Exploring the mysterious forest within us

Unraveling the mysterious forest within us can impact our health and the way we live © Joana Carvalho, IGC 2021

They become part of us as soon as we come into this world and stay with us through our whole lives. They are millions of bacteria that inhabit the human body, establishing an interdependent relationship with it, its host. They need us and we need them. It might seem weird to talk about bacteria in these terms, given that most of the time that we refer to them it is not for the best of reasons. However, not all bacteria cause diseases. Some might even be the reason why some diseases do not get a chance to develop.

While pathogenic bacteria, such as Legionella or Salmonella, are virulent, meaning that they have the capability of causing serious imbalances in our organism and leading to death, commensal bacteria live in harmony with human beings, even acting as a protective barrier against the pathogenic ones. And it is in the gut that one of the most diverse and important communities of commensal bacteria lives. Its name is microbiota. “I think of this community like a forest with different species”, simplifies Karina Xavier, principal investigator at the Instituto Gulbenkian de Ciência (IGC), a specialist in microbiology and responsible for the work team dedicated to microbiota. “We have almost the same number of bacteria in our gut as we have human cells in our bodies. And we have evolved for millions of years with this community”.

After all, what are the roles of microbiota? Besides working as a protective barrier to the entry of pathogenic ones, they have roles as important as guaranteeing nutrients. “We consume vegetables and fibers and we benefit a lot from them, but if we did not have bacteria we would not be able to absorb them. They are essential in the production of vitamins, compounds that we cannot produce”, the researcher points out. “They are also important to stimulate our immune system to fight infection”. Given all of this, the scientific community is realizing that the preservation of the microbiota has a tremendous impact on our health, on the way we live, and on how we could live better.

“We are witnessing a revolution of the microbiota. There are more and more researchers interested in this subject”, says the researcher. There are two reasons behind this revolution, the first being the improvement of technologies for the identification of bacteria. “Ten years ago, we had to culture bacteria to study them. Now we only need to extract DNA from an organ or the environment, to determine which bacteria are present, and use them”, claims Karina Xavier. The second reason is the investment of research groups in cooperation and mutual help. “Every couple of years we organize a summer school at the IGC with international researchers where we talk about advances in the field and build bridges”, adds the researcher. And what is the role of Karina’s group in this matter? It is impossible to highlight just one.

 

An endless conversation

Karina Xavier’s academic path started at the Faculty of Sciences in Lisbon, where she graduated in Biochemistry. She moved into a doctoral program at the Instituto de Tecnologia Química e Biológica – NOVA, where she studied the metabolism of carbohydrates in hyperthermophilic microorganisms, but she surrendered to bacteria when she moved to her postdoc at Princeton University, in the United States. “In the 2000s, I went to a convention and met a professor (Bonnie Bassler) that was working on a subject that was, at that time, very new: how bacteria communicate with each other through chemical signals”, tells Karina Xavier.

Until then it was thought that bacteria, being unicellular, did not have the capacity of acting as a group. However, a marine bacterium named Vibrio fischeri came to change this paradigm. It lives in symbiosis with squid and it is capable of producing light so that its hosts do not produce a shadow while swimming and escape their predators. However, light is only produced when there is a sufficient number of bacteria, since, for example, one or two bacteria producing light would not be enough to overcome the shadow. It would even be counterproductive to do so.

Observing this behavior, which was more group-like than individual, the group of scientists, of which the Portuguese researcher was part, further investigated this question and realized that this was, in fact, a community behavior. Furthermore, they noticed that this mechanism was not exclusive to this type of bacteria: all bacteria, commensal and pathogenic, are able to communicate with each other. “What each bacterium does with that information differs from one to another. For example, when a pathogenic bacterium invades its host it does not start producing toxins right away because it is low in number and it would not be productive. So, it multiplies silently, and only when it realizes that it exists in high numbers does it attack, producing toxins, clarifies Karina Xavier. This is how they guarantee that the invasion is effective.

This capacity of producing and receiving signals is called quorum sensing. “The chemical signals are similar to pheromones or odors that we produce and allow the bacteria to perceive if it is in a community with high or low density of other bacteria”, points out Karina Xavier, comparing this mechanism with a situation that happens to us, human beings, a lot of times. “If we get on a bus with our eyes closed we can perceive if it is loaded with people or empty through the smells, the noise…” Bacteria are equipped with molecule emitters and receptors that allow them to produce, receive, and respond to signals, leading a perfect and endless conversation.

Let’s then follow the path of a bacterium since it arrives at a certain environment until it colonizes it effectively: after getting installed in an environment, the bacteria starts to absorb nutrients and to do what it knows best, to divide and multiply. While it divides, it emits chemical signals to understand if there is an answer coming from identical bacteria in sufficient quantity to occupy most of the available space – that is, it is not about these bacteria being in enough number, but in high density. When this happens, they alter their gene expression profile to stop behaving like individuals and start acquiring group behaviors. These can include the production of light, nutrients, toxins, among others, depending on the type of bacteria.

 

E. coli, the queen of them all

The bacteria Karina Xavier chose to study at Princeton was Escherichia coli (E. coli), which is present in our gut and is one of the most studied around the world for the ease of cultivating and manipulating it, before technological advances. In the workgroup of the north-American Bonnie Bassler, the Portuguese microbiologist wanted to take a leap forward and understand if, besides the language bacteria use with each other, there was a universal language for all bacteria. “I mixed Vibrios with E. coli, Streptococcus, and bacilli in a test tube and realized that two of them were able to communicate, that is, Vibrios were able to produce a signal that E. coli detected and responded to”, the researcher explains. Nowadays we know that each  bacteria “speaks” a specific language with the bacteria from the same species and a universal language. “This allows, in a complex community like the microbiota, a species to be able to determine, not only its own density but also if it is in a community with lots of organisms from different species, says the researcher.

Since this discovery, E. coli started occupying a central position in the professional life of Karina Xavier, offering her some surprises. “At the end of my journey at Princeton I observed that, as it divided, E. coli increased the molecule that constituted the quorum sensing signal to other bacteria. However, as soon as the quorum was reached, the molecule started to degrade and disappeared”, explained the researcher, affirming that this behavior goes against the rules of quorum detection.

If everything pointed towards the bacteria being, after all, able to work as a team and to communicate with other species, why were these specific bacteria behaving like this?” We are still not sure, but everything indicates that E. coli does this to disturb the language of other bacteria and their ability to perceive if they have reached the quorum or not”, says Karina Xavier. E. coli might be able to manipulate the signals to have an advantage over its peers and to avoid them from accomplishing what they would if they knew that their density was sufficient.

 

When the fire comes

Let’s step back to the image of the microbiota as a forest. We already know that there is no “silence” in there and that each bacteria speaks its own language and the universal one. There are even bacteria able to manipulate others’ conversations to gain an advantage. However, this ecosystem is not always balanced and things do not always work as described. From time to time fires destroy various components of the forest, some in an irreversible way.

“In the placenta we are sterile, but we start being colonized by these bacteria as soon as we go through the birth canal. And then by microbes from our mother’s skin, from milk, and our brothers and cousins. Children’s microbiota is a developing forest. But we have to take care of it throughout our life”, Karina explains. “Bacteria feed on fibers, thus, we have to maintain a healthy diet so that they do not extinguish due to the lack of nourishment”. Habits of extreme hygiene might also cause the loss of diversity. Then there are the antibiotics…” Antibiotics are, according to the researcher, a “bomb that drops randomly” in our microbiota. They aim to eliminate pathogenic bacteria, but, unfortunately, they are not able to filter who the good and the bad guys are. “If it is a prolonged treatment, it can take years until the forest recovers”. 

Current research from Karina Xavier’s group focuses on the recovery process after disturbances, from understanding how it happens and the processes that might help, to the selection of the bacteria whose presence is most important in the first phases of recolonization. For that purpose, Karina Xavier made use of what she had already observed in the test tubes with E. coli and scaled it up to the bacteria’s natural environment, the microbiota, to ask the following question: does quorum detection have any influence on this recovery process?

The team moved on to the modification of bacteria to form three types of E. coli: one that produced a lot of quorum sensing signal, one that degrades the signal, and another that does not produce or degrade the signal. “E. coli is one of the first to colonize the gut because it is not as sensitive to oxygen. One of its main functions is, in fact, to remove oxygen near the epithelium so that other sensitive bacteria are able to colonize.”, she specifies. These bacteria were then introduced in mice with healthy microbiota, but that had suffered disturbances due to a one-month antibiotic treatment. “What we observed was that, in mice that had E. coli with augmented signal production, the group of bacteria that should have been most affected by the antibiotic was protected. And we were the first to show this”, she stated.

The group is now testing the role of quorum sensing in the recovery after another type of disturbance: the diet. “We have mice that are being fed a normal diet, rich in fiber, and mice being fed on a diet with a high content of fat and simple sugars, and a low content of fiber, what we call the unbalanced western-style diet”, says Karina Xavier. Preliminary results indicate that the microbiota loses a lot of its functions when exposed to an unbalanced diet, given that the bacteria lose its nutriment – the fibers – and disappear. Additionally, and as a result of the collaborative work developed with another research group from the IGC, the one from the evolutionary biologist Isabel Gordo, it was possible to understand that, by changing the environment in which they lived, the properties of bacteria also changed: “Bacteria are organisms that evolve rapidly, so if we change their diet they will acquire mutations in order to get food in different ways”.

These studies would not be truly innovative without surprising findings. During these tests, researchers also discovered that, after the fire, a bacterium that is not very well-studied serves as a protective barrier against the attack of pathogenic bacteria, alone. “After the assay, we had different results. Some isolated mice were able to recover their protective capacities, others did not, while those who were grouped recovered more easily. We identified that those who were able to recover had Klebsiella michiganensis, a commensal bacterium that has a very efficient metabolism and competes for nutrients after the exposure to antibiotics, conferring protection”, the researcher explains. The next steps will be to cross this bacterium with the quorum detection mechanisms to understand if, for example, it can be used as a probiotic.

The biggest effort of this team and of the millions of researchers that, across the globe, have tried to unravel the mysteries of the forest that lives inside us is to prove that, for our own good and for that of our species, we have to treat it very carefully, from the way we eat to the way we treat disease.