Investigando a comunidade microbiana no intestino grosso da térmita - Entrevista

So termites in their gut microbes are an example of a, a very fascinating and complex mutualistic symbiosis. So I'm interested in studying termites in these microbes to better understand all the different roles, the different components play as they degrade a very complex food source lignocellulose into products which both the insect and the microbes can use. I'm Jared Ledbetter.

I'm an associate professor of environmental microbiology at the California Institute of Technology, and I participate in our program of environmental science and engineering. I've been at Caltech for six and a half years, but for the last 17 years my primary research interests have been in the study of the microbiology microbial communities, which are resident in the hind guts of diverse termites. Since I first encountered termite gut microbiology as a student in the summer course of microbial diversity, which is taught in Woods Hole, Massachusetts, I've been very interested in really the identity and function of the various different microbial species that you find living in the hind guts of termites.

Termite hind gut communities are comprised of about 250 unique microbial species, which represent all three domains of life. You can identify metagenic ArcHa, anaerobic cellulose, decomposing protozoa, so unicellular eukaryotes, and a wide diversity of bacteria including many, many, many novel species of a group of bacteria called spirochetes. Although there are about 250 different species that live in termites and they are found nowhere else in nature, even after about a hundred years of study, we have a very poor grasp of what is the function of all of these different microbes in what in total is a very fascinating mutualistic symbiosis that occurs between the host insect and the microbial community, which together degrade li cellulose into products which basically fuel the metabolism of both the host insect and these various microbes.

So in the last 16 years, 17 years, I've been very interested in identifying who are these species and what are they doing? How do these species interact with each other in the hind gut and what is their benefit to the host? So termites in their gut microbes are an example of a, a very fascinating and complex mutualistic symbiosis.

So I'm interested in studying termites in these microbes to better understand all the different roles, the different components play as they degrade a very complex food source lignocellulose into products which both the insect and the microbes can use. Another reason for studying termites in their gut microbes is that termites are globally a very important group of animals that play very critical and important roles in global carbon and nitrogen cycles in the tropics and subtropics termites are extremely diverse. That is, there are many, many different termite species and they're very, very abundant.

And as one example of how successful these insects are, they are small but significant sources of global CO2 and global methane. By virtue of how much wood they've converted into these products, termites contribute about 2%of global CO2 and about 4%of global methane, which is actually quite significant for any one group of animals. So by studying the microbes in their activities in these insects, we better understand these insects and the basis for their success globally.

Studying the termites in their gut microbes is also interesting from the standpoint of scale and reproducibility. And at some levels I study termites in their gut microbes because this is a very exciting and complex environment. But the environment of the hind gut, this hind gut ecosystem is very different from many other environments.

The termite hind gut is very small about one microliter in volume and it's very well bounded in the sense that it's lined by the gut epithelium or by the insect itself. But we can define very concretely what are the bounds of that environment. And the environment is very small and we can collect a box of termites and have 200 termites, and on average we have that same environment replicated again and again and again.

If you compare the termite gut environment to, for instance the Sargasso Sea, the Sargasso sea is huge and enormous. It's a thousand miles wide. The boundaries of it are defined, but perhaps only operationally.

It's not a clear cut boundary to the Sargasso sea and there's only one of them. So we can learn a lot about marine sciences by the study of the Sargasso Sea and it's very important to study the sargasso sea. But there are certain complications you have to be able to get on a boat and go there.

If you have a finding about some of the microbes there, you may not know where those microbes are at any one point in time and they could differ in location by miles and of course there's only one of them. So it's hard to study the sargasso sea and perturb it in some way and have a control sargasso sea to compare it to. So by studying in their gut microbes, we actually have an example of a very, of an environment which is amenable to laboratory techniques.

We can reproduce that environment, we can perturb that and compare it to control groups. And we always know within about a millimeter where all of these different microbial groups and microbial species are interacting, they're interacting with this in this one microliter gut environment. So my laboratory, we like to study this microbial community using really any technique available that might help us to reveal the secrets of this community.

Certainly one aspect of this has been to try to grow species which are resident in that microbial community. In vitro in the laboratory 250 species is very complex and there's an unsurpassed clarity to the interpretation of research if you can study a pure culture of a bacterium. Some of our efforts have focused on going into this gut community and trying to cultivate and grow single species from that community and then study the physiology and genetics and genomics of those species in detail and then try to reflect back what we believe that means about the function of that species in that community.

An example of this is our study on termite gut. Spiro keets, Spiro keets are spiral shaped organisms, which are found abundant in almost all termites. In termites you have spirochetes, which are actually fairly closely related to the caused of agent of syphilis, but in all healthy termites you see these SPI keets.

And so for many, many years we knew that these spi keets are probably symbiance doing something positive for the host, but we didn't know what they did. So about 10 years ago, I was successful in growing some of these SPI keets in vitro and now we can study those SPI keets and we've learned a lot about their physiology and what those spirochetes are likely doing in the environment. Before that period of time, we had no idea about what these spirochetes were doing in their environment.

And so we've made many pure culture enabled discoveries by studying these pure cultures. We also use gene-based methods to study this microbial community. There may be a particular gene type which represents a certain physiology, and we can go into the this community and start to ask how many variations of this gene are present and who are the organisms that encode these key genes?

And so we can start to dissect functionally, functionally what is occurring in this hind gut by studying the diversity of genes involved with nitrogen metabolism and key aspects of carbon metabolism, we can also use gene-based mechanisms or gene-based approaches to assess the diversity of organisms in the termite. We can use ribosomal RNA genes as marker genes or as barcodes to really assess how many species are present in this community and perhaps in what abundance and how do our pure cultures actually reflect that diversity. So we use a number of gene-based techniques in my laboratory.

We were also engaged in metagenomics where we're interested in studying the whole genome content of this microbial ecosystem to try and get a an idea of what are all the different metabolisms that are occurring in this, this community. So in my laboratory we study pure cultures. We use PCR enabled gene-based techniques, we use metagenomics, we look at activities, enzymatic activities which are present in this hin gut.

And we also use microscopy to try and learn about where organisms are located in the gut and what other cells are they interacting with. In total, what we wanna do is understand how many species are present in the hind gut, what are they all doing and how are they all interacting with each other? There are a number of of challenges to studying microbial communities which are distinct from other communities in nature.

You know, it's, it's relatively straightforward to go for a walk in the woods or in the Savannah and to observe the different species of plant and animal that you encounter along your walk. And certainly in community ecology there have been many methods developed and approaches taken over the last a hundred years to start to ask questions about how many species a plant, an animal are present in a certain community and with what frequency do you encounter them in that community. And then actually observing some of these organisms in their community over time and to see what sort of adaptations they have to sort of environmental dynamics and environmental insults that they, they face.

You know, you can watch an elephant or a lion on the savanna and observe some of their behavior in their adaptations and how they acquire their, their energy and their food and how do they interact with each other. These are all fairly straightforward to observe when you're studying microbes, organisms that you can see with your eye. It is also fairly straightforward to, to differentiate whether you're dealing with a plant or an animal.

I don't mean as a terms of species, but in terms of what they do. You can look at an organism and you can say almost certainly this is a photo tr, this is an organism that absorbs solar radiation and turns that energy into something useful. Or I'm looking at a mediating animal and that this animal primarily uses protein as its source of energy.

So there are a number of observations you can make as you walk across the Havana community or a forest community. It's much more difficult to make such observations and conclusions when you study microbial communities. The organisms are small and it's not always obvious even if you have a very good microscope, what, how different organisms differ from each other.

That is, if you look at a microbial community per se, it may be very difficult to say how many species there are and what their relative distributions are. It may also be very difficult to look at that microbial community using a microscope and answer the question, what do these organisms do? So a major challenge in microbial communities is to ascertain who's there and what are they doing.

And so we have to use different techniques in the study of microbial communities to extract information from that community about who's there, who's active, what do these organisms do, and how do they respond and what are their adaptations to changes in their environment. Since I first became interested in the study of termite gut microbial communities 17 years ago right around 1990, there have been a number of technological advances in my field at large, which have really enabled me and others to learn much more about microbial communities and for me specifically termite gut communities. In large part, these are gene-based techniques.

When I first study started studying termites, you could use microscopy, you could look into a microbial community such as the termite and you could assess different shapes present in that community and get an approximation of perhaps how many different species were present in that community. But over the last 15, 17 years, so many gene-based techniques have become available and so many genomics techniques have become available that our, our ability to extract information from microbial communities such as the termite hind gut have really changed very rapidly. Now we can use genes as almost barcode markers to identify different organisms and environment.

We can go into a microbial community such as a termite hind gut and ascertain how many different species are present using certain genes as proxies of that diversity. And we can use that gene as a barcode to track whether or not we have those organisms in culture or to use that barcode to track fluctuations in the abundance of different species in that community. Using other gene-based techniques, we can ask, ah, are the genes that encode enzymes for this particular present in this community?

And how many variations, so how many species of organism are there that catalyze that and do we have those organisms and culture? So there have been a number of gene enabled and PCR polymerase chain reaction enabled approaches to study microbial communities, which have radically changed how we understand microbial communities. And these changes have all been manifested over the last 20 years.

The study of termites and their gut microbes is interesting, both from basic research or academic research level and also from a bio technological and applied research level. Termites eat and degrade wood. And for me it's very important to try and understand by what mechanisms do termites in their gut microbes actually attack wood, which is actually nature's first composite material.

It's a tri laminate composite and it's, it's tough stuff. And to really understand how this insect in its gut microbes deaminate this composite and then degrade all of its components into products which can be tapped as fuel both by the insect. And these microbes to me is very fascinating, but it also has some fundamental relevance to biotechnology and an industry.

As a society, we really don't understand completely. We really don't actually understand very much at all how wood is degraded into anything. And there is considerable interest these days in, in reaching a better understanding of how plant lignocellulose would, rice hulls corn cobs the leftovers of sugar cane after you squeeze out the sugar.

There's a lot of interest in understanding how we might take low value lignin cellulose commodities and turn them into something of higher value ethanol, hydrogen, methane, any other product. Really what's important to think about is that termites degrade lius cellulose wood all the time and they turn it into their own biofuel and industrially. There's a lot of interest in understanding what are the enzymes involved with those initial attack on wood.

And perhaps then one would be able to take those enzymes and couple, couple them with other downstream processes to come up with a new process which is different from what occurs in industry now and which is different from what occurs in nature now, but really takes the best out of the best to come up with something new, converting wood into a product of interest. Ethanol or hydrogen or methane or anything else really in my laboratory is primarily interested in, in achieving a, a fundamental understanding of this microbial community. And its interaction with the host and the role the host in the microbial community play in lignocellulose degradation.

But there is considerable interest in, in trying to take some of that information and and tap that in the development of novel industrial processes. I think that there's been a lot of discussion about biofuels in the last two years and this has raised attention of perhaps the need and even promise of, of biofuels research. I have to be honest though, I don't think the level of funding initiatives is commensurate with the interest that this has received.

There is no Manhattan project for biofuels. There has not been any dramatic large scale investment in basic research that would relates to biofuel's research. And because our fundamental understanding of lius cellulose degradation is so poor at this point, I think if we have a biofuel initiative, it has to include focused investment at all levels, fundamental, basic academic research all the way out to industry to make that possible.

And if people want this to happen in two or five or 10 years, we're also gonna have to invest funds so that we have people invest in their time and efforts to make that happen. Over the last 20 years, our ability to use gene-based techniques to study microbial communities has progressed so rapidly. I think we can all dream a little bit about what can happen over the next five to 10 years, and I think there's a very good chance that in 10 years we will have the capacity to know and sequence and analyze the entire genome of each and every species.

In the termite hind gut, there are 250 at least, and I think there'll be an opportunity to have this filing cabinet, if you will, of information where we know the genome content of each one of those species. And that is gonna be a very dramatic finding because only 10 years ago we were sequencing the genomes of very well understood microorganisms for the very first time. And in another 10 years, I think we will be able to go into complex microbial communities and have access to genome information of organisms, which we've never, ever studied in any sort of detail, and be able to use those to leverage studies on these organisms for the first time.