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Uncovering the Trimethylamine-Producing Bacteria of the Human Gut Microbiota Silke Rath1, Benjamin Heidrich1,2, Dietmar H

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Uncovering the Trimethylamine-Producing Bacteria of the Human Gut Microbiota Silke Rath1, Benjamin Heidrich1,2, Dietmar H Rath et al. Microbiome (2017) 5:54 DOI 10.1186/s40168-017-0271-9 RESEARCH Open Access Uncovering the trimethylamine-producing bacteria of the human gut microbiota Silke Rath1, Benjamin Heidrich1,2, Dietmar H. Pieper1 and Marius Vital1* Abstract Background: Trimethylamine (TMA), produced by the gut microbiota from dietary quaternary amines (mainly choline and carnitine), is associated with atherosclerosis and severe cardiovascular disease. Currently, little information on the composition of TMA producers in the gut is available due to their low abundance and the requirement of specific functional-based detection methods as many taxa show disparate abilities to produce that compound. Results: In order to examine the TMA-forming potential of microbial communities, we established databases for the key genes of the main TMA-synthesis pathways, encoding choline TMA-lyase (cutC) and carnitine oxygenase (cntA), using a multi-level screening approach on 67,134 genomes revealing 1107 and 6738 candidates to exhibit cutC and cntA, respectively. Gene-targeted assays enumerating the TMA-producing community by quantitative PCR and characterizing its composition via Illumina sequencing were developed and applied on human fecal samples (n =50) where all samples contained potential TMA producers (cutC was detected in all individuals, whereas only 26% harbored cntA) constituting, however, only a minor part of the total community (below 1% in most samples). Obtained cutC amplicons were associated with various taxa, in particular with Clostridium XIVa strains and Eubacterium sp. strain AB3007, though a bulk of sequences displayed low nucleotide identities to references (average 86% ± 7%) indicating that key human TMA producers are yet to be isolated. Co-occurrence analysis revealed specific groups governing the community structure of cutC-exhibiting taxa across samples. CntA amplicons displayed high identities (~99%) to Gammaproteobacteria-derived references, primarily from Escherichia coli. Metagenomic analysis of samples provided by the Human Microbiome Project (n = 154) confirmed the abundance patterns as well as overall taxonomic compositions obtained with our assays, though at much lower resolution, whereas 16S ribosomal RNA gene sequence analysis could not adequately uncover the TMA-producing potential. Conclusions: In this study, we developed a diagnostic framework that enabled the quantification and comprehensive characterization of the TMA-producing potential in human fecal samples. The key players were identified, and together with predictions on their environmental niches using functional genomics on most closely related reference strains, we provide crucial information for the development of specific treatment strategies to restrain TMA producers and limit their proliferation. Keywords: Trimethylamine, Gut microbiota, Microbiome, Atherosclerosis, Cardiovascular disease, Functional diagnostics Background certain diseases. This is exemplified by trimethylamine The human body hosts a myriad of bacteria that play (TMA), a microbial metabolite that is produced by vari- important roles in host health and disease. It is becom- ous taxa of the gut microbiota from dietary quaternary ing evident that besides particular pathogens, whole amines, mainly choline and carnitine. TMA is absorbed microbial consortia are involved in the development of via the intestinal epithelium and further oxidized to trimethylamine N-oxide (TMAO), which was associated with atherosclerosis and severe cardiovascular disease in * Correspondence: [email protected] – 1Microbial Interactions and Processes Research Group, Helmholtz Centre for several independent studies [1 4]. A mechanistic model Infection Research, Inhoffenstraße 7, Braunschweig 38124, Germany has been formulated proposing that TMAO promotes Full list of author information is available at the end of the article atherogenesis through the formation of foam cells (lipid- © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rath et al. Microbiome (2017) 5:54 Page 2 of 14 laden macrophages) and a reduction of the reverse platform. The developed assays were used to characterize cholesterol transport from the atherosclerotic plaque [5]. the TMA-producing communities in the fecal samples of Two major TMA-synthesis pathways have been de- 50 individuals. scribed with a specific glycyl radical enzyme, the choline TMA-lyase (CutC) and its activator CutD [6], that takes Results choline as a substrate and a two-component Rieske-type Establishing databases for cutC and cntA oxygenase/reductase (CntA/B) [7], which acts on carni- Since any gene-based investigations depend on reference tine and its derivative gamma-butyrobetaine, as the key sequences, the entire diversity spectrum of the key genes enzymes. An additional enzyme complex termed YeaW/ from major pathways involved in the formation of TMA X, which shows close sequence similarity to CntA/B, was identified. Comprehensive databases for cutC and was recently proposed as the key component of a third cntA were established applying a multi-level screening major pathway [8]. Campo and colleagues have shown approach on 67,134 genomes provided by the Pathosys- that the so-called choline utilization cluster, which in- tems Resource Integration Center (PATRIC). CntA and cludes cutC/D among other genes involved in anaerobic yeaW show high similarity, and sequences from both choline metabolism, is widely, but discontinuously, genes were included in our reference set used to con- distributed across various taxa belonging to Firmicutes, struct a single hidden Markov model (HMM) for this Actinobacteria, and Proteobacteria [9]. The authors bio- gene group. All genes comprised in this database will be chemically verified TMA synthesis from choline for a referred to as cntA throughout the study (see also the multitude of the revealed taxa and demonstrated that “Discussion” section). both cutC and cutD are required for the TMA- Figure 1 illustrates the result of the multi-level genome- generating cleavage reaction. Another recent study [10] screening approach considering the following three demonstrated that colonization of the mouse gut with a criteria: (i) similarity to developed HMMs, (ii) conserva- specific consortium of CutC-encoding human isolates tion of specific amino acid sites previously suggested as results in TMA synthesis and subsequent TMAO accu- signatures for respective encoding genes [6, 7], and (iii) mulation in the serum of animals where even minute phylogenetic distance to top-scoring sequences. Addition- concentrations of TMA producers (0.15% of the total ally, synteny with the associated activator gene (cutD)as community) were sufficient for substantial TMA pro- well as the reductase (cntB) that were both shown to be duction from choline. Genes encoding CntA/B and essential for TMA production in the corresponding path- YeaX/Y, respectively, have been described in several taxa ways was investigated. Putative cutC genes were detected from the Gamma- and Betaproteobacteria as well as in a total of 1107 genomes (454 dereplicated protein from a few Firmicutes [7]. Functionality has been shown sequences) that belong to Proteobacteria (in particular for Acinetobacter baumannii and Escherichia coli where Gamma-andDeltaproteobacteria)andFirmicutes (mainly both genes cntA/yeaX and cntB/yeaW are required for Clostridia and some Bacilli), as well as a few Actinobac- the formation of TMA. teria (Fig. 1a, Additional file 1A, Additional file 2A). A Despite this multitude of knowledge on TMA- clear drop in the HMM similarity score was detected after producing bacteria and their importance for human sequence 453 that co-occurred with abrupt changes in health, little information on their abundance and taxo- other parameters investigated including phylogenetic nomic composition in vivo is available. So far, only one distance to the top-scoring sequence depicting a profound study estimates the overall potential of intestinal commu- change in gene sequences below that drop. Hence, a cutoff nities to produce TMA [11], and specific structural in- of 906.4, representing the median HMM score of the two sights into this functional community are largely lacking. sequences that frame that drop, was set, and all entries This can partly be explained by the low abundance of above that cutoff were considered as true cutC sequences. TMA producers and the requirement of specific methods With a few exceptions, they all show synteny with cutD for their quantification, as phylogenetic markers are ex- and display conserved amino acid residues previously pected to be poor predictors for that function, at least for suggested to be characteristic for the respective gene the broadly polyphyletic bacteria exhibiting cutC/D [9]. product [6]. Our results are in line with phylogeny where Thus, the aim of this study was to build a comprehensive cutC sequences form a cluster apart from sequences below framework in order to quantify the TMA-production
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