Structure–function analysis of Lactiplantibacillus plantarum DltE reveals D-alanylated lipoteichoic acids as direct cues supporting Drosophila juvenile growth

  1. Nikos Nikolopoulos
  2. Renata C Matos
  3. Stephanie Ravaud
  4. Pascal Courtin
  5. Houssam Akherraz
  6. Simon Palussiere
  7. Virginie Gueguen-Chaignon
  8. Marie Salomon-Mallet
  9. Alain Guillot
  10. Yann Guerardel
  11. Marie-Pierre Chapot-Chartier
  12. Christophe Grangeasse
  13. François Leulier

  • Curated by eLife

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    eLife assessment

    This is an important study on the role of a bacterial cell wall component, D-alanylated lipoteichoic acid, as a bacteria cue in Drosophila melanogaster-microbiome interactions. Overall, the evidence supporting the conclusions is compelling, with a solid approach combining crystallography with biochemical and cellular assays, that take advantage of both fly and bacterial mutants, to demonstrate a physiological role in juvenile growth promotion. The work will be of broad interest to those studying host-microbe interactions, especially as it is related to immunology and metabolism mediated by the microbiome.

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Abstract

Metazoans establish mutually beneficial interactions with their resident microorganisms. However, our understanding of the microbial cues contributing to host physiology remains elusive. Previously, we identified a bacterial machinery encoded by the dlt operon involved in Drosophila melanogaster ’s juvenile growth promotion by Lactiplantibacillus plantarum . Here, using crystallography combined with biochemical and cellular approaches, we investigate the physiological role of an uncharacterized protein (DltE) encoded by this operon. We show that lipoteichoic acids (LTAs) but not wall teichoic acids are D-alanylated in Lactiplantibacillus plantarum NC8 cell envelope and demonstrate that DltE is a D-Ala carboxyesterase removing D-Ala from LTA. Using the mutualistic association of L. plantarum NC8 and Drosophila melanogaster as a symbiosis model, we establish that D-alanylated LTAs (D-Ala-LTAs) are direct cues supporting intestinal peptidase expression and juvenile growth in Drosophila . Our results pave the way to probing the contribution of D-Ala-LTAs to host physiology in other symbiotic models.

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  1. Version published to 10.7554/elife.84669 on eLife
  2. eLife

    eLife assessment

    This is an important study on the role of a bacterial cell wall component, D-alanylated lipoteichoic acid, as a bacteria cue in Drosophila melanogaster-microbiome interactions. Overall, the evidence supporting the conclusions is compelling, with a solid approach combining crystallography with biochemical and cellular assays, that take advantage of both fly and bacterial mutants, to demonstrate a physiological role in juvenile growth promotion. The work will be of broad interest to those studying host-microbe interactions, especially as it is related to immunology and metabolism mediated by the microbiome.

  3. eLife

    Reviewer #1 (Public Review):

    This work is a follow-up of the work from the same group where the authors showed that Lactiplantibacillus plantarum can enhance juvenile growth by activating the expression of an intestinal protease. They previously showed that this process was mediated by the dlt operon which is involved in the D-Alanylation of teichoic acid.

    In the present study, the authors characterized the structure and enzymatic activity of the first protein encoded by this operon and show that the first gene of this operon encodes for an esterase releasing D-Ala from D-Ala lipoteichoic acids (LTA) and renamed it here DltE. The gene encoding this protein was previously uncharacterized and annotated as a peptidoglycan-binding protein putatively involved in peptidoglycan maturation. With the structure and enzymatic characterization of this protein, this study revealed that this protein does not act as peptidoglycan, but instead releases D-Ala from D-alanylated-LTA.

    The authors use a Drosophila mutant impaired in response to mDAP-Peptidoglycan fragments (affected in the IMD pathway) to show that this mutant still responds to D-Ala-LTAs. This result is important to show that D-Ala-LTAs act as additional cues sensed by Drosophila independent of m-DAP-peptidoglycan by a still unknown sensory pathway. The study convincingly shows that D-Ala-LTA from the gut microbe L. plantarum leads to increase intestinal peptidase expression (intestinal activity) and enhance juvenile larva growth.

  4. eLife

    Reviewer #2 (Public Review):

    The authors conduct a structure-function analysis of an uncharacterized gene, DltE, which was found by a genetic screen to be involved in the growth promotion of Drosophila larvae by Lactiplantibacillus plantarum, a bacterium that is consistently associated with Drosophila. They find that DltE is a D-Ala carboxylesterase that removes D-Ala from lipoteichoic acids in the cell envelope and that D-alanylated lipoteichoic acids stimulate Drosophila larval growth. The result that D-Ala LTA stimulates larval growth is compelling, although some minor experimental details to do with biological replicates are not shown and the tracking of bacterial abundances should be addressed to make the conclusions more solid. Additionally, I think the use of the terms "direct" and "symbiotic" is inappropriate in the manuscript, but this can be resolved by removing them or performing additional experiments.

    The authors make these claims:
    - DltE is not a carboxypeptidase modifying Lp peptidoglycan;
    - DltE is a D-Ala esterase acting upon D-Ala-LTA;
    - only LTAs but not WTAs are D-alanylated in LpNC8 cell envelopes;
    - D-Ala-LTAs, in addition to PG, are direct symbiotic cues supporting
    (1) intestinal peptidase expression and
    (2) juvenile growth in Drosophila.
    I find all of the claims to be well supported by data except the suggestion that these are "direct symbiotic" cues. I think the authors provide the support that D-Ala LTAs are nutritional cues, not symbiotic ones.

    Overall, I find the work compelling.

  5. eLife

    Reviewer #3 (Public Review):

    This work by Nikolopoulos et al. expands on prior studies demonstrating the ability of a member of the Drosophila melanogaster gut microbiome, Lactiplantibacillus plantarum, to support juvenile development in nutrient-limiting conditions. Previously, the authors identified the pbpX2-dltXABCD operon of L. plantarum that when mutated eliminated the growth-promoting ability of the bacterium to flies experiencing malnutrition (protein starvation). To better understand the bacterial components that support this larval development, the authors used a combination of structural, biochemical, and mutational analysis to describe the physiological role of the DltE, a previously uncharacterized gene within the pbpX2-dltXABCD operon. Although annotated as a serine-type D-Ala-D-Ala-carboxypeptidase, this work supports its role instead as a D-ala esterase that acts upon D-alanylated lipoteichoic acids, which are directly sensed by the host to induce peptidase expression and support juvenile growth in flies.

    Overall, the data is compelling, and the conclusions are well-supported. The multiple methods used to examine and support their findings - the combination of structural and biochemical analyses, and the use of both bacterial and fly mutants to substantiate and demonstrate physiological relevance was elegant in execution.

    The identification of a role for this bacterial cell component is exciting as it has not previously been appreciated as a bacterial-derived signal in fly immunity and/or metabolism. This work adds to the growing evidence for the breadth and diversity of bacterial metabolites and products that underlie fly-microbiome interactions and may have implications in other animal-microbe interactions, especially L. plantarum-mediated host growth promotion in other models including mammals.

    An intriguing aspect of the work is the evidence of a bifurcation of this bacterial signal on immunity and metabolism, with the pathway regulating the latter yet unknown. Likewise, determining how these cell components are sensed by the host will also be of future interest. Another unknown that may limit the implications of this study is the ubiquity of D-ala LTA production among D. melanogaster-associated L. plantarum strains and whether this is a common or rare signal/role.

  6. Version published to 10.1101/2022.09.14.507969v1 on bioRxiv