Safety/Listeriolysin and Invasin

Questions or Concerns?

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What are Listeriolysin and Invasin?

  • Listeriolysin O (LLO) is a protein from the bacterium Listeria monocytogenes, which is a Risk Group 2 human pathogen. LLO is a virulence factor, because it helps Listeria infect human cells.

LLO is a pore-forming protein from the hemolysin family. It is most active at pH 5.5, the pH of the phagosome. When human cells have engulfed Listeria cells by phagocytosis, LLO becomes active and breaks open the phagosome, allowing the bacteria to live in the cytoplasm. When Listeria live intracellularly, then extracellular immune system components such as antibodies.

  • Invasin is a protein from the bacterial genus Yersinia. Members of the Yersinia genus include Y. pestis (Black Plague, a Risk Group 3 human pathogen) and its relatives Y. enterocolitica and Y. pseudotuberculosis (Risk Group 2 pathogens). Invasin is a virulence factor, because it helps Yersinia species invade human cells.

The Invasin protein on the surface of a Yersinia cell interacts with beta-1-integrin receptors on the surface of eukaryotic cells. This triggers a signal transduction pathway, leading to endocytosis of the whole bacterium.

What safety risks are associated with these parts?

Bacteria transformed with either Listeriolysin or Invasin are invasive microorganisms with the ability to hide from the immune system, and they must be treated with great caution.

With Listeriolysin alone, bacteria can invade macrophages, which are a crucial part of the human immune system. Because macrophages naturally engulf bacteria by phagocytosis, the only remaining step is for Listeriolysin to break open the phagosome, and then the bacteria are free to live inside the cytoplasm.

Listeriolysin and Invasin are most dangerous when they are combined together. Genetically engineered bacteria that express both Listeriolysin and Invasin are able to invade any beta-1-integrin expressing cell type (not just macrophages), and they can penetrate to the cytoplasm at high efficiencies.

While this invading ability is often very useful for a synthetic system, it is also an extremely dangerous ability for engineered cells to have. If engineered cells can hide from the human immune system and continue to grow and thrive, then their ability to cause disease is much, much greater. Granting this ability to an innocuous germ might turn it into a pathogen. Granting this ability to a moderately dangerous pathogen might turn it into an extremely dangerous one.

Note that enteroinvasive E. coli have a similar ability to invade and colonize human cells. Recall that E. coli is both a dangerous pathogen and a "safe" lab strain. Lab strains such as K-12 are safe in part because they lack the ability to invade human cells. Consider: if you transform Listeriolysin and Invasin into lab-strain E. coli, you may have just recreated a dangerous pathogenic strain of E. coli!

For more information, see:
Agapakis et al. Towards a synthetic chloroplast. PLoS One, April 20, 2011. DOI: 10.1371/journal.pone.0018877

How can I use Listeriolysin and Invasin safely?

iGEM does not ban the use of Listeriolysin or Invasin, whether separate or together. However, if any participants wish to work with these parts, we require that they observe strict safety procedures. You must avoid contact with any bacteria that have been transformed with Listeriolysin or Invasin. Basic protective equipment, such as lab coats, gloves, and safety glasses, is absolutely required when handling these parts. Biosafety Level 2 containment is required when using Listeriolysin and Invasin together, and it is highly recommended when using either of them individually.

Additionally, you must take steps to prevent the Listeriolysin/Invasin genes from "escaping" to wild bacteria via horizontal gene transfer. All waste that might contain Listeriolysin/Invasin must be thoroughly autoclaved before disposal.

For the 2014 competition, teams must complete a Check-In form and receive approval before acquiring or using Listeriolysin or Invasin. Additionally, it is appropriate to examine both the immediate and the long-term safety concerns associated with these parts, in the Human Practices component of your project.

(Different countries have different laws about laboratory biosafety. Consult with your faculty advisor and lab manager, and contact whoever is in charge of laboratory biosafety at your institution.)

I am making a new part that contains Listeriolysin or Invasin. What should I do?

First of all, thank you for contributing to the Registry! To help keep the Registry safe and informative, if you create new parts that contain Listeriolysin or Invasin (or both), we recommend that you include the "Safety Flag" template on the main page for those parts. To include the Safety Flag, copy this line and paste it at the beginning of your page:

{{Template:SafetyFlag|reason=[[Safety/Listeriolysin and Invasin | Listeriolysin and Invasin parts]]}}

I am making a new part that does not contain Listeriolysin or Invasin, but my new part is dangerous for a different reason. What should I do?

If you are creating a new part that is dangerous for a different reason, please consult the Safety Committee (email safety AT igem DOT org). We will discuss the part with you, and make a decision about whether your new part requires a Red Flag or not.