Companies spend huge resources going to the far reaches of the Earth to search for the next blockbuster. But we need look no further than our own intestines, which are populated with thousands of bacterial species that are constantly producing and releasing small, bioactive molecules.
Small molecules—the bread and butter of pharmaceutical companies—are compounds of low molecular weight (under 3,000 daltons) and diverse chemical composition. Examples of such molecules are the steroid and small-peptide hormones of higher organisms, with a molecular weight around 300 daltons, which have many important biological functions. The term hormone (from the Greek: excite, arouse) was coined in 1905 by British physiologist[1. E.H. Starling, “The chemical correlation of the functions of the body. Lecture I,” The Lancet, 2:339-41, 1905.] Ernest Starling to describe the chemical messengers produced in an organ or gland of the body that travel to distant organs to exert their physiological effects. In humans, the critical functions of small-molecule hormones include modulation of the immune system, the development of sexual characteristics, the response to stress, metabolism, and mineral balance, among others.
Although much of our knowledge about bioactive small molecules comes from the study of mammalian and plant hormones, it is now known that bacteria can also produce, sense, and respond to a variety of small-molecule signals that enable them to act coordinately. Through the research of Alexander Tomasz on the acquisition and incorporation of foreign DNA by Streptococcus pneumoniae and Woodland Hastings’ studies of Vibrio fischeri luminescence, science had its first hints that diffusible molecules played an important role in the lifestyle of microbes.[2. K.H. Nealson et al., “Cellular control of the synthesis and activity of the bacterial luminescent system,” J Bacteriol, 104:313-22, 1970.],[3. A. Tomasz, “Control of the competent state in Pneumococcus by a hormone-like cell product: an example for a new type of regulatory mechanism in bacteria,” Nature, 208:155-59, 1965.] Decades of subsequent research revealed that these hormone-like compounds are produced by these bacteria at basal levels and accumulate as their numbers increase. Both Tomasz’s and Hastings’ groups noticed that after reaching a threshold concentration, these molecules could regulate the incorporation of exogenous DNA and the production of light, activities that are only useful when undertaken by a population. (Bacteria can take up DNA individually, but when it is taken up by a group, the community can increase its chances of survival due to its greater genetic diversity.) Although the consequences of such discoveries were not fully appreciated at the time—it was unclear whether this behavior would be found in all bacteria—the observations formed the foundation for studies of bacterial communication. It is now widely accepted that many bacterial species use small chemical compounds to communicate with others of the same species, other bacterial species, and their hosts. Because this phenomenon is dependent upon a threshold cell density, bacterial communication has been termed “quorum sensing.”[4. W.C. Fuqua et al., “Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators,” J Bacteriol, 176:269-75, 1994.]
The chemical repertoire used by bacteria to communicate is diverse, and new bioactive molecules continue to be discovered. Nonetheless, our knowledge of these molecules is limited. Studies of bacterial signaling have focused mostly on laboratory-grown, pure cultures of microorganisms. In the natural environment and in their hosts, microbes live in association with a multitude of other species and are constantly presented with opportunities for competition and cooperation.
One of the best-known natural examples is the nitrogen-fixation–driven symbiosis between Rhizobia and their legume hosts. Many chemical signals act to promote the establishment of this mutually beneficial relationship. For example, the growing root exudes flavonoids, which induce the surrounding Rhizobia to migrate to the root surface. There, quorum sensing occurs through the production of acyl-homoserine lactones, culminating in the production of microbial nodulation factors that act on the root and finally result in an endosymbiotic relationship. Although there is a wealth of information about chemical signaling in soil, many other complex microbial populations exist in nature, and it is certain that microbial signaling plays important roles in these communities.
At elevated concentrations, microbial signals can act as antimicrobials. In general, however, microbes produce antibiotics in the environment at concentrations that do not affect their growth.[5. E.B. Goh et al., “Transcriptional modulation of bacterial gene expression by subinhibitory concentrations of antibiotics,” PNAS, 99:17025-30, 2002. Free F1000 Evaluation ] It is probable, then, that the main function of these chemicals is to modulate bacterial gene expression rather than to poison.[6. J. Davies, “Are antibiotics naturally antibiotics?” J Ind Microbiol Biotechnol, 33:496-99, 2006.] Nevertheless, the study of chemicals derived from microbes and other organisms has provided us with more than just information about the roles that small molecules play in biological systems. Whenever possible, humans have taken advantage of these natural products for therapeutic purposes, the classic example being the fortuitous discovery of the antibiotic penicillin. In addition to antibiotics, natural products have been used as anticancer and anti-inflammatory compounds.[7. K.H. Lee, “Discovery and development of natural product-derived chemotherapeutic agents based on a medicinal chemistry approach,” J Nat Prod, 73:500-16, 2010.],[8. O. Werz, “Inhibition of 5-lipoxygenase product synthesis by natural compounds of plant origin,” Planta Med, 73:1331-57, 2007.] Many have been isolated from microorganisms, but others have been derived from plants and marine life. The discovery that the human body is made up of extremely complex ecosystems suggests that it, too, could be used as a rich source of new bioactive molecules.
The wealth withinAt birth, humans are colonized by complex communities of microbes. These communities, which are established within the first year of life, have been termed microbiota, microflora or microbiome. The human microbiome is extremely rich, containing upwards of 1014 cells,[9. I. Sekirov et al., “Gut microbiota in health and disease,” Physiol Rev, 90:859-904, 2010.] and is essential to our health. Microbes colonize our skin, our gastrointestinal (GI), genitourinary, and respiratory tracts. Researchers have estimated that the collection of microbial genes in our bodies exceeds our own genes by a factor of 100, which means that the human genome is predominantly prokaryotic.
It is probable, then, that the main function of these chemicals is to modulate bacterial gene expression rather than to poisonAlthough virtually every body surface that is exposed to the environment harbors microbes, the gastrointestinal tract is by far the most heavily colonized site. More than 1,100 bacterial species have been genetically identified in the human gut; each individual carries an estimated 160 distinct species, only about one-tenth of which appear to be shared by everyone.9,[10. A. Vacharaksa, B.B.Finlay, “Gut microbiota: metagenomics to study complex ecology,” Curr Biol, 20:R569-71, 2010.] This offers a tremendous opportunity for the evolution of multiple microbe-microbe and host-microbe interactions, many of which are conveyed through the activity of small signaling molecules.
It has been known for a while that commensal organisms can use diffusible signals to interact with their hosts. Bacteroides thetaiotaomicron, a prominent member of the human gastrointestinal microbiota, produces signals that can control host gene expression. By doing so, the bacterium controls the availability of nutrients in its surroundings to favor its growth. More recently, this intestinal commensal has also been shown to communicate with pathogens by producing a yet to be identified signal that can control virulence-factor production by enterohemorrhagic Escherichia coli, a pathogenic strain of E. coli that can cause serious illness and death in humans. Colonization by B. thetaiotaomicron, and possibly by other bacterial species, may be an important tool used by mammals to control infection by virulent bacteria.
These interactions are not always initiated by the microbiota. Host-produced small molecules can also have profound effects on both commensals and pathogens. The mammalian stress hormones epinephrine and norepinephrine have been shown to affect commensal microbial populations in the gastrointestinal tract. Although the mechanism involved is not known, these hormones can favor the growth of specific species of the human microbiome. In addition to their roles in normal physiology, these mammalian stress hormones can also influence the production of virulence factors by invading pathogens. For instance, upon sensing intestinal epinephrine and norepinephrine, enterohemorrhagic E. coli activates the production of its type III secretion system, a major virulence factor. Also, Campylobacter jejuni, which commonly causes food poisoning, can respond to norepinephrine by improving its ability to enter host cells.
An even more daring possibility is that some of these molecules could be used to manipulate the physiology of remote organs and systems.Although data on the importance of the human microbiome to health has been accumulating over the past few years, new evidence suggests that its contributions are not always local. The microbiota exerts an impact on host tissues and organs far away from the gut. Changes in intestinal commensal populations have been correlated with many diseases of remote organs, such as diabetes, asthma, obesity, cancer, autism, and even depression.9 The fact that these organs are not in direct contact with gut commensals suggests that chemical signals may be involved. For instance, it has been known for a while that antibiotic usage is correlated with an increased risk of allergic diseases, and it has been suggested that this is due to effects on the intestinal microbiota. Although microbial interactions with the human immune system are crucial in producing these pathologies, it is also possible that the intestinal microbiota affect systemic metabolites by a more general mechanism. Indeed, William Wikoff of the Scripps Research Institute and colleagues have recently shown that intestinal microbes can have a significant impact on mammalian blood metabolites, particularly those involved in amino acid metabolism, suggesting that the influence of gut microbes on the human body may be largely dependent on the activities of small molecules.10 Although some of these effects may be brought about directly by microbial signals, other mechanisms that propagate messages initiated by gut bacteria may also be at play.
The mammalian gut plays an important role in the excretion of waste, and many host-produced small-molecule metabolites that circulate in the gut are a result of this waste-production process. The GI tract is also loaded with other small molecules, such as hormones and bile acids, that not only have significant impact on the GI tract itself but also on other organs through reabsorption. In other words, the molecules with potential bioactivity may not necessarily come only from the microbiota, but also from our own waste products.
Some of these small molecules originating from the host and from the microbes are also likely to have important functions in keeping the relationship between hosts and their commensals in balance. Since these molecules have important functions in human health and disease, they could be mined as therapeutics aimed at maintaining or reestablishing homeostasis and preventing or curing diseases. Not least, the gut may be a source of new antibiotics. It is known that the microbiota exerts important effects on the maturation of the mammalian immune system, and the small molecules in the intestine could be used to modulate these relationships in controlled ways. An even more daring possibility is that some of these molecules could be used to manipulate the physiology of remote organs and systems. It is possible that the molecules produced by some of the microbes associated with protection against certain diseases could be used to remediate or prevent those illnesses. While the potential for applications is enticing, careful studies must be designed and performed to demonstrate that changes in bacterial populations and the molecules they produce during disease are in fact causing the pathological process rather than changing in response to it.
Stalling between discovery and drug developmentOne of the limitations of this potential source of bioactive small molecules is that, like bacteria isolated from deep-sea vents and other exotic locales, the majority of the microbial species present in the mammalian gut still cannot be cultured in the laboratory. Working out a bacterium’s ideal growth requirements is no simple matter. This has forced the use of culture-independent methods to study microbial communities in and on humans.
Most studies rely on metagenomics, or the unbiased sequencing of DNA fragments isolated from mixed microbial populations. This is useful from a phylogenetic standpoint because it allows us to determine the exact microbial composition of a given sample, but it tells us very little about functions of the compounds produced or the interactions between them. Based on analysis of the gut metagenome—the combination of all genes present in the GI tract—we can predict that many biological reactions are yet to be discovered. More recently, the intestinal environment has been studied using other culture-independent methods such as metatranscriptomics and metaproteomics, which focus on the unbiased analysis of messenger RNA or proteins, respectively. Altogether, these explorations of the microbial diversity in the GI tract suggest that significant phylogenetic diversity remains to be explored. These studies have provided much information about the composition of microbial communities in the mammalian gut, but it is only recently that we have begun to decipher the molecular functions of these assemblages.
With the overwhelming amount of genetic material present in the human gut comes the potential for an immensurable source of bioactive small molecules. As a result of recent advances in techniques of chemical separation and structural elucidation, particularly methods for high-throughput analysis of complex samples, we now have tools to probe this chemical lexicon. One such technique is Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS), which has recently been used in pioneering high-throughput studies of the mammalian metabolome.[11. W.R. Wikoff et al., “Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites,” PNAS, 106:3698-703, 2009. F1000 ID 1157492, Free F1000 Evaluation ]FTICR-MS is an extremely sensitive and accurate method for the rapid detection and relative quantification of thousands of small molecules in complex samples. We have used FTICR-MS to study the intestinal metabolome and find that thousands of small molecules are present in the mammalian gut. Interestingly, the levels of the majority of these molecules are affected by antibiotic treatment, suggesting that the intestinal microbiota modulates the chemical composition of the intestinal lumen (Antunes and Finlay, submitted for publication). Additional metabolic studies of samples from healthy and disturbed ecosystems will allow predictions of causative associations between chemical variations and specific disease states.
But even these “-omic” studies, informative as they may be, will need to be verified and tested in basic laboratory experiments to tease apart the specific host-microbe interactions. There is a huge distance between beholding the vast landscape of molecules with potential human applications and finding potential disease interventions. The results from survey studies will need to be used to develop hypotheses that can then be tested using single molecules (natural or synthetic) to treat conditions in animal models, giving us insight not only into the healthy state, but also into how it can be maintained or restored during disease.
That DNA, RNA, and proteins are central to life is irrefutable. However, the functioning of living organisms not only depends on these molecules but, in most cases, extends to incorporate the end products of multiple metabolic pathways. In other words, in most cases it is the small molecules that are the crux of biological function. While other sources of bioactive molecules should not be ignored, harnessing the bacterial chemicals in our own gut may yield molecules that have already been shaped for very specific interactions through years of coevolution between humans and their microbial partners. Without identifying and studying these molecules, we will not fully understand the functions of metabolic pathways and the interconnections among them. Nor will we be able to fully comprehend the complexities of any biological system. The study of small molecules should be undertaken not only with an intellectual view toward understanding the molecular intricacies of life in more detail, but also with a practical view of benefiting from what these molecules may have to offer.