Phylopeptidomics: A Step Closer to Adopting the Microbiome Data into Clinical Practice Lucia Grenga (Presenter) CEA Presenter Bio: I am a highly motivated and accomplished scientist, possessing both a strong background in genetics and molecular microbiology and extensive expertise in multi-omics data generation and analysis. As Senior Research Associate at the John Innes Institute (UK) I applied cutting-edge systems biology techniques based on next-generation sequencing and proteomics to probe previously unexplored aspects of bacterial ribosomal regulation. I recently joined the CEA Laboratory “Innovation tecnologique pour la Détection et la Diagnostic” (FR) where my research activity focus on the characterization of the human microbiome and, through the application of innovative tandem mass spectrometry -based approaches, on the development of improved methodologies for diagnosing and treating microbiome related diseases.

Authors: L. Grenga, G. Steinmetz, O. Pible and J. Armengaud
CEA Marcoule, DRF-Li2D, Bagnols-sur-Cèze, France

By influencing a variety of host processes, the microbiome plays a crucial role in human health and disease. Here we present the application of an innovative tandem mass spectrometry -based approach to the study of the microbiome structure and dynamics. Named phylopeptidomics, this new strategy allows the rapid identification of the components of the microbiome, assessment of their biomass contributions, their deep functional characterization and the simultaneous identification of the resistance arsenal and/or toxin production of the present organisms. Coupled with metabolomic analyses, the phylopeptidomics methodology could drive, through a better understanding of the host-microbiome crosstalk, to the development of new diagnostic tools and/or therapeutic strategies.

Introduction

The human microbiome plays a crucial role in human health and disease. Microbiome-host interactions are complex and dynamic, and influence a variety of host processes including immune, hormonal, and neural pathways. Changes in the microbiome may result in the dysregulation of host homeostasis and in an increased susceptibility to diseases such as autoimmunity, neuroinflammation and bacterial infections. Here we present a new mass spectrometry -based approach to characterize the composition of the microbiome at different phenotypic dysfunctional states of these diseases, to understand the complex interaction of host with the commensal microbiota and extend the horizon of new personalized treatment. Named phylopeptidomics, this new methodology allows, without a priori knowledge, the rapid identification of the components of the microbiome, its functional characterization and the simultaneous identification of the resistance arsenal and/or toxin production of the organisms present. Here we demonstrate the potential of phylopeptidomics for assessing the microbiome structure by applying the methodology to microbial communities from different human body sites. Phylopeptidomics opens the door for the application of mass spectrometry to the microbiome characterization as a diagnostic tool.

Methods

Sample processing. Protein extraction protocols have been optimized for the analysis of the human microbiome at different area of human body such as lung and gut.

Metaproteomics data were recorded by high-resolution tandem mass spectrometry using a Q-Exactive HF mass spectrometer (Thermo) incorporating a high-field Orbitrap analyser and coupled to an UltiMate 3000 LC system (Dionex-LC Packings).

Data processing. The recorded MS/MS spectra were searched against a generalist database such as NCBInr. Metaproteomic data were deconvoluted into precise and quantitative signals by using the phylopeptidomics strategy. Briefly, the spectrum to peptide matches (PSMs) were assigned with taxonomical information prior to the subsequent deconvolution of these signals to identify and quantify the number of different organisms explaining the whole signal, taking into account the expected ratio of shared peptides between closely-related taxa.

Results

We demonstrated the potential of phylopeptidomics to assess the microbiome structure, by applying the methodology to microbial communities from different human body area such as gut and lung. The analysis of stool samples and bronchoalveolar lavages proved that the methodology allows not only a taxonomical view of the organisms present but also their relative quantification by considering the number of PSMs belonging to the each given taxon. Moreover, functional insights have been obtained from the analysis of the identified and quantified proteins and pathways.

Conclusions & Discussion

Our results demonstrate that by applying the phylopeptidomics methodology to shotgun proteomic data we can characterize a given microbiome and is therefore a valuable strategy to gain functional information and a complement to genomic studies. Moreover, the integration of the knowledge obtained with the application of phylopeptidomics with the insights gained by analysing the microbiome with other -omic technologies such as metabolomics, could provide a better understanding of the complex interplay that exists between the microbiome and its host and could open new frontiers for the development of new diagnostic tools and/or therapeutic strategies.