Microbiomes, Footprints of Ancient Lineages of Life

The term “meta-organism” has been recently defined as “the host organism and those components of its associated microbiome to which function has been either ascribed or for which there are reasonable grounds to suspect it.”¹. Today, multiomic approaches represent a method to unravel the complex nature of host-microbiome interactions across multiple functional layers, especially in disease². Amongst the various “omic” approaches, Metabolomics is unparalleled in its ability to capture and summarize mechanistic relationships underlying host-microbe interactions. In this blog we will address the innate biochemical virtuosity of prokaryotes underlying the concept of meta-organism and learn how Metabolomics can provide an invaluable research tool to enhance multiomic microbiome-related research workflows.

Introduction 

Microbes like Archaea and Bacteria constitute the most ancient lineages of life on our planet and microbial fossil studies suggest microbes were already present at least 3.95 billion years ago³  and that biofilms, which are complex microbial communities, were already present 3.25 billion years ago⁴. To put this into perspective, hominids have co-evolved with microbes for only about 0.2% of the time that microbes have been present on the planet.  

“Replication and metabolism define life as we currently understand it” ⁵. During their first 3.95 billion years of evolution, microbes evolved with all possible combinations of carbon utilization and energy generation with representatives amongst the photoautotrophs, photoheterotrophs, chemoautotrophs, and chemoheterotrophs and they appear to be the champions of metabolic virtuosity. Due to this metabolic virtuosity, microbes have played a key role in shaping the evolution of life on Earth leading to the beautiful complexity of life forms we know today. For example, strong evidence suggests that Eukaryotic photosynthesis originated from endosymbiosis of cyanobacterial-like organisms, which evolved into plastids such as the chloroplasts of the plants we see today⁶. Mitochondria are another example of eukaryotic organelles that are derived from microbial endosymbiosis⁷. Through coevolution these endosymbionts have become entirely interdependent with their hosts¹ evolving into the complex structure of the eukaryotic cells we know today.  

Towards the Metaorganism 

We have now entered a new era of awareness where we recognize hosts as meta-organisms acknowledging the metabolic intertwinement underlying the very complex regulatory relationship between ourselves and our microbiomes. In fact, in a recent definition by Berg et al., the term “meta-organism” was defined as “the host organism and those components of its associated microbiome to which function has been either ascribed or for which there are reasonable grounds to suspect it.”¹

Although small metabolites from the maternal microbiome have been shown to modulate fetal development⁸, humans are born virtually sterile and our first true interaction with the microbial world takes place during birth⁹. Microbes like members of our microbiota must make contact with the host, compete with other microbes, evade the host immune system, and change their physiology to enable long-term host association. These interactions have shaped host-microbe co-evolution leading to metaorganisms. In fact, the overwhelming diversity and metabolic specialization of microbes, combined with their capacity to colonize virtually all abiotic and biotic environments appears to be naturally predisposed for intra-specific interaction.

Experimental Methodologies to Study the Physiological Role of Human Microbiomes 

It is now understood that all eukaryotes are meta-organisms and must be considered together with their microbiota as an inseparable functional unit¹⁰. In fact, modern microbiome research is intrinsically multidisciplinary in nature stemming from classic microbial ecology and spanning from ecology, agronomy, nutrition, biotechnology, veterinary science to human medicine.  

The undisputable role of human microbiomes and microbiotas as key modulators of physiology and health have made them key therapeutic targets. For example, the importance of a healthy gut microbiome is highlighted by the recently described gut—brain¹¹, gut-liver¹², gut-lung¹³, and gut-skin axes¹⁴, making the gut microbiome a central focus for human health. However, when considering the inter-individual variability and dynamic nature of microbiomes we must ask ourselves whether precision medicine approaches are not just an opportunity, but rather a necessity.  

Today, multiomic approaches unravel the complex nature of host-microbiome interactions in disease across multiple functional layers². Nevertheless, integrating and analyzing such complex datasets poses significant challenges. There are a variety of techniques and experimental methods that are routinely applied to study microbiomes. The majority of modern microbiome studies employ multiomic and phenomic approaches to address the multiple underlying levels of biology involved in host-microbiome interactions. Methodologies often combine classic culturomics, taxonomical composition analysis relying on metabarcoding approaches such as 16S rRNA sequencing, the study of metabolic potential relying on approaches such as metagenomic shotgun sequencing, and the study of microbial activity and function using approaches such as metatranscriptomics, metaproteomics, and metabolomics. In particular, metabolomics is uniquely positioned to capture and summarize all of the effects deriving from genomics, transcriptomics, proteomics, and epigenomics as well as the environment and the effects of diet and the microbiomes thereby offering the ultimate representation of a meta-organism’s phenotype. 

Some Applications of Metabolomics in Microbiome Research

Metabolomics, the study of small molecules or metabolites present in biological systems, is a fundamental technique to advance microbiome research. Crucially, Metabolomics can be used to study both the function of microbiomes as well as the effects on host homeostasis and more generally health. Metabolomics can be used to detect and identify changes in the metabolic output of complex microbial communities as well as reveal both systemic and organ-specific changes in host metabolite profiles deriving from host-microbiota interactions. As such, metabolomics has the potential to help address a plethora of biological questions in microbiome research.

Characterizing dysbiosis

One of the most logical applications of metabolomics in human health is the characterization of the functional output of dysbiotic microbiotas associated with different phenotypes and conditions. For example, a recent article by Bose et al. used a Gulf War Chronic Multisymptom Illness (GWI) model in mice combined with whole-genome sequencing to characterize the functional dysbiosis associated with this pathology and the exacerbations of symptoms caused by a Western diet. The researchers then used heterogenous co-occurrence network analysis to study the bacteriome–metabolomic association¹⁵. Network analysis revealed important associations of gut bacterial species with metabolites and biochemical pathways with the potential to be used as biomarkers or therapeutic targets to improve symptom persistence in Gulf War Veterans. 

Characterizing the effects of microbiome manipulation

The possibility to understand the functional output of a microbiome in a given physicochemical niche opens the door to its beneficial manipulation. We are all familiar with some interventions and common strategies used to manipulate the function of microbiomes; antibiotics, pre-, pro- and postbiotics, live biotherapeutics (LBPs), fecal and intestinal microbial transplants etc. A recent study using metabolomics investigated an alternative “surgical” strategy to modulate and study microbiome function using phages¹⁶. The results showed that phage predation in the gut microbiota has a strong impact on the host, as manifested by modulation of the gut metabolome. Given the substantial role of microbial metabolites in mediating interactions between bacteria and the host, the connection between phages and microbial metabolites presents an intriguing therapeutic opportunity. 

Characterizing the role of microbiomes in response to treatment

A large number of studies have shown that the gut microbiome can impact the response to disease treatment¹⁷. For example, it is well-established that the gut microbiome can modify medication responses via drug metabolism, as in the case of L-dopa for Parkinson’s disease¹⁸. Furthermore, drugs can lead to alterations in the gut microbiome that can influence treatment efficacy¹⁹. Importantly, a number of studies suggest the composition and function of the fecal microbiota can influence the response to checkpoint inhibitor-based cancer immunotherapy^20,21,22. For example, in a recent study by Hsu et al., researchers identified some bacterial species are strong metabolic markers of response to treatment in melanoma patients treated with a combination of checkpoint inhibitors¹⁶.

Studying the skin microbiome

The skin is a complex and active organ which modulates the cutaneous barrier and opposes external challenges. Improvements in sequencing methodologies have accelerated the characterization of the skin microbiome in both healthy individuals and ones suffering from a range of skin pathologies. Over 200 genera from 18 different phyla have been characterized from the skin. Metabolite-driven skin-microbe interactions play important roles in skin physiology including barrier function, aging, skin diseases, wound pathology, and infection. For example, skin microbes can modulate immune function via pattern recognition receptors such as NOD2 and Toll-like receptors (TLRs) which trigger an immune response and wound repair.  

Our skin surface is colonized at birth and rapidly evolves during the first months of life shaping and training our immune system. For example, a recent study from Roux et al. employed a multiomic approach for microbiome and metabolome analysis of skin surface samples collected from the volar forearm of healthy infants aged 3-6-months to address the role of the skin microbiome in skin physiology in healthy infants. Researchers found the skin surface metabolome influences bacterial community composition and diversity. The skin microbiome of early infants was found to aggregate around three distinct communities characterized by their metabolite microenvironment²³.

Microbiome and nutrition

The gut microbiota serves as a crucial link between diet and host physiology. The composition and function of the gut microbiota is significantly influenced by diet, crucially determining the nutrients available to the host from microbial metabolism. Changes in the gut microbiota induced by diet often perdure for long periods with enduring effects on the host. Moreover, several diet-induced host pathologies are mediated by alterations in the gut microbiota. Overall, both the diet and the gut microbiome combined play a significant role in influencing behavior, impacting mental health, cognitive function, and overall mood. For example, a study by Olson et. al. demonstrated the link between the Ketogenic diet and the gut microbiota in modulating host metabolism and seizure susceptibility in mice²⁴. The researchers found the Ketogenic diet alters gut microbiota composition promoting select microbial interactions and reducing bacterial gamma-glutamylation activity. The study also showed a significant decrease in peripheral GG-amino acids and elevated bulk hippocampal GABA/glutamate ratios which were related to the protective anti-seizure effect of this diet.

Drug development- focus on safety and toxicity 

Metabolomics analysis can add value throughout the entire drug development continuum aiding in disease characterization,consequent biomarker and target identification, assessment of efficacy, elucidating mechanism of action(MOA), toxicology and safety, the development of personalized medicine approaches, the optimization of clinical trials, and drug repurposing. Most importantly, metabolomics can provide an unparalleled understanding of the crucial role our microbiomes play during therapy. For example, in a cornerstone publication looking at drug-induced off-target effects, Zgoda-Pols et al. used metabolomics analysis to identify two new sensitive microbe-derived markers of kidney toxicity, p-cresol sulfate and 3-indoxyl sulphate²⁵. The systemic changes in the levels of these two metabolites measured using metabolomics have proven crucial in evaluating the impact of new therapies on kidney function during pharmaceutical development. 3-indoxyl-sulphate originates from the metabolism of l-tryptophan to indole by gut bacteria which is absorbed into the blood and converted in the liver to indoxyl sulfate in blood and eventually accumulates in urine. P-Cresol sulfate is another tyrosine metabolite produced by the gut microbiome. In chronic kidney disease, increased production of P-Cresol sulfate in the gut, along with decreased clearance from the kidneys, results in toxicity that can affect multiple organs in the body.

Immune modulation

The crosstalk between a host and small microbial metabolites is vital for maintaining immune balance and ensuring overall health. Disruptions in this crosstalk can lead to a variety of diseases, highlighting the importance of a healthy and balanced microbiome. Metabolomics offers the unique possibility to monitor the levels of a plethora of these metabolites both in microbiomes and their hosts.

Examples of Microbially-Derived Immune Modulators

Short-Chain Fatty Acids (SCFAs) are microbially-derived immune modulators. SCFAs like acetate, propionate, and butyrate are produced through the fermentation of dietary fibers by gut bacteria. SCFAs have been shown to have immunoregulatory functions (34) including modulating the production of regulatory T cells, regulating gut barrier function, and reducing the production of pro-inflammatory cytokines through G-protein-coupled receptors (GPCRs)²⁶.  

Microbially-derived indole derivatives represent another class of important immune modulators. Compounds like indole-3-aldehyde produced from the metabolism of tryptophan by gut bacteria can activate the aryl hydrocarbon receptor (AhR) on immune cells, promoting the differentiation of Tregs and maintaining mucosal homeostasis²⁷. Another microbially-derived indole derivative and strong antioxidant, indole-3-propionic acid, can modulate immune responses by influencing the function of intestinal immune cells²⁸.  

Secondary bile acids which are metabolized by gut bacteria from primary bile acids can modulate immune responses by activating the farnesoid X receptor (FXR) and the Takeda²⁹ G protein-coupled receptor 5 (TGR5)), which influence the production of anti-inflammatory cytokines and regulate intestinal inflammation. 

Microbially-derived vitamins and cofactors like vitamin K2 can influence the immune system by modulating the activity of various immune cells³⁰. For example, vitamin B12 is essential for the proper functioning of immune cells, including lymphocytes and macrophages³¹. 

Quorum Sensing Molecules such as the N-Acyl Homoserine Lactones (AHLs), which are produced by Gram-negative bacteria can also modulate the immune response³² by affecting the behavior of immune cells such as macrophages and neutrophils³³. Autoinducer metabolites like the Gram negative AHLs are also used by the bacteria themselves to regulate sociomicrobiology and decide whether to switch on specific metabolic pathways³⁴. 

Conclusions 

Microbial metabolites are integral to the crosstalk between the microbiotas of our microbiomes and the host’s homeostasis. Their production and activity are influenced by diet, the environment, the composition of the gut microbiota, and overall health status. As such, metabolomics is critically important in microbiome studies as it provides a comprehensive analysis of the small molecules and metabolites produced by microbiotas and the host metabolites and pathways modulated by microbial small metabolites. Today, multiomic workflows integrating data from various “omics” fields can provide a comprehensive understanding of biological systems and help address the host-microbiome interactions across multiple functional layers, especially in disease, opening the door to novel therapeutic avenues. 

Defining Terms 

Microbiome 

“The microbiome is defined as a characteristic microbial community occupying a reasonable well-defined habitat which has distinct physio-chemical properties. The microbiome not only refers to the microorganisms involved but also encompass their theatre of activity, which results in the formation of specific ecological niches. The microbiome, which forms a dynamic and interactive micro-ecosystem prone to change in time and scale, is integrated in macro-ecosystems including eukaryotic hosts, and here crucial for their functioning and health.” ¹ 

Microbiota 

“The microbiota consists of the assembly of microorganisms belonging to different kingdoms (Prokaryotes [Bacteria, Archaea], Eukaryotes [e.g., Protozoa, Fungi, and Algae]), while “their theatre of activity” includes microbial structures, metabolites, mobile genetic elements (e.g., transposons, phages, and viruses), and relic DNA embedded in the environmental conditions of the habitat.”¹

Meta-organisms  

“The term metaorganism is used to refer to the host organism and those components of its associated microbiome to which function has been either ascribed or for which there are reasonable grounds to suspect it.”

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