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Spore surface display of enzymes used for molecular diagnosis by RT-LAMP
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Orientador(es)
Resumo(s)
O uso de enzimas em larga escala para aplicações industriais ou diagnóstico
molecular é limitado devido ao alto custo de produção e purificação e à baixa estabilidade
das mesmas. Estas dificuldades limitam a utilização destas enzimas por laboratórios com
orçamentos limitados. O LAMP (Loop-mediated isothermal amplification) é um teste de
amplificação de ácidos nucleicos desenvolvido nos anos 2000 como alternativa mais
barata e simples ao PCR na deteção de patogénios, o teste mais utilizado no diagnostico
molecular. A principal enzima utilizada no LAMP é a polimerase de DNA Bst do Bacillus
stearothermophillus. No caso da deteção de patogénios que têm RNA como o seu material
genético, é necessário a adição de uma transcriptase reversa à reação de LAMP (RT LAMP). LAMP não necessita de ciclos de temperatura e por isso consegue ser
implementado em laboratórios com poucos recursos. De modo a tornar esta técnica mais
acessível, seria de interesse desenvolver uma plataforma simples e robusta para a
produção e purificação da Bst e da transcriptase reversa. Esta ideia poderia ser posta em
prática com a apresentação destas enzimas à superfície de um organismo de forma a
contornar purificações, aumentar a sua estabilidade e permitir a sua reutilização. Uma das
opções mais bem estudada são os esporos de Bacillus subtilis, que foram usados com
sucesso no passado para a apresentação de diversas proteínas à sua superfície com vista
a diversas aplicações em biotecnologia e biomedicina.
Este estudo foca-se na apresentação das enzimas utilizadas em RT-LAMP, a Bst
e a MashUp-RT, na superfície dos esporos de Bacillus subtilis. De modo a obter uma
maior quantidade de enzima e aumentar a exposição destas, as proteínas escolhidas como
âncora foram CotZ e CotY. Ambas são componentes abundantes da crosta, a camada mais
exterior do esporo. Conseguimos apresentar ambas as enzimas à superfície do esporo,
com um impacto mínimo no rendimento da produção de esporos e na sua estrutura e
propriedades. É importante notar que, quando CotZ foi utilizada como âncora, várias
proteínas do manto externo do esporo estavam ausentes, sugerindo que esta é importante
para manter a integridade do manto externo. Também foi avaliado se esta metodologia
poderia ser utilizada na deteção de material genético de patogénios. Apenas a enzima
MashUp-RT apresentou atividade enzimática. Curiosamente, embora os esporos
recombinantes apresentando Bst não mostrassem atividade de DNA polimerase, a
atividade residual de transcriptase reversa da Bst comercial foi estabilizada na presença
de esporos selvagens.
Tanto quanto sabemos, este é o primeiro estudo sobre a apresentação de enzimas
utilizadas no diagnóstico molecular à superfície do esporo.
The use of enzymes in a wide scale for industrial applications or molecular diagnosis is limited, due to high production and purification costs and often their low stability. This can make access of these enzymes difficult to laboratories with scarce economic resources. Loop-mediated isothermal amplification, or LAMP, is a nucleic acid amplification method developed in the 2000’s as a less expensive and simple alternative to PCR, which is widely used in molecular diagnostics. The main enzyme used in LAMP is a DNA polymerase, Bst, from Bacillus stearothermophillus. For the detection of pathogens in which RNA is the genetic material, a reverse transcriptase is added to the LAMP reaction (RT-LAMP). LAMP does not require temperature cycling and therefore can be easily implemented in low-resource settings. To make LAMP even more accessible, it would be of interest to develop a platform enabling simple, robust, and inexpensive production and purification of the reverse transcriptase and Bst. This could be achieved by simply displaying the enzymes on the surface of organisms to obviate the need for purification steps, increase their stability and allow their reuse. A well-studied option is the surface of the Bacillus subtilis spore, which has been used in the past for the display of enzymes or antigens in view of various biotechnology and biomedicine applications This study is focused on the display of the RT-LAMP enzymes, Bst and a variant of the reverse transcriptase from feline immunodeficiency virus, MashUp-RT, at the surface of Bacillus subtilis spores. Aiming at obtaining a high enzyme concentration and increasing their exposure to the environment, the anchor proteins chosen for this study were CotZ and CotY. Both are abundant components of the crust, the outermost layer of the spore. We were able to display Bst and MashUp-RT at the spore surface, with a minimal impact on the yield of spores, their composition and functional properties. Importantly, we note that when CotZ was used as the anchor, several proteins of the outer coat were absent from spores, suggesting that CotZ is important to maintain the integrity of the outer coat. We also evaluated whether this system could be used to detect the genetic material of pathogens. Only MashUp-RT showed enzymatic activity. Interestingly, although the recombinant spores displaying Bst did not show DNA polymerase activity, the residual reverse transcriptase activity of the commercial enzyme was stabilized by wild type spores. To our knowledge, this isthe first report on the the use of spore display of enzymes used in molecular diagnostic testing.
The use of enzymes in a wide scale for industrial applications or molecular diagnosis is limited, due to high production and purification costs and often their low stability. This can make access of these enzymes difficult to laboratories with scarce economic resources. Loop-mediated isothermal amplification, or LAMP, is a nucleic acid amplification method developed in the 2000’s as a less expensive and simple alternative to PCR, which is widely used in molecular diagnostics. The main enzyme used in LAMP is a DNA polymerase, Bst, from Bacillus stearothermophillus. For the detection of pathogens in which RNA is the genetic material, a reverse transcriptase is added to the LAMP reaction (RT-LAMP). LAMP does not require temperature cycling and therefore can be easily implemented in low-resource settings. To make LAMP even more accessible, it would be of interest to develop a platform enabling simple, robust, and inexpensive production and purification of the reverse transcriptase and Bst. This could be achieved by simply displaying the enzymes on the surface of organisms to obviate the need for purification steps, increase their stability and allow their reuse. A well-studied option is the surface of the Bacillus subtilis spore, which has been used in the past for the display of enzymes or antigens in view of various biotechnology and biomedicine applications This study is focused on the display of the RT-LAMP enzymes, Bst and a variant of the reverse transcriptase from feline immunodeficiency virus, MashUp-RT, at the surface of Bacillus subtilis spores. Aiming at obtaining a high enzyme concentration and increasing their exposure to the environment, the anchor proteins chosen for this study were CotZ and CotY. Both are abundant components of the crust, the outermost layer of the spore. We were able to display Bst and MashUp-RT at the spore surface, with a minimal impact on the yield of spores, their composition and functional properties. Importantly, we note that when CotZ was used as the anchor, several proteins of the outer coat were absent from spores, suggesting that CotZ is important to maintain the integrity of the outer coat. We also evaluated whether this system could be used to detect the genetic material of pathogens. Only MashUp-RT showed enzymatic activity. Interestingly, although the recombinant spores displaying Bst did not show DNA polymerase activity, the residual reverse transcriptase activity of the commercial enzyme was stabilized by wild type spores. To our knowledge, this isthe first report on the the use of spore display of enzymes used in molecular diagnostic testing.
Descrição
Palavras-chave
Microbiologia Bacillus subtilis Esporos Crosta Display-esporos RT-LAMP