Beneficial host - microbiota symbiosis upon chronic undernutrition: Commensal Bacteria Envelope and Host Linear Growth Promotion – SymEnvLop

A set of techniques spanning genetics, biochemistry, molecular biology and in vivo assays are being used for this project.
Cell wall components are being purified (peptidoglycan (PG) and teichoic acids (TA) including wall teichoic acid (WTA) and lipoteichoic acid (LTA)) from wild-type (WT) and several isogenic strains mutated in key cell envelope components. PG is analysed using RP-UHPLC and MS and TA will be analysed by GC/MS and HPAEC-PAD for composition analysis or MS and NMR for fine structural analysis. Following purification, the compounds are supplemented to Drosophila larvae facing chronic undernutrition to evaluate their impact for growth promotion and development, in order to narrow down the molecule responsible for the phenotype.
We are generating strains overexpressing pbpX2 and dltD, the putative enzymatic effectors encoded in the operon and strains with point mutations in the catalytic sites of pbpX2 and dltD are being engineered accordingly with structural data obtained through crystallography and structure-function analysis.
Concerning the biochemical activity of PbpX2 and DltD, we are now conducting tests on purified and synthetic substrates.

We have constructed several mutants on pbpX2-dlt operon and genes involved in teichoic acids (TA) biosynthetic pathways in order to obtain strains depleted for lipoteichoic acids (LTA) and wall teichoic acids (WTA). Strains were tested for their ability to promote Drosophila growth. All strains are impaired in their ability to promote growth but ?tagO. The quantity of d-Ala released by the strains was determined through HPLC, confirming the results previously published. In addition, we obtained intermediary amounts of d-alanine released from ?pbpX2 and ?ltaS. We have extracted peptidoglycan (PG) and analyzed its structure from the WT strain LpNC8 and 10 derived mutants. A reference muropeptide profile has been established by UHPLC and LC-MS/MS for the LpNC8 PG. The muropeptide profiles for 10 have been also established. Moreover, we have purified WTA from LpNC8 and the three mutants, ?dltop, ?pbpX2 and ?ltaS for composition analysis and NMR structure determination. Both structures were found identical and consist of chains made of ribitol-phosphate subunits, each substituted with two glucose. We embarked in the structure-function analysis of the proteins encoded by the pbpX2-dlt operon (initially focusing on PbpX2, DltA and DltD) and of PbpX1. After several optimization steps, we successfully cloned, expressed and purified, in a two steps protocol, all the extracellular catalytic domains of PbpX2, PbpX1, DltD and the full length DltA protein. Crystallization hits were obtained for all targets and following crystallization conditions optimizations, diffraction data were collected at SOLEIL synchrotron for all protein crystals at high resolution. All structures were solved using molecular replacement approach. In total 7 different structures were successfully solved so far. All the structures now available for PbpX2 allowed us to a rather detailed structural analysis. Comprehensive analysis and comparison of the other structural data are currently performed.

The first 18 months of the project allow us to determine the relative importance for the Drosophila growth promotion phenotype of the 2 types of TA present within Lp the cell envelope.
We determined LpNC8 WTA composition and structure: chains are made of ribitol-phosphate subunits, substituted with two glucose. Unexpectedly, no D-Ala substituents were found on the ribitol-phosphate chains. Preliminary results suggest that D-Ala substituents are only located on LTA chains.
After several optimization steps, the crystal structure of PbpX1, PbpX2, DltA and DltD were obtained.
Accordingly with the progress we made so far, we are in a good position to understand the role of teichoic acids for bacterial physiology and Drosophla growth promotion. Further analysis of LTA and WTA structures will provide clues on how those structures are synthetized and concomitantly how they are substituted with d-Alanines. Progress made with the structure-function analysis of the proteins under study will allow us to resolve their role in PG or TA synthesis, in particular PbpX2, and their biochemical activity be determined.

Poster presentation :

Renata C. Matos, Octobre Clocher, Pascal Courtin, Marie-Pierre Chapot-Chartier and François Leulier. Lactobacillus plantarum cell envelope and Drosophila linear growth promotion. New approaches and concepts in Microbiology EMBO Symposium July 2019. Heidelberg, Germany & The Great Wall Symposium. September 2019. Paris, France.

Ste´phanie Ravaud, Nikos Nikolopoulos, Virginie Gueguen-Chaignon, Renata C. Matos, François Leulier and Christophe Grangeasse. Structural and functional characterization of two atypical carboxypeptidases PBPX1 and PBPX2 in Lactobacillus plantarum. The Great Wall Symposium. September 2019. Paris, France.

Nikos Nikolopoulos, Stéphanie Ravaud, Renata Matos, François Leulier, Christophe Grangeasse. Structural and functional mechanisms underlying host/intestinal microbiota interaction. LS-CCP4 Data Collection and Structure Solution Workshop Online, November 30th - December 11th 2020

The mutualistic interaction that binds animals to their resident microbial communities (i.e. microbiota) profoundly shapes many aspects of their biology. One such important aspect is juvenile growth especially upon chronic undernutrition. Here we aim to elucidate the molecular mechanisms that forge the mutualistic interaction between the juvenile host and its gut microbiota, and delineate the genetic and regulatory networks of mutualism in juvenile growth both in microbes and hosts. To this end, gnotobiotic animal models (i.e with a defined and controlled microbiota) are powerful host models to study symbiosis and host physiology in an integrative manner. Using gnotobiotic drosophila and mice models, we previously demonstrated a causative link between microbiome activities and juvenile animal growth promotion. Using these models, we now aim to probe the molecular dialog engaged between the host and its microbiome, which underlies microbiota-mediated enhanced juvenile growth