Firefly light gives rise to sensor that detects cellular alterations

An enzyme cloned from an insect found by Brazilian researchers – and genetically modified – makes it possible to monitor intracellular acidity and could be used to study diseases and drugs.

By André Julião  |  Agência FAPESP – The gene encoding an enzyme from a firefly, discovered at the Sorocaba campus of the Federal University of São Carlos (UFSCar) in Brazil, has given rise to a biosensor capable of detecting pH changes in mammalian cells – which could be useful, for example, in studying diseases and assessing the toxicity of a drug candidate.

The luciferase from the species Amydetes vivianii changes color from bluish-green to yellow and red as acidity decreases in fibroblasts, the most common cell type in connective tissue. And it does so with great intensity and stability, something that had not been achieved with other luciferases tested by the research group.

The work, supported by FAPESP, was published in the journal Biosensors.

Luciferases are enzymes found in bioluminescent organisms that produce light by oxidizing luciferin, a compound that must also be present in the process. The luciferase now being studied comes from a species discovered in 2006 by one of the study’s coordinators, Vadim Viviani, coordinator of the Biochemistry and Bioluminescent Technologies Laboratory at UFSCar. The enzyme was cloned by his research group in 2011.

“Inside the cell, changes in pH can indicate processes such as homeostasis, proliferation and cell death, among others. Our technique has the potential to study diseases or drug toxicity, for example,” points out Vanessa Bevilaqua, first author of the article and postdoctoral fellowship recipient at the School of Medical and Health Sciences of the Pontifical Catholic University of São Paulo (PUC-SP), Sorocaba campus.

The work was carried out in laboratories coordinated by Viviani at UFSCar and by Eliana Duek at PUC-SP. Duek is also supported by FAPESP.

Other luciferases tested by the group produced reddish light that changed color less at the 36 °C temperature at which mammalian cells function.

“The new luciferase has been modified to be better expressed in mammalian cells. In addition to having a wider range of light color change, it has greater stability and provides a stronger glow [intensity]. The technique is non-toxic and does not depend on an external light source, as is the case with fluorescence, another way of using light to study cells,” Viviani explains.

In addition to capturing bioluminescence images with photodetection cameras, the group’s tests also allowed the light emitted by luciferases in mammalian cells to be photographed even with a smartphone camera.

The glow was intense for the first 30 minutes, and then began to fade. Although weaker, it continued for at least 12 hours – although it could only be detected with advanced photodetection equipment.

“With this, it’s possible to use the color of the light to indicate the pH inside cells, including human cells, and infer whether there’s cellular stress or some other effect related to acidity. It’s something unprecedented and something we developed entirely in Brazil,” Viviani adds.

The work is part of the project “Development of bioluminescent inputs for immunoassays, environmental analysis and bioimaging”, supported by FAPESP and coordinated by Viviani.

Other co-authors are Gabriel Pelentir, a doctoral fellowship recipient at UFSCar, and Moema Hausen, a professor at PUC-SP.

History

In previous studies, the researchers had already developed the use of luciferase from another firefly, of the genus Macrolampis, to indicate the pH of bacterial cells (read more at: agencia.fapesp.br/20609).

However, when tested in mammalian cells, the bioluminescence emitted by the Macrolampis luciferase was very reddish and varied little with changes in pH, lacking stability above 36 °C and reducing the efficacy required for this cell type.

During the pandemic, Viviani’s group also developed an immunoassay to detect COVID-19 based on the luciferase of Amydetes vivianii, which glows when in contact with antibodies to SARS-CoV-2 (read more: agencia.fapesp.br/36427).

With the work now completed, the laboratory led by the UFSCar professor, which already had a unique infrastructure for bioluminescence studies and applications, has also acquired the capacity to perform bioluminescent tests on mammalian cells.

This will allow it to carry out both new studies with this luciferase, as well as to test others that are part of the laboratory’s collection, which the researcher has collected over more than 30 years, mainly from Brazilian insects (read more at: agencia.fapesp.br/29066, agencia.fapesp.br/31797 and agencia.fapesp.br/50536).

“With this development, we’ve opened up a range of possibilities, from bioassays for drug and cosmetic toxicity to the effect of biomaterials on human cells, and even new ways of studying cancer cells,” Bevilaqua concludes.

The article “Selection and Engineering of Novel Brighter Bioluminescent Reporter Gene and Color-Tuning Luciferase for pH-Sensing in Mammalian Cells” can be read at: www.mdpi.com/2079-6374/15/1/18.