium. If the studied protein expressed from a compatible plasmid binds to its target site in the lacI promoter, it will interfere with lacI transcription and lead to increased ß-galactosidase activity. The method was tested with two zinc finger proteins, with the lambda phage cI857 repressor, and with CRISPR-dCas9 targeted to the lacI promoter. The I-Block assay was shown to work with standard liquid cultures, with cultures grown in microplate and with colonies on X-gal indicator plates.


Assuntos
Bioensaio , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Transcrição Genética , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Sistemas CRISPR-Cas , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Repressores Lac/deficiência , Repressores Lac/genética , Plasmídeos/química , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
14.
Inorg Chem ; 59(4): 2288-2298, 2020 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-31986027

RESUMO

Cancer is the uncontrolled growth of abnormal cells via malignant cell division and rapid DNA replication. While DNA damaging molecules can cause cancer, their role as anticancer drugs are very significant. For this purpose, the novel series of paraben substituted spermine bridged(dispirobino) cyclotriphosphazene compounds 2-6 were synthesized for the first time, and their structures were characterized by various spectroscopic techniques. The solid-state structures and geometries of compounds 2-6 were determined using single-crystal X-ray structural analysis. In addition, it was confirmed by TGA that all compounds 1-6 showed high thermal stability. Two methods were used in order to investigate DNA interaction properties of the targeted molecules. While biosensor-based screening test that measures DNA hybridization efficiency on a biochip surface, the agarose gel electrophoresis method examines the effect of compounds on plasmid DNA structure. The results collected from the automated biosensor device and agarose gel electrophoresis have indicated that compounds 1, 5, and 6 showed higher DNA damage than the compounds 2-4. According to the biosensor results, compounds 1, 5, and 6 showed 85%, 69%, and 77% activity, respectively.


Assuntos
DNA/química , Compostos Organofosforados/química , Parabenos/química , Plasmídeos/química , Espermina/análogos & derivados , Técnicas Biossensoriais , Dano ao DNA , Eletroforese em Gel de Ágar , Compostos Organofosforados/síntese química , Parabenos/síntese química , Espermina/síntese química
15.
Sci Adv ; 6(3): eaax5032, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31998834

RESUMO

While immunotherapy holds great promise for combating cancer, the limited efficacy due to an immunosuppressive tumor microenvironment and systemic toxicity hinder the broader application of cancer immunotherapy. Here, we report a combinatorial immunotherapy approach that uses a highly efficient and tumor-selective gene carrier to improve anticancer efficacy and circumvent the systemic toxicity. In this study, we engineered tumor-targeted lipid-dendrimer-calcium-phosphate (TT-LDCP) nanoparticles (NPs) with thymine-functionalized dendrimers that exhibit not only enhanced gene delivery capacity but also immune adjuvant properties by activating the stimulator of interferon genes (STING)-cGAS pathway. TT-LDCP NPs delivered siRNA against immune checkpoint ligand PD-L1 and immunostimulatory IL-2-encoding plasmid DNA to hepatocellular carcinoma (HCC), increased tumoral infiltration and activation of CD8+ T cells, augmented the efficacy of cancer vaccine immunotherapy, and suppressed HCC progression. Our work presents nanotechnology-enabled dual delivery of siRNA and plasmid DNA that selectively targets and reprograms the immunosuppressive tumor microenvironment to improve cancer immunotherapy.


Assuntos
Biomarcadores Tumorais , Fenômenos Imunogenéticos , Terapia de Alvo Molecular , Nanopartículas , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/terapia , Nanomedicina Teranóstica , Animais , Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores/metabolismo , Fosfatos de Cálcio/química , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Sistemas de Liberação de Medicamentos , Técnicas de Transferência de Genes , Terapia Genética , Humanos , Imunoterapia , Lipídeos/química , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Nanopartículas/química , Nanopartículas/ultraestrutura , Nanotecnologia , Neoplasias/patologia , Plasmídeos/administração & dosagem , Plasmídeos/química , Plasmídeos/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Transdução de Sinais
16.
Methods Mol Biol ; 2119: 183-199, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31989525

RESUMO

Identification of the protein complexes associated with defined DNA sequence elements is essential to understand the numerous transactions in which DNA is involved, such as replication, repair, transcription, and chromatin dynamics. Here we describe two protocols, IDAP (Isolation of DNA Associated Proteins) and CoIFI (Chromatin-of-Interest Fragment Isolation), that allow for isolating DNA/protein complexes (i.e., nucleoprotein elements) by means of a DNA capture tool based on DNA triple helix (triplex) formation. Typically, IDAP is used to capture proteins that bind to a given DNA element of interest (e.g., a specific DNA sequence, an unusual DNA structure, a DNA lesion) that can be introduced at will into plasmids. The plasmids are immobilized by means of a triplex-forming probe on magnetic beads and incubated in nuclear extracts; by using in parallel a control plasmid (that lacks the DNA element of interest), proteins that preferentially bind to the DNA element of interest are captured and identified by mass spectrometry. Similarly, CoIFI also uses a triplex-forming probe to capture a specific chromatin fragment from a cultured cell line that has been engineered to contain multiple copies of the DNA element of interest.


Assuntos
Cromatina , Proteínas de Ligação a DNA , DNA , Campos Magnéticos , Plasmídeos/química , Cromatina/química , Cromatina/isolamento & purificação , DNA/química , DNA/isolamento & purificação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Células HEK293 , Humanos
17.
Biochemistry ; 59(7): 892-900, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-31977191

RESUMO

Colibactin is a genotoxic gut microbiome metabolite long suspected of playing an etiological role in colorectal cancer. Evidence suggests that colibactin forms DNA interstrand cross-links (ICLs) in eukaryotic cells and activates ICL repair pathways, leading to the production of ICL-dependent DNA double-strand breaks (DSBs). Here we show that colibactin ICLs can evolve directly to DNA DSBs. Using the topology of supercoiled plasmid DNA as a proxy for alkylation adduct stability, we find that colibactin-derived ICLs are unstable toward depurination and elimination of the 3' phosphate. This ICL degradation pathway leads progressively to single strand breaks (SSBs) and subsequently DSBs. The spontaneous conversion of ICLs to DSBs is consistent with the finding that nonhomologous end joining repair-deficient cells are sensitized to colibactin-producing bacteria. The results herein refine our understanding of colibactin-derived DNA damage and underscore the complexities underlying the DSB phenotype.


Assuntos
Reagentes para Ligações Cruzadas/farmacologia , DNA/metabolismo , Peptídeos/farmacologia , Policetídeos/farmacologia , Reagentes para Ligações Cruzadas/química , DNA/química , DNA/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , Reparo do DNA , Desoxirribonuclease IV (Fago T4-Induzido)/química , Escherichia coli/química , Peptídeos/química , Plasmídeos/química , Policetídeos/química
18.
Nucleic Acids Res ; 48(1): 486-499, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31745563

RESUMO

Cross-species pathway transplantation enables insight into a biological process not possible through traditional approaches. We replaced the enzymes catalyzing the entire Saccharomyces cerevisiae adenine de novo biosynthesis pathway with the human pathway. While the 'humanized' yeast grew in the absence of adenine, it did so poorly. Dissection of the phenotype revealed that PPAT, the human ortholog of ADE4, showed only partial function whereas all other genes complemented fully. Suppressor analysis revealed other pathways that play a role in adenine de-novo pathway regulation. Phylogenetic analysis pointed to adaptations of enzyme regulation to endogenous metabolite level 'setpoints' in diverse organisms. Using DNA shuffling, we isolated specific amino acids combinations that stabilize the human protein in yeast. Thus, using adenine de novo biosynthesis as a proof of concept, we suggest that the engineering methods used in this study as well as the debugging strategies can be utilized to transplant metabolic pathway from any origin into yeast.


Assuntos
Adenina/biossíntese , Vias Biossintéticas/genética , Carboxiliases/genética , Cromossomos Artificiais Humanos/química , Peptídeo Sintases/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sistemas CRISPR-Cas , Carboxiliases/metabolismo , Cromossomos Artificiais Humanos/metabolismo , Teste de Complementação Genética , Engenharia Genética/métodos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Peptídeo Sintases/metabolismo , Filogenia , Plasmídeos/química , Plasmídeos/metabolismo , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
19.
Nucleic Acids Res ; 48(1): e1, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31612958

RESUMO

Multiplex genetic assays can simultaneously test thousands of genetic variants for a property of interest. However, limitations of existing multiplex assay methods in cultured mammalian cells hinder the breadth, speed and scale of these experiments. Here, we describe a series of improvements that greatly enhance the capabilities of a Bxb1 recombinase-based landing pad system for conducting different types of multiplex genetic assays in various mammalian cell lines. We incorporate the landing pad into a lentiviral vector, easing the process of generating new landing pad cell lines. We also develop several new landing pad versions, including one where the Bxb1 recombinase is expressed from the landing pad itself, improving recombination efficiency more than 2-fold and permitting rapid prototyping of transgenic constructs. Other versions incorporate positive and negative selection markers that enable drug-based enrichment of recombinant cells, enabling the use of larger libraries and reducing costs. A version with dual convergent promoters allows enrichment of recombinant cells independent of transgene expression, permitting the assessment of libraries of transgenes that perturb cell growth and survival. Lastly, we demonstrate these improvements by assessing the effects of a combinatorial library of oncogenes and tumor suppressors on cell growth. Collectively, these advancements make multiplex genetic assays in diverse cultured cell lines easier, cheaper and more effective, facilitating future studies probing how proteins impact cell function, using transgenic variant libraries tested individually or in combination.


Assuntos
Bioensaio , Biblioteca Gênica , Plasmídeos/química , Transgenes , Animais , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HT29 , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Células NIH 3T3 , Oncogenes , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinases/genética , Recombinases/metabolismo , Recombinação Genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
20.
Nucleic Acids Res ; 48(1): 212-230, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31665437

RESUMO

When replication forks encounter template DNA lesions, the lesion is simply skipped in some cases. The resulting lesion-containing gap must be converted to duplex DNA to permit repair. Some gap filling occurs via template switching, a process that generates recombination-like branched DNA intermediates. The Escherichia coli Uup and RadD proteins function in different pathways to process the branched intermediates. Uup is a UvrA-like ABC family ATPase. RadD is a RecQ-like SF2 family ATPase. Loss of both functions uncovers frequent and RecA-independent deletion events in a plasmid-based assay. Elevated levels of crossing over and repeat expansions accompany these deletion events, indicating that many, if not most, of these events are associated with template switching in postreplication gaps as opposed to simple replication slippage. The deletion data underpin simulations indicating that multiple postreplication gaps may be generated per replication cycle. Both Uup and RadD bind to branched DNAs in vitro. RadD protein suppresses crossovers and Uup prevents nucleoid mis-segregation. Loss of Uup and RadD function increases sensitivity to ciprofloxacin. We present Uup and RadD as genomic guardians. These proteins govern two pathways for resolution of branched DNA intermediates such that potentially deleterious genome rearrangements arising from frequent template switching are averted.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Proteínas de Bactérias/química , Replicação do DNA , DNA Bacteriano/genética , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Transportadores de Cassetes de Ligação de ATP/deficiência , Adenosina Trifosfatases/deficiência , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ciprofloxacino/farmacologia , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Genoma Bacteriano , Plasmídeos/química , Plasmídeos/metabolismo , Origem de Replicação , Deleção de Sequência
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