March 1, 2015 (Vol. 35, No. 5)
Richard A. A. Stein M.D., Ph.D.
Scratching the Surface of the Microbiome Has Already Revealed Incredibly Rich Host-Microbe Interactions
To envision the abundance of the unseen majority inhabiting our planet, it is sufficient to remember that the bacterial biomass is estimated to exceed that of all animals and plants combined.
With respect to the human body, bacterial cells outnumber human cells by a factor of ten, leaving little doubt that we are more bacterial than human. As recent strides in biotechnology and life sciences reveal, capturing the structure and the function of the microbiome is an indispensable prerequisite for understanding health and disease.
“The big tidal wave of interest has been in the bacterial microbiome, partly because bacteria are quickly and inexpensively typed. While this is more complicated for viruses, the viral microbiome will also catch up,” insists Herbert W. Virgin IV, M.D., Ph.D., professor and chair of the department of pathology and immunology at the Washington University School of Medicine.
In a recent study, Dr. Virgin and colleagues showed that in mice treated with broad-spectrum oral antibiotics and subsequently inoculated orally with murine norovirus, the antibiotic prevented the establishment of a persistent enteric viral infection in most animals. “It was not known that antibiotic control of bacteria could have a secondary consequence on viral persistence,” explains Dr. Virgin.
In vivo, this effect was specific to the intestine, and in a cell culture system, the antibiotic did not prevent viral replication. “A key element of this finding was that antibiotics were used, in our experimental setting, as a pretreatment, and it is important to note that there is no evidence that an established viral infection could be treated with antibiotics,” he adds.
A higher viral dose overcame the effect of the antibiotics, suggesting that host pathways that regulate viral replication could be involved in preventing the establishment of the persistent viral infection in the intestinal tract. “We wanted to identify the host gene that is involved, and this turned out to be the host molecule interferon lambda,” explains Dr. Virgin. Interferon lambda was able to cure the established infection independent of adaptive immunity, reducing viral shedding 100-fold within two days.
“This suggested that there may be ways to stimulate the innate immune system itself to eliminate viral infections in the intestine,” says Dr. Virgin. When the gut microbiome was eliminated by antibiotic treatment, the viral infection disappeared in wild-type mice, but not in mice lacking the interferon lambda receptor, and restoring the gut microbiome by fecal transplantation helped rescue viral replication.
One potential explanation for these results is that the intestinal bacterial microbiome could help the persistence of noroviruses by controlling the lambda interferon pathway. “Key challenges will be to explore these findings in humans and to understand the precise molecular mechanisms, and this will take some time and a lot of work that we are very excited to pursue,” notes Dr. Virgin.
The air is full of microbiomes of human origin, and the contribution to the air microbiome differs across populations, according to Patrick Lee, Ph.D., assistant professor at the school of energy and environment at the City University of Hong Kong.
Dr. Lee and colleagues conducted a comprehensive analysis of bioaerosols in 7 of the 10 lines from the Hong Kong subway network, one of the busiest subway systems in the world, and concomitantly surveyed the air bacterial microbiome at several locations citywide.
“The air microbiome is a dynamic ecosystem, and especially under certain conditions, humans are changing it,” Dr. Lee claims. The analysis revealed a more diverse subway microbial habitat than previously thought. The subway bacterial microbiome included representatives from several ecological habitats, including soil, water, and vegetation, along with representatives of the commensal human skin and oral flora, and its structure varied with the structure of the nearby outdoor microbiomes.
The most common bacteria found in the subway were human skin commensal species. During afternoon and evening hours, the subway bacterial microbiome was more diverse than during the morning hours, and microbial diversity varied with several parameters, including temperature, humidity, and carbon dioxide levels. Further analyses indicated that closely connected subway lines shared more microbial communities than lines located further apart.
“Because the concentration of microbes in the air is low, large volumes need to be collected for these analyses,” says Dr. Lee. Currently, collecting an air sample requires about two hours, and results are obtained after about a week. “As we come up with better ways to collect the samples, and the bottleneck in terms of the downstream sequencing is reduced, we hope to improve turnaround times and make this an online, real-time detection system.”
Humans constantly shed bacteria from their body into the environment and shape the microbial ecosystem on surfaces and in the air and, at the same time, the air microbiome impacts people. “We are currently mapping out the interrelationships between these two ecosystems, to better understand the interactions between them,” says Dr. Lee.
Cecil M. Lewis, Jr., Ph.D., associate professor of anthropology at the University of Oklahoma, believes our understanding of aspects of human biology in the last few years has dramatically changed thanks to microbiome science. One of the major efforts in Dr. Lewis’ lab is the study of microbial populations on coprolites, which are fecal material retrieved from archeological samples.
“A lot of our work proposes to understand the ancestral state of the human microbiome,” says Dr. Lewis. Dr. Lewis and colleagues performed next-generation sequencing of the 16S rDNA V3 region on samples collected at three archeological sites in the United States, Chile, and Mexico, and revealed that coprolite samples are informative about the distal gut microbiome.
“Work has shown that individuals with a more traditional lifestyle, such as the ones from rural populations, have a potentially commensal Treponema strain lost in urban peoples; our work has found this same commensal bacterium in ancient microbiome samples from Mexico that are over a thousand years old,” explains Dr. Lewis.
These findings indicated that ancient microbiomes are different from existing cosmopolitan microbiomes, but are more closely related to current rural microbiomes. “Now that we have established that rural microbiomes have something distinct, we would like to understand why we are seeing these differences,” says Dr. Lewis.
Dissecting the functional role of individual bacteria that make up the microbiome is one of the major efforts in the field. “There are several pathways to study function,” Dr. Lewis explains. One of these, metagenomics, involves using DNA extracted from ecological samples to identify individual genes and characterize them functionally.
“Metagenomics is incredibly powerful, but even though it informs about genes that are present, it cannot tell much about which of these genes are active,” continues Dr. Lewis. An alternative strategy, which is more informative about the functional potential of the individual microbial representatives within any ecosystem, is collecting information about proteins themselves.
“The metaproteomic approach can identify the genes that are expressed and provides a much more direct way to study function, but it is nowhere close to being as advanced as metagenomics is today,” notes Dr. Lewis.
Another strategy involves examining metabolites, a larger category of products that also includes proteins, to capture the metabolic activity of a system under various conditions or dynamically, over time. “Looking at these byproducts of metabolism provides a powerful line of evidence about function, but there are limitations of how the data can be analyzed,” says Dr. Lewis.
New Generation of Antibiotics
GlaxoSmithKline has a broad interest in the microbiome, and the company is closely watching the space to identify opportunities that could improve its understanding of disease, reports James R. Brown, Ph.D., director of computational biology, infectious disease at the company.
Dr. Brown and colleagues recently examined the effect of GSK1322322, a new antibacterial compound that inhibits bacterial peptide deformylase, on the human gut microbiota. The very specific mechanism of action of this compound, and the fact that it belongs to a new generation of antibiotics, eliminated potential confounding effects as a result of antimicrobial resistance due to previous clinical exposure.
“The purpose of this study was to understand if there are any downstream effects of this antimicrobial compound on the gut microbial community,” explains Dr. Brown. “While long-term antibiotic use has long been known to perturb the microbiome, very few longitudinal clinical studies have explored this in depth.”
In a randomized, double-blind, placebo-controlled clinical trial that enrolled 62 healthy volunteers, Dr. Brown and colleagues used next-generation sequencing on bacterial 16S rRNA at the pre- and post-dosing time points to compare the placebo, intravenous and the combined oral-intravenous administration of this compound.
“The oral, but not the placebo or intravenous formulation of the drug changed the gut microbiome,” says Dr. Brown. Microbiome changes involved a significant decrease in the relative abundance of certain bacterial species and an increase in the abundance of others. Functional analyses revealed that the most pronounced changes occurred in genes involved in antibiotic resistance and metabolism.
In addition to the intestinal microbiome, perturbations in the microbial populations from other organs present clinical importance for disease management. “As part of collaborative projects, we are looking at how the lung microbiome changes in patients with chronic obstructive pulmonary disease and asthma, and how these changes may further exacerbate these and other chronic diseases,” explains Dr. Brown.
“We have a longstanding interest in understanding how the intestinal microbiota shapes host physiology,” says John F. Rawls, Ph.D., associate professor of molecular genetics and microbiology at the Duke University Medical Center.
The intestinal epithelium is situated on the frontline of host-microbiome interaction. The importance of the human and animal gut microbial communities for host health and disease has been reported for years, and one accepted mechanism is the regulation of host gene expression in the intestinal epithelium by bacteria. “But the upstream mechanisms by which microbes regulate host gene expression in the intestinal epithelium was not well understood,” points out Dr. Rawls.
Chromosomal regions important for controlling gene expression in a given cell type often exist as open chromatin that is depleted of nucleosomes and therefore accessible to binding by transcription factors. To examine the contribution of the microbiome to shaping the open chromatin landscape in the intestine, Dr. Rawls and colleagues examined DNA-Seq and RNA-Seq datasets from epithelial cells at multiple locations along the intestinal tract in mice raised in the presence or absence of microbes.
As expected, the commensal microbiome was able to modify the transcriptional landscape in intestinal epithelial cells in the ileum and colon. In contrast, open chromatin landscapes in the ileum and colon appeared to be nearly identical in mice grown in the absence or in the presence of bacteria. “This came as a surprise,” says Dr. Rawls. The data pointed toward the possibility that microbes could control gene expression via transcription factors that act on a relatively static and open chromatin landscape.
Using bioinformatics approaches, Dr. Rawls and colleagues searched the open chromatin near microbially regulated genes for the enrichment in known transcription factor binding motifs. This revealed that open chromatin was enriched for a specific set of transcription factors near genes upregulated by the microbiome, but for a different set of transcription factors near genes downregulated by the microbiome.
“It appears that microbes may regulate gene expression in the intestinal epithelium not by remodeling the open chromatin landscape, but instead by regulating the activity or abundance of distinct transcription factors,” he explains.
While these studies used intestinal epithelial cells, they did not provide information about regulatory mechanisms that may be operational in other organs. “It will be important to determine if different cell types use distinct regulatory logic to translate microbial cues into appropriate changes in gene expression, and how these strategies may be altered in disease states,” Dr. Rawls notes.
One of the most dynamic areas in life sciences, the microbiome is reshaping existing concepts and catalyzing the development of new ones. While recent years witnessed major advances, it is becoming increasingly clear that in terms of understanding our interaction with microbes, we are merely scratching the surface.
Technological developments and interdisciplinary endeavors, promising an unprecedented level of scrutiny, are ideally positioned to unveil the microbial choreography that is constantly unfolding in every ecosystem of our planet.
Unique Microbial Signature
“The microbiome encompasses not only bacteria, but also fungi and viruses, which all live together with us,” says Bart De Spiegeleer, Ph.D., professor of pharmaceutical sciences at Ghent University. Many of the functions of the human microbiome are still insufficiently understood but, increasingly, the microbiome has been implicated in chronic, inflammatory, and metabolic diseases. Some of these conditions evidently also harbor a genetic component. “The microbiome is constantly influenced by many more factors than we can imagine, and this occurs over long periods that may start even before birth,” adds Dr. De Spiegeleer.
While the importance of the microbiome-host interaction for health and disease has been appreciated for a long time, investigating the underlying mechanisms has been lagging behind for several reasons. “Understanding the mechanisms of action is clearly an interdisciplinary challenge, involving different research groups with different backgrounds, knowledge, and experience,” explains Dr. De Spiegeleer.
A major research effort in Dr. De Spiegeleer’s lab is focusing on microbiome-produced peptides and their communication with mammalian host cells. “Initial in vitro studies on colon and breast cancer cells have shown that some of these quorum-sensing peptides stimulate invasion and angiogenesis, which are hallmarks of metastasis,” he adds. These findings may open the possibility to survey and therapeutically modulate the microbiome to implement prophylactic interventions.
For microbiome research, the road from bench to bedside, while rewarding, promises to be challenging, particularly considering the bidirectional and intricate host-microbiome interactions. Recent studies that examined the human microbiome revealed that each individual harbors a unique microbial signature, similar to a fingerprint. During the continuing interaction with other ecosystems, this microbial signature is being actively reshaped.
“It is no longer unthinkable that the microbiome at large will be included as a bio-characteristic characterizing a person, much like the DNA profile that can be used in personalized diagnostics and therapeutics,” says Dr. De Spiegeleer.