An ally treaty with bacteria

An ally treaty with bacteria — A conversation with Dr. Alex Rodrigo.

By ANDRÉS ALBA     5th June 2015

c2c12 cells on a bacterial fibronectin biofilm

c2c12 cells on a bacterial fibronectin biofilm

Bacteria are everywhere. They have been found in the Mariana Trench and in manned spacecraft, and each one of us has several hundreds of species living in our body. During the last few years research has shown the amazing ‘friendly’ relations that some bacteria have with humans. Some of them turned out to be harmless or even perform important roles on our skin, bowels or lungs. Many others not so much. Even with antibiotics many infections are dangerous and cause many deaths every year. We have learned to look at bacteria as the bad guys, so even for scientists it can be a challenge to come up and accept ways of using them together with human cells, as a tool to improve the way cells behave.

Dr. Alex Rodrigo started his PhD four years ago, within a newly awarded project that explored a very new concept at the moment. Using bacteria to help mammalian cells. The goal of the project was actually to develop a bacterial population that produces a particular protein very helpful for how cells interact with their environment, and using it as an alternative tool to study cell behaviour. A paper was published in 2013 [1] with the first successful results of the project and another one in 2014 [2]. “At the beginning I learned a lot from Dr. Anas Saadeddin and other researchers in Valencia. Now it has been my turn to train other students and share out tasks and responsibilities,” says Alex. He defended his thesis last month and is now part of a team of PhD student Jake Hay, MSc student Karoliina Hassi, and principal investigator Manuel Salmerón-Sanchez. “I think we are a great team,“ says Alex. “I feel that we all are quite eager to make progress with this. We easily find the time or motivation to learn new techniques or solve the small problems that you need to solve to move forward.”

They work with lactic bacteria, the same kind of bacteria widely used in the dairy industry, to make cheese and yoghurts. Scientist have actually found that lactic bacteria and several other families of bacteria live naturally in our intestinal tract, with complex interactions between them and the surrounding human tissues. “These are non-pathogenic bacteria, meaning that they don’t cause any disease. It is generally easy to work with them in the lab. There are also well know protocols to modify them, to genetically engineer them and make them express or produce something interesting. So they are a good choice for our goals.” If you go in a biological research lab, where cells are grown and studied with great care, and say that you have a few million bacteria that would like to meet the cells, most certainly you will be swiftly banned from entering the lab ever again. Bacteria can be the bane of a cell culture lab, and a lot of resources and efforts in the labs go towards avoiding contamination of the cells with bacteria. So why would someone want to deliberately put bacteria and mammalian cells together? Is there anything the bacteria make that cells will like?

“Our initial concept was to have what we call a ‘living biointerface’ between mammalian cells and the synthetic materials where cells are usually cultured and studied in the lab. When cells are in living tissues, in a human bone or heart for instance, they are always surrounded by proteins and other molecules that are like a gel or dough that keeps them together in a tri-dimensional world. This matrix is usually non-existent or very weak when cells are cultured in the lab in the classical way, because we typically use wells with flat surfaces to grow them and only add media (what they eat and need to survive) but not the matrix they use to ‘feel’ their surroundings.” Prof Salmerón-Sanchez had the idea that bacteria could be used to produce fibronectin, one important protein of this matrix or complex mesh of structural and adhesive proteins which serve as mechanical support and that cells have around them in a living tissue.

An esquematic of the system and the interface between the bacterial and mammalian cell walls

An esquematic of the system and the interface between the bacterial and mammalian cell walls

“It happens that bacteria are very good and quick at producing proteins around them. They use them to build what is called a biofilm that allows them to be more resistant, aggregate and multiply. They are little efficient machines at producing molecules that they deposit around them,” explains Alex. “We have prepared Lactococcus lactis bacteria that produces one particular fragment of fibronectin. Cells are cultured together with the bacteria, expressing fibronectin in their membrane, and therefore in an environment that resembles a little what the cell would encounter in a living tissue.”

Preparing the bacteria has been a long process, even if the techniques to modify them are well known genetic engineering techniques. “We had a lot of help from several researchers in Valencia that gave us the right plasmid and the fibronectin fragment gene. Then we’ve had to check that the bacteria are viable, that the protein is expressed on the membrane, and that it is biologically active. This has taken quite some time, and we still would like to improve the stability of the biofilm for instance.”

Back to the central goal of the project, the bacteria are meant to produce a fragment of a protein that can be used to control how mammalian cells behave. It is quite surprising that only a fragment of this protein, one between the several proteins that make up a living extracellular matrix, would have such an impact on cells.

“The III7−10 fragment of the human fibronectin contains two important sequences, RGD and PHSRN. RGD is an adhesive sequence, an important part of the protein that the cells use basically to move and get information about what to do next. RGD interacts with a wide range of integrins, receptors in the membrane of the cells that work as ‘senses’ for the cell and allow them to transmit signals from the outside to the inside of the cell where their machinery is. The other sequence, PHSRN, increases the specificity of the interaction of the RGD sequence with some particular integrins, such as α5β1 and others. This means that both sequences together play an important role in critical cellular processes such as adhesion, migration, proliferation and differentiation,” explains Alex.

Having solid a proof of concept of the system with living cells can be tricky, because fibronectin is naturally produced by the cells. Also, it is important to be aware that several other parameters affect the way cells proliferate or differentiate, such as the stiffness of the culture substrate, the media used, or the actual cell line. “We are very lucky to have a collaborator—Prof. Mercedes Costell in Valencia—that has developed a knock-out mouse for fibronectin. That means that we can have mammalian cells that don’t produce fibronectin, and when we culture them with our bacteria we can be sure that all the fibronectin comes from the bacteria,” says Alex. “These cells from the knock-out mice are a little more difficult to work with than commercial fibroblast lines that also are modified to not express fibronectin, but with them we have been able to demonstrate that this living biointerface triggers cell adhesion and FAK phosphorylation.” FAK, short for Focal Adhesion Kinase, is a protein that cells produce to help themselves stick to each other and to their surroundings. When FAK is phosphorylated, meaning that the protein receives a phosphate group and as a consequence is ‘switched on’ into a different state, is usually in response to integrin engagement, that is, to the cell receiving signals from the outside. “We have tested several cell lines. For instance the system also triggered myogenic differentiation on C2C12 cells.”

Immuno fluorescence image of C2C12 cells, with actin (red cytoskeleton), vinculin (green focal adhesions) and Dapi (blue nucleus)

Immunofluorescence image of C2C12 cells on bacterial biofilm, with actin (red cytoskeleton), vinculin (green focal adhesions) and Dapi (blue nucleus)

Overall the system looks like a promising new tool to study cell behaviour on surfaces. But, as with everything related to scientific progress and biology, many would like to know if this work could be used to design new health applications, treatments, or helping solve other biomedical challenges. Is there any work in that direction?

“Biomedical research has some of the best minds in the world and the knowledge we have about how things work in our bodies is more and more detailed every year. However the challenges are many if you expect bacteria to be used in a safe and efficient manner against a disease or a medical condition. We cannot risk or rush ideas until all the necessary milestones are well covered. Right now we are trying to have a population of bacteria that expresses other interesting proteins, besides fibronectin, so that these proteins together have a synergistic effect on the cells. This is what we intend to do in the near future, continue to understand how our system works and how robust it is.”

Bacteria have been our sworn enemies since they were linked to disease about 150 years ago. We have now found out that we require their presence in some parts of our body for a normal function, and during the next few decades we could find out that they can be very powerful allies in unexpected ways. Good luck with your work Alex!

References:
[1] Anas Saadeddin et al. Functional Living Biointerphases. Adv Health Mater. 2013 Sep;2(9):1213-8
[2] Rodrigo-Navarro, A. et al (2014) Living biointerfaces based on non-pathogenic bacteria to direct cell differentiation. Scientific Reports, 4(5849)