Transdução fágica: um método para transferir a resistência à ampicilina da E. coli doadora para a receptora
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Overview
Fonte: Alexander S. Gold1, Tonya M. Colpitts1
1 Departamento de Microbiologia, Escola de Medicina da Universidade de Boston, Laboratórios Nacionais de Doenças de Infecções Emergentes, Boston, MA
Transdução é uma forma de troca genética entre bactérias que utilizam bacteriófagos, ou phages, uma classe de vírus que infecta exclusivamente organismos procarióticos. Esta forma de transferência de DNA, de uma bactéria para outra por meio de um phage, foi descoberta em 1951 por Norton Zinder e Joshua Ledererg, que chamou o processo de “transdução” (1). Bacteriphages foram descobertos pela primeira vez em 1915 pelo bacteriologista britânico Frederick Twort, então descoberto independentemente novamente em 1917 pelo microbiologista franco-canadense Felix d’Herelle (2). Desde então, a estrutura e a função desses phages têm sido amplamente caracterizadas (3), dividindo essas pragas em duas classes. A primeira dessas classes são as fábulas líticas que, após a infecção, se multiplicam dentro da bactéria hospedeira, interrompendo o metabolismo bacteriano, lisendo a célula e liberando phage progênero (4). Como resultado dessa atividade antibacteriana e da crescente prevalência de bactérias resistentes a antibióticos, esses frascos líticos têm se mostrado recentemente úteis como um tratamento substituto para antibióticos. A segunda dessas classes são as fálvias lisogênicas que podem se multiplicar dentro do hospedeiro através do ciclo lítico ou entrar em um estado quiescente no qual seu genoma é integrado ao hospedeiro (Figura 1), um processo conhecido como lisogenia, com a capacidade de a produção de phage ser induzida em múltiplas gerações posteriores (4).
Figura 1: Infecção de célula hospedeira por bacteriófago. Adsorção pela phage para a parede celular bacteriana através de interações entre as fibras traseiras e receptor (roxo). Uma vez na superfície celular, o phage é irreversivelmente ligado à célula bacteriana usando a placa base (preta) que é movida para a parede celular pela baia contraltil (amarelo). O genoma phage (vermelho) então entra na célula e se integra ao genoma das células hospedeiras.
Embora a transdução bacteriana seja um processo natural, usando tecnologia moderna foi manipulado para a transferência de genes para bactérias no ambiente laboratorial. Ao inserir genes de interesse no genoma de uma praga lisogênica, como a praga, é capaz de transferir esses genes para os genomas das bactérias e, consequentemente, expressá-los dentro dessas células. Enquanto outros métodos de transferência genética, como a transformação, usam um plasmídeo para transferência e expressão genética, a inserção do genoma phage na da bactéria receptora não só tem o potencial de conferir novos traços a esta bactéria, mas também permite que mutações que ocorram naturalmente e outros fatores do ambiente celular alterem a função do gene transferido.
Em comparação com outros métodos de transferência de genes horizontais, como a conjugação, a transdução é bastante flexível nos critérios necessários para as células doadoras e receptoras. Qualquer elemento genético que possa caber dentro do genoma da praga que está sendo usada pode ser transferido de qualquer cepa de bactérias doadoras para qualquer cepa de bactérias receptoras, desde que ambas sejam permissivas ao phage, exigindo a expressão de receptores phage necessários nas superfícies celulares. Uma vez que este gene é removido do genoma do doador e embalado para o phage, ele pode ser transferido para o receptor. Após a transdução, é necessário selecionar para o receptor bactérias que contenham o gene de interesse devem ser selecionados. Isso poderia ser feito pelo uso de um marcador genético, como uma marca BANDEIRA ou polyhistidina-tag, para marcar o gene de interesse, ou a função intrínseca do gene, no caso de genes que codificam para resistência a antibióticos. Além disso, o PCR poderia ser usado para confirmar ainda mais a transdução bem sucedida. Usando primers para uma região dentro do gene de interesse e comparando sinal a um controle positivo, bactérias que têm o gene de interesse, e um controle negativo, bactérias que passaram pelos mesmos passos da reação de transdução sem phage. Embora a transdução bacteriana seja uma ferramenta útil na biologia molecular, ela tem e continua a desempenhar um papel importante na evolução das bactérias, particularmente no que diz respeito ao recente aumento da resistência a antibióticos.
Neste experimento, a transdução bacteriana foi utilizada para transferir a codificação genética para resistência à ampicilina antibiótico da cepa W3110 de E.coli para a cepa J53 através do bacteriófago P1 (5). Este experimento consistia em dois passos principais. Primeiro, a preparação do phage P1 contendo o gene de resistência à ampicilina da cepa do doador. Em segundo lugar, a transferência deste gene para a cepa receptora por transdução com a phage P1 (Figura 1). Uma vez realizada, a transferência bem sucedida do gene de resistência à ampicilina poderia ser determinada por qPCR (Figura 2). Se a transdução fosse bem sucedida, a cepa J53 de E. coli seria resistente à ampicilina, e o gene conferenciando essa resistência detectável por qPCR. Se não tivesse sucesso, não haveria detecção do gene de resistência à ampicilina e a ampicillina ainda funcionaria como um antibiótico eficaz contra a cepa J53.
Figura 2: A confirmação da transdução bem sucedida por qPCR. Comparando os valores Cq detectados para o gene de interesse da reação de transdução e a reação de controle negativo, e normalizando esses valores em relação a um gene de limpeza, foi possível confirmar que a transdução bacteriana foi bem sucedida.
Procedure
Applications and Summary
The transfer of genes to and from bacteria by bacteriophage, while a natural process, has proved extremely useful for a multitude of research purposes. While other methods of gene transfer such as transformation and conjugation are possible, transduction uniquely uses bacteriophages; not only allowing for gene integration into the host genome, but also for gene delivery to multiple bacteria that are not susceptible to other methods. This process, while especially useful in the laboratory, has also been used in the recently emerging field of gene therapy, more specifically in alternative gene therapy, a therapeutic strategy that utilizes bacteria to deliver therapeutics to target tissues, many of which are not susceptible to other delivery methods and have much clinical relevance (8,9).
References
- Lederberg J, Lederberg E.M., Zinder, N.D., et al. Recombination analysis of bacterial heredity. Cold Spring Harbor symposia Quantitative Biol. 1951;16:413-43.
- Duckworth DH. "Who Discovered Bacteriophage?". Bacteriology Reviews. 1976;40:793-802.
- Yap ML, Rossman, M.G. Structure and Function of Bacteriophage T4. Future Microbiol. 2014;9:1319-27.
- Sulakvelidze A, Alavidze, Z., Morris, J. G. Bacteriophage Therapy Antimicrobial Agents and Chemotherapy 2001;45(3):649-59.
- Moore S. Sauer:P1vir phage transduction 2010 [Available from: https://openwetware.org/wiki/Sauer:P1vir_phage_transduction].
- Kobayashi A, et al. Growth Phase-Dependent Expression of Drug Exporters in
- Escherichia coli and Its Contribution to Drug Tolerance. Journal of Bacteriology. 2006;188(16):5693-703.
- Rocha D, Santos, CS, Pacheco LG. Bacterial reference genes for gene expression studies by RT-qPCR: survey and analysis. Antonie Van Leeuwenhoek. 2015;108:685-93.
- Pálffy R. et al. Bacteria in gene therapy: bactofection versus alternative gene therapy. Gene Ther. 2006 13:101-5.
- O'Neill JM, et al. Intestinal delivery of non-viral gene therapeutics: physiological barriers and preclinical models. Drug Discovery Today. 2011;16:203-2018.
Transcript
Bacteria can adapt quickly to a fast-changing environment by exchanging genetic material and one way they can do this is via transduction, the exchange of genetic material mediated by bacterial viruses. A bacteriophage, often abbreviated to phage, is a type of virus that infects bacteria by first attaching to the surface of the host and then injecting its DNA into the bacterial cell. It then degrades the host cell’s own DNA and replicates its viral genome, whilst hijacking the cell’s machinery to synthesize many copies of its proteins. These phage proteins then self-assemble and package the phage genomes to form multiple progeny. However, due to the low fidelity of the DNA packaging mechanism, occasionally, the phage packages fragments of bacterial DNA into the phage capsid. After inducing the lysis of the host, the phage progeny are released and, once such a phage infects another host cell, it transfers the DNA fragment of its previous host. This can then recombine and become permanently incorporated into the new host’s chromosome, thereby mediating gene transfer between the two bacteria.
To carry out phage transduction in the laboratory requires a donor strain that contains a gene of interest, a recipient strain that lacks it, a phage that can infect both the strains, and a method to select the transduced bacteria. In most cases, this will be a selective solid growth media that supports the growth of transduced bacteria but inhibits the growth of non-transduced ones. To begin, the donor strain that contains the gene of interest is cultured in a liquid growth medium. When all the bacteria are actively dividing in the log phase of their growth, the culture is inoculated with the target phage. After three to four hours of incubation, when nearly all the bacteria have lysed and released the phage particles, the donor phage lysate is inoculated into a freshly grown culture of the recipient bacterial strain. After a brief incubation of one hour, the culture should now contain a mixture of transduced and non-transduced bacterial cells and this is screened for the transduced cells by spreading a fraction of the suspension onto an appropriate selective solid growth media. Upon further incubation, the transduced cells should grow and multiply to yield visible colonies. These colonies can then be selected for further analysis using a variety of methods to further confirm successful transduction, such as colony PCR, DNA sequencing, or quantitative PCR.
Before starting the procedure, put on any appropriate personal protective equipment, including a lab coat and gloves. Next, sterilize the workspace with 70% ethanol and wipe down the surface.
After this, prepare three one-milliliter aliquots of LB salt solution. Now, prepare a donor strain culture by adding 100 microliters of E. coli to a 15 milliliter conical vial containing five milliliters of LB growth medium with 500 micrograms of ampicillin. Then, grow the culture overnight at 37 degrees Celsius with aeration and shaking at 220 rpm. The next day, wipe down the bench top with 70% ethanol before removing the culture from the shaking incubator. Next, dilute the overnight culture one to 100 by adding 10 microliters of donor strain to 990 microliters of fresh LB supplemented with salt solution.
Allow the bacterial dilution to grow at 37 degrees Celsius for two hours with aeration and shaking at 220 rpm. Once the cells have reached early log phase, remove the culture from the incubator, add 40 microliters of P1 phage to the culture and incubate again. Continue to monitor the cells for one to three hours until the culture has lysed. Next, add 50 to 100 microliters of chloroform to the lysate and mix by vortexing. Then, centrifuge the lysate to remove debris and transfer the supernatant to a fresh tube. Add a few drops of chloroform to the supernatant and store it at four degrees Celsius for no more than one day.
To begin the transduction procedure, obtain a one milliliter culture of recipient strain. Next, transfer 100 microliters of donor phage lysate into a 1.5 milliliter microcentrifuge tube and incubate it at 37 degrees Celsius with the cap open for 30 minutes to allow any remaining chloroform to evaporate. While the donor phage lysate incubates, pellet the recipient strain cells via gentle centrifugation. Discard the supernatant and resuspend the cell pellet in 300 microliters of fresh LB containing 100 millimolar magnesium sulfate and five millimolar calcium chloride.
Next, set up the transduction reaction by combining 100 microliters of the recipient strain and 100 microliters of the donor phage lysate in a microcentrifuge tube. Then, set up the negative control by combining 100 microliters of the recipient strain and 100 microliters of the LB with magnesium sulfate and calcium chloride. After incubation, add 200 microliters of one molar sodium citrate and one milliliter of LB to both tubes, and mix by gently pipetting up and down. Then, after the tubes have been incubated for an hour, gently pellet the cells via centrifugation.
After centrifuging, discard the supernatant and resuspend the pelleted cells in 100 microliters of LB with 100 millimolar sodium citrate. Vortex the solutions and pipette the entire transduced sample onto an LB agar plate with 1X ampicillin. Finally, pipette the entire volume of the negative control cell mixture onto an LB agar plate without ampicillin. After incubating the plates overnight at 37 degrees Celsius, use a sterile pipette tip to pick three to four colonies from the transduction plate and streak them onto a new LB agar plate containing 1X ampicillin and 100 microliters of one molar sodium citrate. Repeat this plating method for the negative control on another LB agar plate containing only 100 microliters of one molar sodium citrate. Then, incubate the plates at 37 degrees Celsius overnight to allow colonies free of phage to grow.
The next day, wipe down the bench top with 70% ethanol before removing your plates from the incubator. Using a sterile pipette tip, pick three colonies from the transduction plate and add them each to a separate tube containing five milliliters of LB media. Then, select three colonies from the negative control plate and add them to another tube containing five milliliters of LB media. Grow the cultures overnight at 37 degrees Celsius with aeration and shaking at 220 rpm. After sterilizing the bench top as previously demonstrated, use a DNA miniprep kit to isolate DNA from 4.5 milliliters of each culture according to the manufacturer’s instructions. Then, elute the DNA with 35 microliters of nuclease-free water and measure the resulting concentration by lab spectrophotometer. Finally, prepare glycerol stocks by adding the remaining 0.5 milliliters of both bacterial solutions to 0.5 milliliters of 100% glycerol.
To confirm transduction, first prepare two qPCR master mixes for 24 qPCR reactions. For the first master mix, add 150 microliters of qPCR buffer mix to a microcentrifuge tube and 12 microliters each of a forward and reverse primer designed to amplify the ampicillin resistance gene. Next, prepare a second qPCR master mix by adding 150 microliters of qPCR master mix to a microcentrifuge tube and then adding 12 microliters each of a forward primer and reverse primer designed to amplify a housekeeping gene.
For each qPCR reaction, combine 100 micrograms of experimental DNA from each reaction with 14.5 microliters of qPCR master mix. Now, prepare the remaining reactions as previously demonstrated. Transfer the reactions to a thermocycler preheated to 94 degrees Celsius and then initiate the program. Finally, use the cycle quantification, or Cq, values generated by qPCR to calculate the normalized transduction efficiency of the ampicillin resistance gene.
The cycle quantitation, or Cq, values for the genes of interest were tabulated for each of the negative controls and transduced samples. Low Cq values, typically below 29 cycles, like the transduced samples in this example indicate high amounts of the target sequence.
A housekeeping gene, also tabulated here, is used as a loading control to normalize the amount of DNA in each reaction and as a positive control to ensure the qPCR is working. Provided the same amounts of the housekeeping gene are loaded, it is found at relatively the same rate in each sample.
Next, to calculate the delta Cq value for each sample, subtract the Cq value of the housekeeping gene for each sample from the Cq value of its corresponding target gene. For example, the delta Cq of the first negative control is 13.54. Then, use this value to calculate the normalized transduction efficiency of each sample using the formula shown here. Finally, the average normalized transduction efficiency for each sample group can be calculated.