Unravelling necrotrophy by human gut microbes through powerful single cell labeling and detection methods

Abstract

Microbial necrotrophy, the metabolization of necromass (dead microbial biomass) by living microbial cells, is understudied in the field of human gut microbiome research. Yet, necromass is abundantly available in the human gut. In a recent study, we observed high necromass loads (~60% of the fecal biomass consists of damaged/dead cells) in a cohort of 157 subjects. This dead biomass fraction does not directly contribute to the activity of the microbial community, but it could present an important overlooked nutrient source for the active microbial population. This hypothesis was explored in in vitro batch incubations, as well as, a more complex continuous in vitro gut simulator using various single-cell labelling and detection methods. In a first experiment, we demonstrated necrotrophic growth in in vitro batch incubations by means of Raman microspectroscopy. 100% 13C labeled necromass was supplied to a fresh fecal inoculum in the presence of deuterium (D2O) as a general activity marker, but in the absence of other nutrient sources. After 48 hours, we were able to identify double labeled (13C and D2O) necrotrophic cells, i.e. active cells that had taken up necromass. In a next stage, we used NanoSIMS, an extremely sensitive technique, to probe necrotrophy in the SHIME (Simulator of the Human Intestinal Microbial Ecosystem) in vitro model. 10% 13C and 15N labeled necromass was supplied to the SHIME in the presence of deuterium (D2O) as a general activity marker, in different nutrient conditions. We confirmed label transfer of 13C and 15N from necromass into living cells. Measurements of the 13C enrichment in the produced short chain fatty acid (SCFA) fermentation products through LC-IRMS, moreover, showed that the SCFA 13C over 12C ratio is proportional to the nutrient load. We furthermore, observed a higher 13C fraction in propionate and butyrate compared to acetate. This finding may indicate that necrotrophy is specific to a subset of gut microbes. The microbial community composition was followed up through quantitative microbiome profiling, combining Illumina 16S rRNA gene amplicon sequencing with flow cytometry, enabling the absolute quantification of gut microbial cells and the identification of their physiological state. This analysis confirmed that nutrient load affected the microbial community but this technique does not allow for the characterization of the necrotrophic population. We thereto established a high-throughput FACS-viability staining workflow that enables population level quantification and characterisation of the necrotrophic subpopulation after sorting. The method makes use of bio-orthogonal click chemistry to label proteins and sugars in necromass and study their transfer into living necrotropic cells. Proof of principle for protein transfer was obtained in a gut microbial community derived from one subject. In order to capture the inter-individual variability and diversity within the human gut microbiome, further research will be performed to identify the necrotrophic population from different fecal donors