What effects do specific members of the microbiota have on their host?
What’s in a name? That question is often difficult to answer in the field of gut microbiota studies, where we explore connections between the microbial communities in the gastrointestinal tract (the gut microbiota) and the biology of their host. This is largely because a lot of the tools we use to study the microbiota are good for identifying the organisms in these communities, but are not so great at telling us what those microbes are doing. However, figuring out the function of the microbiota is essential for understanding how animals interact with their microbiota as well as using the microbiota to intentionally shape host biology.
This was the challenge for our recent paper, where we set out to characterize the Turicibacters, a genus of bacteria from the vertebrate gut microbiota. These bacteria have been found in a wide range of animals, including humans and mice, and have been found to be closely linked to host genetics, suggesting (but not proving) important connections to their hosts. Also, Turicibacters have often shown intriguing correlations with specific areas of host physiology, including lipids and neurotransmitters, which could be important if we want to exploit the microbiota to improve these areas of health.
A previous postdoc in the Hsiao lab, Thomas Fung, discovered that Turicibacters benefitted from high levels of the neurotransmitter serotonin in the intestinal tract, likely through a bacterially-novel putative serotonin transporter. However, although this finding was super interesting, there was very little known about any members of the Turicibacter genus; the type strain, Turicibacter sanguinis MOL361, was really the only isolate that had been studied in the 20+ years since the Turicibacters were first described, and even then had only been characterized to any level in in ~3 papers. (Note: recently, Joel Maki and colleagues have begun to characterize another Turicibacter, Turicibacter bilis, so the field is already expanding!).
Digging into Turicibacter biology
With this in mind, we initially set out with a fairly straightforward goal of characterizing more Turicibacters so we could start to understand the mechanisms behind their associations with their hosts . We gathered Turicibacter isolates from human and mouse feces, then sequenced and assembled their genomes and found something surprising: even though they would typically be grouped together by microbiota analysis techniques like 16S rRNA sequencing, these isolates were very different from one another at the genomic level. For example, the genomes of the mouse isolates were ~2/3 the size of the largest human isolate genomes, indicating a huge amount of non-shared DNA between these related bacteria (Fig.1).
Unfortunately, we made these discoveries right before the Covid-19 pandemic disrupted our research for several months. This eventually led to us transitioning to a mix of in-person and remote research, with one goal being that trainees who weren’t allowed back in the lab at the time could continue to be involved in research. To that end, we decided to dive into an interesting finding from Julia Kemis in Federico Rey’s lab at the University of Wisconsin-Madison: Turicibacter sanguinis MOL361 could transform bile acids. Bile acids are a mixture of compounds that animals release into their intestinal tract where they can be chemically transformed by a wide range of bacteria, altering their function. The Rey lab found that MOL361 could transform bile acids, and we decided to try to identify and characterize the Turicibacter genes involved in these processes.
We grew a few of our Turicibacter isolates in a mixture of bile acids, then measured what kinds of bile acid transformations they could perform using liquid chromatography mass spectrometry (LC-MS). Initially, we were really just trying to hone our LC-MS protocol, so we were surprised to find that although all of our isolates altered some bile acids, each genomically distinct group of Turicibacters performed different chemical transformations. As different transformations lead to unique bile acid pools, this could mean that each Turicibacter group was able to generate a different bile acid profile in their host. This agreed with our concurrent analysis of circulating metabolites in mice that were colonized by different Turicibacters, where Turicibacters stimulated unique changes in the animals’ bile acids.
We combed through our assembled Turicibacter genomes looking for homologs of genes predicted to be important for bile modifications and found that they were present in all our genomes, albeit with different genes in different genomic locations for each group of Turicibacter isolates. We then expressed each gene in E. coli and confirmed that they each performed similar but unique bile transformations, largely mirroring the abilities of the Turicibacter each was derived from.
What does this mean for the Turicibacter-host relationship?
Turicibacters are diverse and perform different bile transformations, but what does that mean for the relationship between these bacteria and their hosts? Thomas had found that MOL361 had a big effect on host lipids, and our metabolomics analysis showed that colonization with different Turicibacter isolates stimulated unique lipid profiles in mice, so we decided that host fat was a logical place to look next. Further, bile acids are important for fat digestion and metabolism, so it made sense that Turicibacter bile transformations could have an effect there. We don’t have the ability to genetically modify Turicibacters yet, so we decided to express individual bile-modifying Turicibacter genes in another gut bacterium, Bacteroides thetaiotaomicron, to test their effects on host fat. This led to one final surprise, as we found that colonizing mice with B. thetaiotaomicron that expressed these genes by themselves was enough to globally reshape the fat of a mouse (Fig. 2). Turicibacter bile-modifying genes were especially good at reducing cholesterol, triglycerides, and overall fat tissue in mice, which are all important for both healthy animal metabolism as well as diseases like obesity and cardiovascular disease.
Bacteria in the gut microbiota do a lot of important things for their host animals, but it isn’t always easy to figure out exactly how. We found that gut bacteria from the Turicibacter genus were able to have a big impact on host bile, and subsequently, host lipids and cholesterol. This offers a mechanistic connection between these bacteria and the physiology of their hosts, as well as informs further exploration into connections between host biology, Turicibacters, and other members of the gut microbiota. This project was a lot of fun to work on because it was full of unexpected twists and turns, and we’re looking forward to seeing where these results lead next!