The human body provides a home to microbes of different varieties, including bacteria, archaea, fungi, viruses, protists and microscopic animals. The collective sum of these organisms is known as the human microbiome, Over recent years, the human microbiome has garnered increased research attention, and is now dubbed by many scientists as "an organ in its own right", with a plethora of studies outlining its contribution to human health and disease. The microbes inhabiting our bodies can disturb our energy metabolism and immunity, and therefore significantly influence the development of an array of human diseases.
However, one element of human microbiome research that has not made headlines is the difficulty that scientists face in analyzing how it changes over time in response to certain stimuli.
The most commonly adopted approach to analysis is extracting bacteria from a fecal sample and then sequencing the bacteria's genome. Whilst advances in next-generation sequencing (NGS) techniques have bolstered the field of microbiome research, the issue remains in that crucial information is lost about where and when bacterial changes actually occur in the gut. The picture is incomplete.
In a new study published in Nature Communications, scientists have developed a novel approach that they hope will help to overcome these issues and complete the full picture of the human microbiome physiology. Their solution comes in the form of an oscillating gene circuit containing a distinct set of engineered bacterial genes. These genes have been designed to detect and record changes in the growth of different bacterial populations in the guts of living mice with single cell precision.
Building a repressilator
The three genes in the circuit encode the proteins tetR, cl, and lacl, each of which blocks the expression of one of the other proteins. They are linked in a negative feedback loop, whereby a drop in concentration of one of the repressor proteins below a certain level triggers expression of the protein that it had been repressing. In turn, this blocks the expression of the third protein.
Inserting all three genes into a plasmid and introducing the plasmid into bacteria enables the scientists to record the negative feedback loops. The number of loops that occur acts as a measure for the number of cell divisions that have taken place. Each time the bacteria cells divide, the repressor proteins fall in concentration, and therefore trigger the expression of the second protein in the repressilator cycle.
Regardless of the speed at which the bacteria are growing, the repressilator cycle repeats after 15.5 generations of bacteria, enabling an objective measure of time. The scientists behind the method liken this to a clock or a watch:
"Imagine if you had two people wearing two different watches, and the second hand on one person's watch was moving twice as fast as the other person's," explained first author David Riglar, Ph.D., a former postdoc at the Wyss Institute and HMS who now leads a research group as a Sir Henry Dale Fellow at Imperial College London. "If you stopped both watches after one hour, they wouldn't agree on what time it was, because their measurement of time varies based on the rate of the second hand's movement. In contrast, our repressilator is like a watch that always moves at the same speed, so no matter how many different people are wearing one, they will all give a consistent measurement of time. This quality allows us to more precisely study the behavior of bacteria in the gut."
Lighting up the way for microbiome research
The three proteins were each coupled to a differently colored fluorescent molecule, and an imaging workflow named repressilator-based inference of growth at single-cell level (or, RINGS) was used to track which of the proteins was being expressed at different time points throughout the bacteria's growth.
"As a bacterial colony grows outwards, the repressilator circuit creates these different fluorescent, tree-ring-like signatures based on which repressor protein was active in the single bacterium that started the colony," said Riglar. "The pattern of the fluorescent rings records how many repressilator cycles have occurred since growth began, and we can analyze that pattern to study how growth rates vary between different bacteria and in different environments."
The team of scientists were able to monitor cell division in a variety of different bacterial species in vitro. They then grew the bacteria on extracted samples of mouse intestine (to simulate a complex microenvironment) and exposed the bacteria to an antibiotic (in order to mimic stressful conditions) and observed that the bacteria's repressilator cycle remained constant.
The next steps involved assessing the repressilator's performance in vivo. To achieve this, the researchers administered E.coli containing the repressilator circuit to mice orally, and analyzed bacteria in the fecal samples of the mice. They found that the repressilator system remained active for up to 16 days post-introduction, illustrating that the engineered genes could be maintained in gut bacteria in vivo. Using RINGS, Riglar and team successfully analyzed changes in bacterial growth patterns and found that bacteria whose repressilator circuits were in different stages could be "synchronized" by adding a compound to the mice's drinking water that arrested the repressilator cycle.
To explore the system's function when researching diseased states, the mice were given an inflammation-inducing compound followed by the repressilator-loaded bacteria. After 15 hours, analysis utilizing RINGS illustrated that the bacteria from the inflammation-induced mice had repressilators in a wider range of phases when compared to controls. These findings suggest that inflammation initiates a change in the environment that drives inconsistency in bacterial growth.
"This repressilator allows us to really probe the intricacies of bacterial behavior in the living gut, not only in both healthy and diseased states, but also spatially and temporally," said corresponding author Pamela Silver, Ph.D., who is a Core Faculty member at the Wyss Institute and the Elliot T. and Onie H. Adams Professor of Biochemistry and Systems Biology at HMS. "The fact that we can re-synchronize the repressilator when it's already in the gut, as well as maintain it without the need to administer selective antibiotics, also means that we can study the microbiome in a more natural state with minimal disruption."
The scientists note that in addition to aiding our understanding of the microbiome dynamics, the repressilator tools can serve as a platform for synthetic-biology-based diagnostics for a wide variety of applications in the gut. One example they provide is the creation of a system that is programmed to initiate a gene transcription cascade at a specific point in the circadian rhythm, or a diagnostic test that measures how much time has elapsed following the detection of a certain biomarker.
"Not only does this research solve a specific problem related to monitoring dynamic changes in microbiome physiology within the living gut, it provides a platform that could lead to entirely new types of diagnostics and even time-dependent therapeutics," concludes Wyss Founding Director Donald Ingber, Ph.D.
Reference: Riglar et al. 2019. Bacterial variability in the mammalian gut captured by a single-cell synthetic oscillator. Nature Communications. DOI: https://doi.org/10.1038/s41467-019-12638-z.