Dynamics of microbiome from an aquatic sentinel species upon toxicants – Dyn-Microbiome

In order to study the role of the microbiota in an ecotoxicological context, this project proposes the development of an innovative metaproteomic approach, as yet little used in this field. The major advantage of this approach is its ability to provide information in a single, relatively short analysis cycle concerning (i) the taxonomic composition of the microbiota, (ii) its metabolic functions beneficial to the host, and (iii) biomarkers of the health status of the host organism. Analyses are performed on gammarid intestines from laboratory or in situ experiments. A protocol for extraction and proteolysis of proteins on gel has been set up. The digestates obtained represent a mixture of peptides which are then analyzed by high-resolution tandem mass spectrometry coupled to liquid chromatography (LC-MS/MS). The LC-MS/MS data are then processed by a set of bioinformatics tools, known as the metaproteomics pipeline, developed within the Li2D team. This pipeline relies on querying generalist databases and a database derived from host RNA sequencing. Queries against these databases are performed in several cascading steps to increase the rate of assignment of MS/MS spectra to peptides, whether taxon-specific or not, and thus determine the composition of the microbiota. This pipeline also enables functional annotation of microbiota or host proteins, associating them with the molecular and cellular processes or metabolic pathways in which they are involved. In this way, this information helps to understand the function of the microbiota, as well as the physiological state of the host.

The first phase of the project involved the development of a comparative metaproteomics pipeline. An initial analysis campaign investigated the effects of the fungicide tebuconazole, detected in aquatic environments, on the taxonomic composition of the microbiota and its functions, as well as on the health status of the host organism. Male gammarids were subjected to three exposures under controlled laboratory conditions: 0, 50 and 200 µg/L. After 15 days of exposure, individuals (n=15 per condition) were fasted for 24 hours, during which time feces were collected. The organisms were then sacrificed and the (empty) intestines dissected for analysis. The metaproteomic data generated initially concerned the intestinal samples. The pipeline confirmed the taxonomic identification of 16 genera (4 eukaryotes, 10 bacteria and 1 archaea) grouped under 9 phyla: Arthropoda, Chordata and Streptophyta for eukaryotes, Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria for bacteria, and Candidatus Thorarchaeota for archaea. As expected, the majority of specific peptides concern host organisms belonging to the phylum Arthropoda. On the other hand, only a small number of peptides were detected that were specific to the microbiota. In terms of the taxonomic structure of the microbiota, differential statistical analyses revealed no significant difference between exposed individuals and the control condition. Functionally, tebuconazole affected certain microbiota processes relating to cell motility (from 50 µg/L), transport and catabolism, terpenoid and polyketide metabolism, and drug resistance (200 µg/L).

In view of these initial results, the low quantity of microbiota-specific peptides and the absence of any marked effect of tebuconazole on the taxonomic structure of the microbiota could be due to the type of sample analyzed. As the intestines are emptied beforehand to collect faeces, they do not contain sufficient information about the microbiota. It would therefore be interesting to carry out these analyses on samples of full intestines or faeces, in order to increase the microbiota signal and potentially observe the effects of tebuconazole on the structure of the microbiota. If the choice of feces proves more relevant, this would enable us to move towards non-invasive sampling methods, avoiding animal sacrifice.

The first four publications to emerge from this work concern the use of metaproteomics in environmental diagnostics and the improvement of the metaproteomics methodology:
DOI: 10.1002/pmic.202200055
DOI: 10.1128/msystems.00589-22
DOI: 10.1111/1462-2920.16238
DOI: 10.1186/s40168-024-01766-4

Host microbiota is a key factor for animal health and its sensitivity to contaminants. The “Dyn-Microbiome” project will decipher the nature and role of host-associated microbiota in the modulation of contaminant toxicity for an aquatic sentinel animal, the Gammarus fossarum fresh-water amphipod, largely used for biomonitoring in France. We developed an innovative metaproteomics approach that allows establishing the microbiota taxonomical composition, quantifying the respective biomasses, deciphering its main metabolic pathways and host molecular biomarkers. This technology complemented with metagenomics will provide evidence on i) whether shifts on the microbiota parameters are induced by model toxicants (metals, polycyclic aromatic hydrocarbons, antifungals and antibiotics), ii) evaluate detrimental long-term consequences of dysbiosis for the host, and iii) propose novel molecular biomarkers of exposition.