What is a microbiome and describe its significance.
A microbiome is a community of microorganisms occupying a specific environment or ecological niche. One of the most thorough ways to identify a specific microbiome is through the identification of the genomes of community members. Humans host a number of microbiomes. They occupy distinct niches within the human body. The human intestinal tract represents one of these microbiomes. As many as 1000 different species of microorganisms comprise the intestinal microbiome of human beings. This community is important in several ways: they assist in digestion, improve energy uptake from food, produce vitamins and aid in the development of the immune system.
Not surprisingly, microbiomes are an important topic in health studies. The Human Microbiome Project (HMP) is a U.S. National Institutes of Health Roadmap for Medical Research. The HMP is looking for a "core" microbiome. They are attempting to find aspects of human microbiomes that are universal. Studies to date have largely focused on clinical samples and model organisms. Our research compliments this objective by examining the evolution of microbiome ecosystems in samples unaltered by modern advances in medicine or contexts blurred by globalisation.
What prompted you to undertake this line of research?
The short story, there were three factors prompted me to conduct this research: 1) an understanding of the challenges involved in sequencing ancient genomes; 2) a realisation that the human microbiome evolution is still poorly understood; 3) the resources available to me, including the impressive infrastructure provide by the University of Oklahoma (OU) and the collaboration of several essential scholars, including Raul Tito and Lauren Cleeland from my lab, biologists Karl Reinhard and Agustín Jiménez Ruiz from the University of Nebraska, who provide contextual data and access to the coprolite samples, and the additional wet lab support provided by Bruce Roe's group at the OU Advanced Center for Genome Technology, including Fares Najar, Simone Macmil, Graham Wiley, Chunmei Qu and Ping Wang.
The long story, my graduate work at the University of New Mexico included an ancient DNA analysis of human skeletons and mummies, but during my postdoc at the University of Michigan's Human Genetics Department, I put my wet lab work temporarily on the shelf to focus more intensely on statistical approaches to genetic research. But I kept my eye on what was being published in ancient DNA.
When I started my faculty position in the Department of Anthropology in 2007, OU provided excellent labs, including a lab devoted to ancient DNA. I was once again able to contribute to ancient DNA research. What I needed was an interesting and challenging project. I was particularly interested in evolutionary based projects, which would influence (or enhance) current health focused research.
A number of well-established labs have been sequencing ancient genomes, but the challenges they faced sparked an idea. For example, groups from Canada, Denmark and Germany are involved in sequencing the Neanderthal and Mammoth genomes, which will inevitably provide interesting information about evolution. However, this impressive work is not without challenges. One particular difficulty is that ancient bacteria within ancient samples are often sequenced alongside the ancient Neanderthal and Mammoth genomes. This represents a detrimental increase of cost, in both time and money, for the sequencing and data analysis of ancient projects.
I decided to turn this detriment into a benefit, by focusing on the bacteria within ancient samples. I learned more about human microbiome projects. I learned that intestinal microbiomes were frequently studied from human fecal samples. This is when everything fell into place.
I realised that an ancient human microbiome project was theoretically possible: 1) we know that ancient bacterial DNA can be sequenced because these sequences are abundantly retrieved by ancient DNA studies that typically would prefer to avoid them; 2) we can get ancient feces, these are called coprolites which have been studied by archaeologists and biologists for years; 3) my ancient DNA lab and the genome centre run by Bruce Roe provided the necessary infrastructure to start collecting and analysing the data. The question remained, if we get ancient microbiome data, will it be clearly meaningful. In other words, would our data fit the expected intestinal bacterial profile of a human gut, or would we obtain something completely unexpected?
How can the investigation of ancient human microbiomes help shed light on modern ones? Moreover, how can it help establish a core human microbiome?
The question of whether there is a core microbiome remains. One potential confounding factor is that industrialised nations are on the receiving end of a global economy, where largely non-local diets are commonplace. It is possible that human microbiomes studied from clinical samples are too heterogeneous for systematic studies of microbiome evolution. If we want to understand the microbial ecology that co-adapted with our ancestors, then looking at our ancestor's microbiomes is a good strategy.
How do human microbiomes evolve theoretically? How have they evolved in reality? Can you speculate on how human microbiomes might evolve in the future, considering the modern world, technology, and transport?
These are questions we are currently trying to address. We can say that human microbial ecologies face selective pressures influence by diet, medical practises, and the host genome. As for the future of microbiomes, it is hard to speculate when there is still so little we understand about the microbiomes of the past and present. I suspect we will identify increasing variation within populations and decreasing variation between populations as long as the global economy remains viable. But then again, our current obsession with antimicrobial treatments may result in a significant loss human microbiome variation.
It is also possible that a global core human microbiome existed even in prehistoric times. Scientists have speculated that a typical bacterial species has few geographic constraints, commonly referred to as the "everything is everywhere" hypothesis. This hypothesis suggests that any geographic structure observed in microbial ecosystems is attributed to natural selection within a specific environment and not to exposure to a specific bacterial species. Our published study did not address whether "everything is everywhere", but it did raise a question about the role of natural selection.
How can you tell whether ancient coprolites are human?
If DNA has preserved well enough, then some of the host cells will be present within the coprolites. We use a chemistry that specifically targets human genomes (human specific PCR primers), particularly a genomic region informative of aspects of geographic ancestry. In this case, we target the human mitochondrial genome. Because, these coprolites were from prehistoric Mexico, the human mitochondrial DNA retrieved should have variation characteristic of Native Americans. We found this to be true. We also found that our two coprolite samples originated from two different individuals, evidence by two different Native American mitochondrial types.
Can you describe how you approached studying microbiomes and what you discovered?
All the initial preparation of the samples was performed in our ancient DNA laboratory, which is a positive pressure clean room with filtered air and antimicrobial UVC lighting. This reduces the chance of DNA contamination. We followed several standard procedures for controlling for contamination in this lab, but we added an additional control by a novel use of DNA tags.
Once we confirmed sufficient DNA in our samples, we began the necessary preparation for high throughput sequencing using the 454/Roche GS FLX pyrosequencing system. Special tags, called multiplex identifiers, were added to the DNA fragment prior to the ancient DNA samples being removed from the ancient DNA laboratory. This provided a unique method of assessing DNA contamination that occurred during the later stages of pyrosequecing.
The DNA sequences represented a near random sampling of the DNA extract. Bacterial genomes are by far the most abundant in the sample; thus, they are sequenced preferentially. From here we analysed the sequences in two ways. One, we attempted to determine the bacteria represented. Two, we attempted to determine the genes represented. We then asked, do the bacteria and gene profiles match that expected of modern human intestinal microbiomes? The answer was yes! This alone was exciting, as it demonstrated that ancient microbiomes can be studied. We also found that these two ancient microbiomes, despite originating from two different people, were very similar, and that this similarity could not be attributed to chance.
With respect to microbial geography, as mentioned earlier, we did not address the question of whether "everything is everywhere", but we did raise questions about the role of Natural Selection in microbiome evolution. Within a gut, genes from different bacterial species are thought to provide redundant functional roles within the microbial ecosystem, such as carbohydrate or protein metabolism. If this is the case, then natural selection may impose pressures for particular functional attributes, but may be less of a factor when it comes to which bacterial species assumes that role. This means that presence of a particular bacterial species within these ecologies may evolve more stochastically, while the functional profile approaches a steady state with the selective pressures.
What challenges do you face researching ancient human microbiomes?
We characterised two extinct human microbiomes when there is still very little known about modern human microbiomes. Generally, this is not how ancient DNA research works. Typically, ancient studies are grounded on a foundation provided by modern studies. There are fantastic modern studies out there, for example, results from Gorden's lab at Washington University. Nevertheless, the work on human microbiomes is still at a very early stage. Our research demonstrates that extinct microbiomes can be studied. Because these extinct microbiomes are not confounded by the processes that happened from modern globalisation, they may help lay a foundation for identifying the "core" microbiome. But, this type of research is expensive - now we need to convince the granting agencies.
The coprolites we studied were ideal, a cave that was relatively inaccessible by non-human mammals, very good DNA preservation, and no evidence for soil contamination. We have many coprolites in my lab, and several of them are from undisturbed contexts with good DNA preservation. The biggest challenge is soil contamination. Some of the bacteria typical of the gut are also found in soil. We have samples where soil contamination is less of a concern, including some that were taken directly from a mummy's lower intestine, so these samples are next on our agenda. Nevertheless, soil contamination is a primary concern.
What is next for your laboratory in terms of research? More microbiomes?
We have some great coprolites from Texas and southern Peru/northern Chile, thanks to collaborations with Kristin Sobolik (University of Maine) and Art Aufterheide (University of Minnesota), respectively. We are excited to examine these samples. Also, we are adding a contemporary study thanks to collaborations with Morris Foster, Paul Spicer and Paul Lawson at the University of Oklahoma and Beatriz Lizarraga at the University of San Marcos, in Lima, Peru.
More information about his research can be found at www.anthdna.com or www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0003703
(Reporting by Marc Landas)
