RESUMO
MB-003, a plant-derived monoclonal antibody cocktail used effectively in treatment of Ebola virus infection in non-human primates, was unable to protect two of six animals when initiated 1 or 2 days post-infection. We characterized a mechanism of viral escape in one of the animals, after observation of two clusters of genomic mutations that resulted in five nonsynonymous mutations in the monoclonal antibody target sites. These mutations were linked to a reduction in antibody binding and later confirmed to be present in a viral isolate that was not neutralized in vitro. Retrospective evaluation of a second independent study allowed the identification of a similar case. Four SNPs in previously identified positions were found in this second fatality, suggesting that genetic drift could be a potential cause for treatment failure. These findings highlight the importance selecting different target domains for each component of the cocktail to minimize the potential for viral escape.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Ebolavirus/imunologia , Doença pelo Vírus Ebola/virologia , Evasão da Resposta Imune/genética , Imunização Passiva , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Sequência de Bases , Ebolavirus/genética , Ebolavirus/patogenicidade , Epitopos/química , Epitopos/imunologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/mortalidade , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Macaca mulatta , Dados de Sequência Molecular , Mutação , Ligação Proteica , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Estudos Retrospectivos , Análise de Sobrevida , Nicotiana/genética , Replicação ViralRESUMO
Excision of the conjugative transposon CTnDOT from the chromosome of Bacteroides spp. involves four CTnDOT-encoded proteins: IntDOT, Xis2c, Xis2d, and Exc along with a host factor. These proteins form excisive intasomes on the attR and attL sites which undergo synapsis and recombination to form the attDOT and attB sites. We recently developed an in vitro intramolecular excision reaction where the attL and attR sites are on the same plasmid. This reaction requires IntDOT, Xis2c, Xis2d, and is stimulated by Exc. We used this reaction to identify the binding sites of the IntDOT, Xis2c, and Xis2d. In this paper, we show that three of the six arm-type sites are absolutely required for excision. Furthermore, we also identified two binding sites for Xis2d and two possible binding sites for Xis2c on the attR site. We also showed that IntDOT interacts cooperatively with the Xis2c and Xis2d proteins on the attR site.
Assuntos
Proteínas de Bactérias/metabolismo , Bacteroides/genética , Conjugação Genética/genética , Elementos de DNA Transponíveis/genética , Proteínas de Membrana/metabolismo , Plasmídeos/genética , Recombinação Genética/genética , Sítios de Ligação/genética , Pareamento Cromossômico/genética , Conjugação Genética/fisiologia , Primers do DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Mutagênese , Recombinação Genética/fisiologiaRESUMO
The Bacteroides conjugative transposon, CTnDOT, is an integrated conjugative element (ICE), found in many human colonic Bacteroides spp. strains. It has a complex regulatory system for both excision from the chromosome and transfer and mobilization into a new host. It was previously shown that a cloned DNA segment encoding the xis2c, xis2d, orf3, and exc genes was required for tetracycline dependent activation of the P(tra) promoter. The Xis2c and Xis2d proteins are required for excision while the Exc protein stimulates excision. We report here that neither the Orf3 nor the Exc proteins are involved in activation of the P(tra) promoter. Deletion analysis and electromobility shift assays showed that the Xis2c and Xis2d proteins bind to the P(tra) promoter to activate the tra operon. Thus, the recombination directionality factors of CTnDOT excision also function as activator proteins of the P(tra) promoter.
Assuntos
Proteínas de Bactérias/metabolismo , Bacteroides/genética , Conjugação Genética , Elementos de DNA Transponíveis/genética , Óperon/genética , Bacteroides/efeitos dos fármacos , Sequência de Bases , Conjugação Genética/efeitos dos fármacos , Ensaio de Desvio de Mobilidade Eletroforética , Deleção de Genes , Glucuronidase/metabolismo , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Tetraciclina/farmacologiaRESUMO
Excision from the chromosome is the first step during the transfer of conjugative transposons (CTns) to a recipient. We previously showed that the excision of CTnDOT is more complex than the excision of lambdoid phages and CTns such as Tn916. The excision in vivo of CTnDOT utilizes four CTnDOT-encoded proteins, IntDOT, Xis2c, Xis2d, and Exc, and a host factor. We previously developed an in vitro excision reaction where the recombination sites attL and attR were located on different plasmids. The reaction was inefficient and did not require Exc, suggesting that the reaction conditions did not mimic in vivo conditions. Here, we report the development of an intramolecular excision reaction where the attL and attR sites are located on the same DNA molecule. We found that Exc stimulates the reaction 3- to 5-fold. The efficiency of the excision reaction was also dependent on the distance between the attL and attR sites and on the sequences of the overlap regions between the sites of the strand exchanges. Substrates with identical overlap sequences recombined more efficiently than ones with heterologous overlap sequences. This was surprising, because the integration reaction is not sensitive to heterology in the overlap regions of the attDOT and attB sites.
