Background

Complex assemblages of microbes live in and on humans, colonizing every surface exposed to the external environment. These communities have received several denominations over the decades, including normal flora, microbiota, and, more recently, microbiome (Sekirov et al., 2010; Kashyap et al., 2017). In humans, these vast microbial communities colonize our skin, respiratory tract, genitals, gastrointestinal tract, and many other sites. By far, the most heavily colonized site is the gastrointestinal tract, where trillions of microbes, encompassing several hundred species, coexist peacefully with their hosts. Some of these species knowingly live in symbiotic associations with the human organism, where both parts benefit from the interactions. For others, the relationship may be purely commensal, where the parts coexist without causing any harm to each other, but without providing or obtaining any benefit (Sekirov et al., 2010; Kundu et al., 2017).

The human gastrointestinal microbiota presents significant diversity in their composition, and stands out as a complex environment where interactions between different microbes as well as between microbes and host cells constantly occur. Bacteria are known to produce a plethora of bioactive small molecules, such as antibiotics, bacteriocins, pigments, secondary metabolites, quorum sensing signals, and many others (Antunes and Ferreira, 2009; Antunes et al., 2010; Antunes et al., 2011a). In such a complex environment as the intestinal microbiome, it is almost imperative to consider the production and accumulation of such molecules. These small molecules may represent by-products of metabolic activities or signals with specific roles, and can be produced both by the host itself as well as the microbes living in that environment; in many cases these small molecules are the tools used by these organisms to interact. Using high-throughput mass spectrometry-based metabolomics, we have previously shown that thousands of small molecules can be found in the lumen of the mammalian intestinal tract, and that the gut microbiome is involved in the production of many of them (Antunes et al., 2011b). In order to ascertain the signaling potential of small molecules from the gut metabolome, we have also extracted these molecules and tested their ability to modulate growth and gene expression of an enteric pathogen. As shown by our previous results, Salmonella enterica serovar Typhimurium responds to these molecules, and modulates the expression of over one hundred genes in response to bioactive small molecules from human feces (Antunes et al., 2014). Interestingly, many of the genes regulated by the fecal extract are required for the pathogenesis of Salmonella, such as those involved in the invasion of non-phagocytic host cells. More recently, we were able to purify and identify small aromatic compounds as the culprits for the regulation of Salmonella genes by the human gut metabolome (Peixoto et al., 2017). Here, we describe in detail the methods used by our group to obtain small molecules from the human gut metabolome and test them against Salmonella for various biological activities. A workflow of the procedures described herein can be found in Figure 1.


Figure 1. Workflow of the procedures described in this protocol