Researchers investigate a widespread hitchhiker in the human gut

DNA sequencing is faster, more affordable, and more widespread than ever before, especially in studies of the gut microbiome. Sequences from bacteria of the gut microbiome have now been characterized in countless studies from thousands of people all over the world. So, how could it be possible that a small “genetic hitchhiker,” highly prevalent in microbiomes from people in the western world, has gone unnoticed for so long?

In a recent publication in Cell, Emily Fogarty PhD, a former UChicago graduate student in the laboratory of A. Murat Eren (Meren), PhD, describes pBI143: an enigmatic genetic element that’s remarkably conserved in the gut microbiomes of people living in industrialized countries, without any clear benefit to its bacterial hosts.

The Meren Lab, a former UChicago laboratory now based at the Helmholtz Institute for Functional Marine Biodiversity in Oldenburg, Germany, was primed to make this sort of discovery. The lab’s research focus is to understand how microorganisms, both in nature and in human systems, interact with one another and their environments.

“Meren was re-analyzing some gut microbiome data and came across an anomaly,” Fogarty said. “He found that one of the sequences of DNA in these gut samples was astoundingly abundant—we’re talking three to four orders of magnitude more abundant than expected.”

It was during Fogarty’s Meren lab rotation that she first investigated this strange piece of DNA.

“I spent my first couple weeks in the lab learning the techniques needed to analyze DNA sequencing data,” she said. “By the end of those two weeks, I was hooked.”

A plasmid prevalence paradox

Variation in the DNA sequences of microorganisms allows them to rapidly adapt and interact with changing environments and acquire new useful traits. While humans mostly acquire this variation through sexual reproduction, bacteria predominantly rely on genetic mutations and horizontal gene transfer: the uptake of genetic material from the environment or interfacing microorganisms and viruses.

Plasmids are key agents of horizontal gene transfer. These small, circular pieces of DNA replicate independently of their bacterial hosts. Plasmids typically encode traits beneficial to bacteria, such as antibiotic resistance. However, some plasmids don’t seem to contain any advantageous traits at all.

pBI143 is one such “cryptic” plasmid. It is impressively tiny, encoding only two genes: one for replication and one for transferring between different bacterial hosts.

In a screen of more than 4,500 human gut microbiome samples across 23 countries, Fogarty and coauthors found pBI143 in nearly 75% of all samples–an enormous fraction for something studied so little. However, its presence was not uniform. pBI143 was widespread in the guts of individuals who lived in relatively industrialized countries, such as Japan and the United States, but rarely detected in non-industrialized countries such as Madagascar or Fiji.

Further, pBI143 appeared as three distinct versions, each characterized by tiny variations in the gene responsible for its replication. Aside from these small variations, the DNA sequence of this plasmid was consistent. These observations suggested that this bare-bones genetic element was subjected to strong evolutionary selection.

pBI143 is efficient and versatile

Fogarty’s massive genomic screens left her with unanswered questions. What explains the uneven distribution of pBI143 in the industrialized world, and what is the relevance of the different plasmid sub-types?

Individuals living in industrialized and non-industrialized nations have distinct gut microbiome compositions owing to differences in diet and lifestyle. For example, people from industrialized nations tend to have a much higher proportion of gut bacterial species belonging to the genus Bacteroides, one of the primary hosts of pBI143.

While this explains why this plasmid is typically found in the western world, Fogarty wanted to know if the three different versions of pBI143 were specific to certain bacterial species–a question she couldn’t answer using genomic data alone. Turning to samples collected at the UChicago Duchossois Family Institute, Fogarty investigated if different versions of pBI143 were present in different species of bacteria.

“I went in with the idea in mind that I was going to see a nice grouping of version one, version two, and version three in these different bacterial species,” Fogarty said. “We did not see that at all.”

Instead, she saw that the version of pBI143 within an individual had no correlation to the bacterial species in the gut. However, pBI143 was extremely specific to the individual from whom the sample originated. This observation, Fogarty remarked, likely speaks to the efficiency of pBI143 to move to different hosts once it’s introduced to the human gut.

“What this result indicated to me is that this plasmid is capable of extremely rapid transfer. There seems to be some type of priority effect; once it is acquired in one bacterial host cell, it transfers within most of the population of available cells within the gut,” she said.

The genetic hitchhiker

It was now clear that pBI143 was a highly efficient genetic element that was strongly selected for in the microbiome. But the forces driving the overwhelming prevalence of this newfound plasmid, likely the most abundant mobile element in the human gut, remained a mystery.

Fogarty faced two possibilities. Perhaps pBI143 confers an unknown benefit to its bacterial hosts, which would explain its widespread prevalence. Alternatively, this plasmid could act more like a virus or a “genetic hitchhiker”: a free agent shuttling between disparate bacterial hosts, offering nothing in return.

To investigate this, Fogarty decided to observe the effects of pBI143 in a Bacteroides species within mice.

“This was a difficult process,” said Fogarty. “The real challenge was figuring out how to insert the plasmid into cells without disrupting its native structure.”

Using a creative experimental plan developed with help from UChicago investigators Mark Mimee, PhD and Laurie Comstock, PhD, Fogarty incorporated pBI143 into a larger plasmid vector introduced those into bacterial cells, then performed a series of nuanced selections. These steps resulted in two groups of bacteria that were genetically identical: one with the native pBI143, one without. The survival of the two bacterial populations was then measured in mice.  

To Fogarty’s surprise, pBI143 seemed to have a slight negative impact on the survival of the bacteria, suggesting that it does not confer an advantage to its host in this system.

While puzzling, this result prompted crucial questions in our understanding of cryptic plasmids. It is still not known why cryptic plasmids exist in the first place. In fact, this work was one of the first to experimentally test the function of cryptic plasmids.

One theory is that pBI143 overcomes negative consequences by hopping between bacterial hosts. When transferring between bacteria, it may occasionally pick up additional genetic material– a hypothesis supported in Fogarty’s genomic data, where a few pBI143 sequences contained additional genes. These extra genes, even if exchanged at low frequencies, may offer benefits to the bacterial host. In controlled environments like the mouse models, however, pBI143 may slow the growth of its bacterial hosts.

“Our running theory is that pBI143 shifts between the naïve 2-gene state, where it acts parasitically, and the larger state with varying additional genetic material,” Fogarty said. “When it transfers the additional genetic material between populations, it’s very possible that it confers benefits to the bacterial host, the effect of which depends on what the additional genetic cargo is in that instance.”

Redefining the microbiome

While our understanding of the cryptic plasmids is still preliminary, Fogarty and her research team have established that they are more familiar than previously thought. For example, in a screen of genomic data from inflammatory bowel disease patients, Fogarty found that pBI143 had increased the copies of its genes, a consistent feature of well-known non-cryptic plasmids in times of host stress.

This quality of pBI143, they found, makes it a useful indicator for cell stress in inflammatory bowel disease, suggesting its potential in clinical applications. The sheer abundance of this plasmid means that it can serve powerful tool for detecting human-associated bacteria in other settings, like contaminated water samples.

As technological advances in DNA sequencing continue, cryptic plasmids will likely receive more attention for their role in the human gut. In fact, Fogarty says that her deep dive of pBI143 reframed her idea of the human gut microbiome, informally defined as the collection of bacteria, archaea and fungi inhabiting our digestive tracts.

“Plasmids will often carry fitness benefits that are really critical in a specific environment. If they are shared across many individuals, that is something that should be accounted for in the idea of a ‘core microbiome,’ because it is going to have an important impact on the microbes that live in that environment,” she said.

“I think expanding the definition of the core microbiome to include plasmids and other mobile genetic provides a more holistic view.”

Research in this press release was supported by University of Chicago International Student Fellowship, NIGMS R35 GM133420, National Science Foundation Graduate Research Fellowship under grant number 1746045, National Science Foundation Graduate Research Fellowship under grant number 1746045, Swiss National Science Foundation (NCCR Microbiomes - 51NF40_180575), and NIH NIDDK grant (RC2 DK122394) and University of Chicago start-up funds.