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Posted by Fernanda on May 16th, 2018  ⟩  0 comments

Sandra’s haunting journey with a deadly flesh-eating parasite started a few years ago while Sandra was enjoying her last day of vacation at a tropical paradise. That day, Sandra decided to walk through the sandy beach one last time. Just as she was about to return to her hotel room, she felt a sting on her left leg and quickly forgot about it. Soon after, the bite developed into a small itchy lump. A month later, the red, itchy lump was still there. Then the lump became a painful ulcer, an open sore in her leg, that slowly kept growing. That day at the beach, Sandra had been bitten by a sandfly infected with leishmaniasis, a disease that can be extremely painful and sometimes fatal for humans.

Fortunately, dangerous diseases, such as leishmaniasis, are being combated by a new emerging field called synthetic biology that is defined as “the design and construction of new biological parts, devices, and systems, or the re-design of existing, natural biological systems for useful purposes,” and requires the merging of biology, mathematics and engineering. But, how is synthetic biology ultimately battling disease?

Synthetic biology is changing how we study disease by redesigning a powerful biological system, the inducible gene expression system. Normally, genes encoded by DNA are transcribed into mRNA by the cell’s molecular machinery. This mRNA is then translated into proteins. This process of manufacturing protein products from DNA is the very definition of a gene expression system. And often, these systems have switches that we can manipulate and control to activate expression of any gene we want by introducing an artificial inducer. It is this introduction of a synthetic activator that makes the gene expression system inducible. We are then able to use the cell’s machinery and switches to generate an artificial system that we can turn on anytime and use to construct complex genetic circuits and networks.

Today, one of the most important inducible gene expression systems being used in synthetic biology is the induction of the lac operon by the compound isopropyl β-D-1-thiolgalactopyranoside (IPTG). IPTG mimics allolactose, a lactose metabolite that activates transcription of the lac operon, and is often used to induce expression of genes under regulation of the lac operator. In the absence of IPTG, the lac repressor (encoded by the lacI gene) binds to the lac operator (lacO), preventing transcription. However, when IPTG is present, it binds to the lac repressor, releasing it from the lac operator in an allosteric manner, allowing the transcription of genes in the lac operon.


This powerful ability to induce gene transcription has made IPTG a technology that has revolutionized biology across many fields, including the study and treatment of diseases. Indeed, the creation of disease models for the study of the pathology observed in complex and devastating human illnesses, including leishmaniasis, is necessary and is becoming achievable with the emergence of synthetic biology and IPTG induction.

Leishmaniasis, the flesh eating disease Sandra contracted, impacts many human populations around the world. This complex infection is not actually caused by the sandfly, but by its protozoan parasite Leishmania. Leishmania has two distinct forms: the amastigote and the promastigote. During transmission, an infected sandfly bites a mammal and passes the promastigote, which is then engulfed by mammalian macrophages. Once in macrophages, the promastigote can either be killed by oxidative mechanisms or it can begin to interfere with the macrophage’s intracellular signaling leading to progression of the disease.

That day at the beach, a chain of events began occurring under the surface of Sandra’s skin to protect the parasite. For its survival, Leishmania interferes with intracellular signaling by ultimately changing the macrophage’s phenotype which leads to progression of disease, and results in ulcerative lesions, mucosal lesions and infection of organs including spleen, liver, and bone marrow, and death. The mechanism behind this signal interference requires the modulation of Nuclear factor-KB (NFKB) and protein kinase C (PKC), two signaling molecules that are essential for an organism’s immune and inflammatory responses. In the case of PKC, leishmaniasis changes the activity of two PKC isoforms: PKC-ς and PKC-α. On one hand, this parasite increases PKC-ς ‘s activity by changing its affinity for substrate in the presence of intracellular ceramide, a lipid whose production increases during infection. On the other hand, this increase in ceramide interferes with PKC-α’s catalytic action by preventing its binding with co-modulators calcium and di-acyl glycerol (DAG). Because NFKB’s activation is dependent upon PKC activity, leishmaniasis’s interference with PKC activity results in aberrant NFKB signaling.

To counteract this cellular signaling manipulation by leishmaniasis, a group of synthetic biologists adopted a similar approach. Milsee Mol and her team engineered PKC to contain domains of both the α and ς isoforms giving rise to PKC_ας under the regulation of the lac repressor and induced by IPTG, resulting in NFκB signaling modulation. They observed that induction of gene expression with IPTG in peritoneal macrophages carrying the engineered PKC_ας led to a change of gene expression in macrophages from an anti-inflammatory phenotype to a pro-inflammatory phenotype, in vitro.

Because inflammation is part of many diseases’ pathology, this finding may present a future way to use IPTG to treat multiple diseases simultaneously, without the addition of other medication or medication that has debilitating side effects.

Another way biologists are currently using IPTG to combat disease is in the regulation of synthetic gene networks in 3-D materials, generating a microenvironment where cellular mechanisms and behavior can be manipulated and studied. In these microenvironments, the biomaterial presents the genetic inducer, IPTG, to cells that contain synthetic gene circuits and are grown and cultured within the biomaterial. This technique is especially exciting because we can have additional ways of controlling gene expression due to materials being able to release inducers, such as IPTG, in many different ways. In addition, we know that cells gain different characteristics and may differentiate into different cells depending on the type of material they are grown in.

In one study coupling synthetic biology and materials science, Tara Deans and her research team aimed to couple inducers, such as IPTG, into biomaterials and create 3-D environments using three different types of materials: polycaprolactone (PCL) electrospun fibers, polyethyleneglycol (PEG) hydrogels, and poly lactic-co-glycolic acid (PLGA) sponges. They observed that IPTG could diffuse in these materials where cells were growing, resulting in gene expression in vitro. Furthermore, Deans and her team also found that modifying the biomaterial by attaching a sugar ring or a peptide sequence affects the structure of cells and how genes are expressed after induction with IPTG. They also found that implanting PLGA sponges containing CHO cells into the abdomen of mice and allowing them to drink water containing IPTG resulted in induction of gene expression. Thus, Deans et al. showed that IPTG is a powerful tool that can be attached to biomaterials and used in to manipulate gene expression networks both in vivo and in vitro and may help in establishing disease models and therapeutic treatments.

As evident in the studies described here, the generation of complex genetic circuits and microenvironments requires the ability to control expression of multiple genes independently of each other. In the past, scientists developed various inducible systems such as the IPTG-inducible lac promoter system. However, this system often interferes with other inducible systems, impeding the creation of a complex genetic circuit. One inducible system that is inhibited by IPTG is the arabinose-inducible araBAD promoter system (PBAD). In this system, when arabinose is not present, the dimeric AraC protein contacts two half-sites on DNA (I1 and O2), creating a DNA loop, which interferes with RNA polymerase binding. Once arabinose binds to AraC, the dimer’s position changes and binds to half-sites I1 and I2, allowing transcription from the PBAD promoter. IPTG inhibition prevents the use of both systems in the same cell.

Fortunately, Lee et al. generated a mutant library of arabinose-binding regulatory protein AraC and identified mutants showing insensitivity to IPTG, leading to the generation of a PBAD system that can be used with the IPTG/lac operon system. So, these two systems can control gene expression in a gene circuit at the same time and independently of each other lending flexibility to their use in the study of cell signaling in cellular microenvironments.

The re-design and application of IPTG induction of gene expression in synthetic biology is certainly promising for our pursuit of understanding biological mechanisms and disease. Perhaps in the near future, our ability to manipulate gene expression, to engineer novel proteins, and to generate cellular microenvironments will help individuals like Sandra heal faster and lead to a better life.

References

Deans, T. L., Singh, A., Gibson, M., & Elisseeff, J. H. (2012). Regulating synthetic gene networks in 3D materials. Proceedings of the National Academy of Sciences, 109(38), 15217-15222. doi:10.1073/pnas.1204705109.

Jensen, P. R., Westerhoff, H. V., & Michelsen, O. (1993). The use of lac-type promoters in control analysis. European Journal of Biochemistry,211(1-2), 181-191. doi:10.1111/j.1432-1033.1993.tb19885.x.

Lee, S. K., Chou, H. H., Pfleger, B. F., Newman, J. D., Yoshikuni, Y., & Keasling, J. D. (2007). Directed Evolution of AraC for Improved Compatibility of Arabinose- and Lactose-Inducible Promoters. Applied and Environmental Microbiology, 73(18), 5711-5715. doi:10.1128/aem.00791-07.

Mol, M., Kosey, D., Bopanna, R., & Singh, S. (2017). Transcription Factor Target Gene Network governs the Logical Abstraction Analysis of the Synthetic Circuit in Leishmaniasis. doi:10.1101/151779.

Mol, M., Patole, M. S., & Singh, S. (2014). Immune signal transduction in leishmaniasis from natural to artificial systems: Role of feedback loop insertion. Biochimica Et Biophysica Acta (BBA) - General Subjects,1840(1), 71-79. doi:10.1016/j.bbagen.2013.08.018.

Olivier, M., Brownsey, R. W., & Reiner, N. E. (1992). Defective stimulus-response coupling in human monocytes infected with Leishmania donovani is associated with altered activation and translocation of protein kinase C. Proceedings of the National Academy of Sciences,89(16), 7481-7485. doi:10.1073/pnas.89.16.7481.

Parasites - Leishmaniasis. (2013, January 10). Centers for Disease Control and Prevention. Retrieved May 7, 2018, from https://www.cdc.gov/parasites/leishmaniasis/index.html.

Singh, V. (2014). Recent advancements in synthetic biology: Current status and challenges. Gene,535(1), 1-11. doi:10.1016/j.gene.2013.11.025. 


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