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

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 LTAs but not WTAs are D-alanylated in Lactiplantibacillus plantarumNC8 cell envelope and demonstrate that DltE is a D-Ala carboxyesterase removing D-Ala from LTA. Using the mutualistic association of L. plantarumNC8 and Drosophila melanogaster as a symbiosis model, we establish that D-Ala-LTAs are direct symbiotic cues supporting intestinal peptidase expression and juvenile growth in Drosophila. Our results pave the way to probing the contribution of D-Ala-LTA to host physiology in other symbiotic models.


50
Metazoans establish mutually beneficial interactions with their resident microorganisms 1 . These interactions contribute to different aspects of host physiology including juvenile growth, a post-natal developmental process marked by rapid body-size increase and organ maturation 2 . Juvenile growth results from the integration of environmental cues with the organism's intrinsic genetic potential, driven by energy and nutritional demands. 55 Harsh environmental conditions, notably nutrient deprivation, result in linear and ponderal growth failure (i.e stunting) 3 as well as alteration in gut microbiota maturation 4 which can be mitigated by microbial interventions and/or microbiota-directed nutritional interventions 5 .
Despite some recent advances, the understanding of how gut microbes buffer the deleterious effect of undernutrition and contribute to healthy juvenile growth remains elusive. 60 Drosophila melanogaster (referred here as Drosophila) is a valuable experimental model to study the physiological consequences and underlying mechanisms of host-commensal bacteria interactions 6,7 . Bacterial strains associated with Drosophila influence multiple physiological processes including juvenile growth, and in several occasions, the underlying symbiotic cues, i.e. bacterial molecules directly impacting host functionalities, has been 65 identified. For instance, amino acids produced by symbiotic bacteria can inhibit the production of the neuropeptide CNMamide in the gut, which shape food foraging behavior by repressing preference for amino acids 8 . Bacterial metabolite such as acetate produced by strains of Ap is necessary to support juvenile growth 9 by altering the epigenome of enteroendocrine cells and stimulating the secretion of the intestinal hormone Tachykinin 10,11 . In the context of microbe-70 mediated Drosophila juvenile growth promotion 12 , peptidoglycan (PG) fragments from Lp cell walls are directly sensed by PeptidoGlycan Recognition Receptors (PGRPs) in Drosophila enterocytes. This recognition signal, via the IMD/NF-kappaB pathway, promote the production of intestinal peptidases which helps juveniles optimizing the assimilation of dietary proteins to support their systemic growth 13 . 75 Recently, in an effort to further characterize the bacterial machinery involved in Lpmediated juvenile growth promotion, we identified through forward genetic screening the pbpX2-dltXABCD operon as an important determinant of Lp-induced Drosophila larval growth 14 . The first gene of the operon, pbpX2 (here renamed dltE for D-Ala-LTA Esterase, see below), is uncharacterized and annotated as a serine-type D-Ala-D-Ala-carboxypeptidase 80 putatively involved in PG maturation cleaving the terminal D-Ala residue of the peptide stem in newly made muropeptides. Remarkably, Lp peptidoglycan precursors do not contain a terminal D-Ala in peptide stems but a terminal D-lactate (D-Lac) 15 raising the question of the biochemical activity of DltE. On the other hand, the remaining dltABCD encode a multi-protein machinery responsible for the D-alanylation of Teichoic Acids (TA) in diverse gram-positive 85 bacteria 16 and in Lp 17 . TAs are anionic polymers localized within the gram-positive bacteria cell wall, representing up to 50% of the cell envelope dry weight and present in two forms: Wall-TA (WTA) which are covalently bound to PG and Lipo-TA (LTA) which are anchored to the cytoplasmic membrane 18 . Study of a Lp NC8 isogenic strain deleted for the entire dlt operon revealed that this operon is indeed essential to D-Ala esterification to the cell envelope (most 90 likely on TAs) and that purified D-alanylated cell envelopes triggered intestinal peptidase expression and support Drosophila juvenile growth 14 . These results suggested that TA modifications are important symbiotic cues shaping commensal-host molecular dialog.
However, which type of TAs and whether their modification is directly involved remains unaddressed and an indirect influence of the D-alanylation process on PG maturation and PG 95 sensing by the host could not be excluded.
To address these standing questions, we investigate here the structure, the biochemical activity and the physiological role of DltE. We also study the impact of DltE on TA structure and modifications in Lp cell envelope and test the relative contribution of purified PG, WTA and D-alanylated-LTA (D-Ala-LTA) from Lp cell envelope to support Drosophila growth. Our 100 results establish that DltE is not a carboxypeptidase modifying Lp peptidoglycan but rather a D-Ala esterase acting upon D-Ala-LTA. After characterizing the chemical nature of LTAs and WTAs, we show that only LTAs but not WTAs are D-alanylated in Lp NC8 cell envelopes and we demonstrate that D-Ala-LTAs, in addition to PG, are direct symbiotic cues supporting intestinal peptidase expression and juvenile growth in Drosophila. 105

Structure of the extracellular domain of DltE
DltE is annotated as a putative serine type D-Ala-D-Ala carboxypeptidase. However, sequence 110 comparison with canonical members of this protein family revealed low sequence identity (Extended Data Fig. 1). Additionally, while the catalytic S-X-X-K motif (Motif 1) is conserved in DltE, the second (Y-X-S) and third (K/H-T/S-G) motifs that complete the active site are altered with only the catalytic Tyr and the Gly residues being conserved, respectively. This suggests a modified active site environment with potentially different catalytic and/or substrate 115 binding mechanisms. Supporting this, we showed that the extracellular catalytic domain of DltE (DltEextra 34-397) is not able to bind penicillin although this property is a hallmark of serine type D-alanyl-D-alanine carboxypeptidases that belongs to the PBP (Penicillin binding proteins) protein family (Extended Data Fig. 2).
These features prompted us to determine the structure of the extracellular domain of DltE 120 (DltEextra) by X-ray crystallography (Supplementary Table 1). The domain consists of two subdomains, a α-β sandwich (residues 76-126 and 246-397) that form a five strand (β1-β5) antiparallel β-sheet flanked by 5 α-helices (α2, α9-α12) and a α-helix rich region (residues 127-245) folding in 6 α-helices (α3-α8) ( Fig. 1a and Extended Data Fig. 1). DltE is structurally homologous to the D-Ala-D-Ala carboxypeptidase R61 (DDCP) from Streptomyces (Fig.  125 1b,c) 19,20 that exhibits the classical β-lactamase fold of PBPs (Fig.1b). The positions of the active site S-X-X-K motif ( 128 SIQK 131 ), containing the catalytic Ser128 and Lys131 residues, and the catalytic Tyr213 from the second motif are conserved (Fig. 1a,b and Fig. 2). These last two residues are expected to function as a general base during the acylation step and function as a relay mechanism for the transfer of a proton from the incoming substrate to the departing 130 catalytic serine. In contrast, several striking features distinguish DltE from DDCP and more generally from PBPs and DD-carboxypeptidases. The β3-strand that usually bears the third motif and defines one side of the catalytic cavity is not conserved in terms of length and position 19 (Fig. 1c). Second, DltE contains an extra α-helix (α1) at the N-terminus of the catalytic domain (Fig. 1c) as well as three major structural differences affecting the regions 135 lining the sides of the active site of DDCP and responsible for its cavity-like shape (Fig. 1c, residues longer in DDCP in which it forms an antiparallel β-sheet of 3 small strands that follows the main 5-stranded β-sheet; (iii) the loop between α8 and α9 (residues 257-283 in DltE) connecting the two subdomains is slightly shorter in DltE and adopts a different position (Fig.   1c). This last difference is of particular interest because this loop was described as the Ω-like loop in β-lactamases and PBP proteins 21,22 and shown to play a key role in the maintenance of 145 the active site topology and in the enzymatic activity ( Fig. 1e and Extended Data Fig. 1). The global architecture of the active site cavity of DltE is therefore reshaped and different from that of DDCP enzymes, adopting a deeper cleft topology largely opened on both sides of the protein ( Fig. 1d-g). Together with its inability to bind penicillin, such structural features strongly suggest that DltE possesses an alternative substrate recognition mode and/or a different 150 substrate specificity than PBPs.

DltE is not active on the peptidoglycan stem depsipeptide
Besides the apo form structure, we obtained two other DltE structures in complex with tartrate or TCEP (Tris(2-carboxyethyl)phosphine hydrochloride), two compounds arising from the 155 crystallization conditions (Supplementary Table 1 and Fig. 2a,b). Intriguingly, these two molecules are anchored by an interaction network that involves 8 different residues, among which the conserved active site nucleophile Ser128 and the base Tyr213 (Ser 62 and Tyr 159 in DDCP; Fig. 2a,b,e). Importantly, the catalytic Ser128 with the hydroxyl group of its side chain positioned at only 2.7 Å away from a carboxylic group of TCEP and Tartrate is ideally 160 placed as it would activate a substrate for the enzyme acylation event as described for DDCP 19 .
This suggests that the tartrate and TCEP molecule may mimic the nature and position of the natural substrate of DltE in its active site and that the acylation/deacylation step of the catalytic reaction is probably preserved in DltE (Fig. 2b,c). In addition, tartrate or TCEP established a series of interactions with the structural elements specific to DltE (see above) and not found in 165 DDCP (Fig. 2e). Notably, a Tyr residue (Tyr338 in DltE) interacting with TCEP or tartrate replaces the conserved Arginine (Arg285 in DDCP) in carboxypeptidases. This arginine is key for the recognition of the terminal carboxylate of the peptide substrate and the carboxypeptidase activity 19 (Fig. 2b,d,e). Furthermore, the interactions involving motif 2 and 3 that stabilizes the substrate in DDCP and in particular the penultimate D-Ala residue of the peptide substrate, are 170 also largely altered (Fig. 2b,d,e). In DltE, the hydrophobic contact with TCEP or tartrate is only maintained with the conserved Tyr213 and Gly348 of motif 2 and motif 3, respectively. Instead, residues specific to DltE (Tyr338, His347, Arg345 and Leu349) stabilize the TCEP or tartrate molecules (Fig. 2a,b). Taken collectively, our structural data suggest that DltE would not be able to recognize the D-Ala-D-Ala terminal end of a stem peptide as DDCP does and that DltE 175 would not be a DD-carboxypeptidase.
In L. plantarum peptidoglycan precursors, a D-Lac residue rather than the more common D-Ala is present in position 5 of the peptide stem with an ester bond between D-Ala 4 and D-Lac 5 15 . Tartrate and TCEP molecules having chemical groups that are also found in D-Lac (Extended Data Fig. 3), we hypothesized that the DltE structural features described above could 180 reflect its ability to act as a carboxylesterase trimming the terminal D-Lac in mature peptidoglycan. However, two enzymes, namely DacA1 and DacA2 homologous to the D-Ala-D-Ala-carboxypeptidase DacA (PBP3) of S. pneumoniae 23 could be potentially responsible for the D-Lac hydrolysis. Thus, we generated a Lp NC8 mutant deficient for dacA1 and dacA2 and analyzed the peptidoglycan composition. As shown in Extended Data Fig. 4a, we detected the 185 presence of disaccharide-depsipentapeptide (GlcNAc-MurNAc-L-Ala-D-Gln-mDAP-D-Ala-D-Lac). As D-Lac residues are not detected in WT strain 24 , this indicates that DacA1 and/or DacA2 could behave as carboxylesterases removing the terminal D-Lac. To confirm this, we purified the disaccharide-depsipentapeptide from the ∆dacA1∆dacA2 strain and measured the ability of the purified recombinant catalytic domains of DacA1 and DltE to catalyze D-Lac 190 hydrolysis. We observed that DltE was not able to cleave the ester bond between D-Ala 4 and D-Lac 5 from the disaccharide-depsipentapeptide, whereas purified DacA1, efficiently did (Fig.   3a). These results indicate that DltE is not a carboxylesterase active on peptidoglycan stem depsipentapeptide.

DltE is a D-Ala esterase acting on D-alanylated-LTA
Since DltE is not active on peptidoglycan and because dltE gene is part of the dlt operon in Lp NC8 , we hypothesized that DltE could have a D-Ala esterase activity on D-alanylated teichoic acids. To assess whether DltE could act on WTA and/or LTA, we first characterized their structure by NMR in Lp NC8 and determined their alanylation levels. Multidimensional NMR 200 spectroscopy analysis revealed that WTA from Lp NC8 contained two main repetition units composed of Rbo-phosphate chains substituted by two α-Glc residues either in C-2 and C-4 positions (major unit) or in C-3 and C-4 positions (minor unit) (Extended Data Fig. 4b).
Remarkably, no Ala substituents were detected. WTA purified from ΔdltXABCD were structurally identical from those purified from Lp NC8 , which supports that the DltXABCD 205 complex is not involved in the D-alanylation of WTA. In contrast to WTA, NMR analysis established that LTA from Lp NC8 is constituted by repetitive units of Gro-phosphate chains that were either unsubstituted (Unit A) or substituted with either Ala (Unit B), α-Glc (Unit C) or Ala-6-αGlc (Unit D) groups at C-2 position of Gro residues (Fig. 3b). Relative quantification of NMR signals associated with individual units showed that Unit A was the major form with 210 A/B/C/D ratio of 62:19:15:4. NMR analysis of LTA extracted from ΔdltXABCD mutant confirmed the absence of Ala residues characterized by the absence of signals GroB-2 and GlcD-6 that typify the presence of Unit B and D in WT LTA, respectively (Fig. 3b). Taken together these results establish that L. plantarum NC8 cell envelope carries LTAs with a distinctive pattern of structure and substitution and that only LTAs are D-alanylated. In 215 addition, these observations confirm that the Dlt machinery is necessary to the D-alanylation of Lp LTAs.
Given that only LTAs are alanylated in L. plantarum NC8 , we started testing DltE ability to bind LTA through microscale thermophoresis (MST) experiments. As shown in Fig. 3c, DltEextra was able to efficiently bind LTA. Then, we examined the activity of DltE on D-Ala-LTA 220 purified from Lp NC8 . To achieve this, we analyzed the relative amounts of Ala esterified to Gro (Gro-Ala) and free Ala released from LTA upon incubation with or without DltEextra. Both compounds were detected in the control sample as a result of spontaneous release of D-Ala from LTA, when the test was performed without DltE ( Fig. 3d and Extended Data Fig. 5). Upon incubation of LTA with DltE, we observed a clear decrease (56 % decrease) of Gro-Ala and a 225 concomitant increase of free D-Ala. This effect was enhanced (64 % decrease) with a double amount of DltEextra in the test. In contrast, no difference with the control sample was observed for LTA incubated with DltE S128A catalytic mutant protein or with DacA1 (Fig. 3d). These results establish that DltE possesses a D-Ala esterase activity that cleaves the ester bond between the substituting D-Ala and Gro in LTA chains. 230 To gain structural insights into this activity, we soaked DltE crystals with LTA extracted from the ΔdltXABCD mutant and determined the 3D structure of such enzyme-substrate complexes (Supplementary Table 1). We observe that the cleft harbors an additional, elongated density compatible with the presence of a 2-mer unit of the LTA molecule (Fig. 2c). Half of the electron density is located at the same place as the tartrate and TCEP ligands (Fig. 2) and would 235 correspond to one unit of glycerol phosphate. It is stabilized by the same interaction network described above for tartrate and TCEP that share similar chemical groups, i.e. hydroxyl, carboxyl and phosphate with LTA ( Fig. 2d, Extended Data Fig. 3). The second half of the electron density extends further on the catalytic cleft of DltE in the vicinity of Glu290 (in helix α9) and Tyr351 (between β3 and β4), forming thus a negatively charged subsite. The latter is 240 not conserved in DDCP and replaced by a hydrophobic subsite that was proposed to recognize the aliphatic portion of the peptide (Extended Data Fig. 6a,b). These structural features are in agreement with the ability of DltE to bind and hydrolyse D-Ala-LTA and together with the biochemical data, they confirm that DltE is a D-Ala esterase acting on D-Ala-LTA.

245
The D-Ala esterase activity of DltE contributes to D-alanylation of the cell envelope and is required to sustain Drosophila juvenile growth We previously reported that the machinery encoded by the dlt operon (including dltE) is necessary to support D-Ala esterification in L. plantarum cell envelope and sustain Drosophila juvenile growth 14 . As previously observed upon mild alkaline hydrolysis, D-Ala was released 250 in appreciable amounts from Lp NC8 cells 14 (Fig. 4a) whereas no D-Ala was released from an isogenic mutant deleted for the entire dlt operon 14 . However, we observed a significant reduction (around 70%) of D-Ala esterified to LTAs in ΔdltE mutant cells and an even higher reduction (around 93%) for dltE S128A catalytic mutant cells. These results indicate that the esterase activity of DltE, in addition to the activities of DltX, A, B, C and D contributes to the 255 D-alanylation machinery of LTAs in bacterial cells.
We next wondered if an active DltE protein is necessary for L. plantarum support to Drosophila juvenile growth. To this end, we compared juvenile growth (larval size at Day 6 after egg laying) and developmental timing (i.e day of 50% population entry to metamorphosis) of germfree animals and ex-germ-free animals associated at the end of embryogenesis with either Lp NC8 260 or ΔdltE and dltE S128A mutants. Both mutations did not affect the ability of Lp to thrive in the fly niche but significantly altered the ability of Lp to support larval growth and developmental timing ( Fig. 4b-d). These results demonstrate that DltE activity is required to sustain Drosophila juvenile growth and together with the structural and biochemical insights on DltE activity point to the importance of D-Ala-LTAs as symbiotic cues supporting Drosophila growth. 265

D-Ala-LTAs are necessary symbiotic cues supporting Drosophila intestinal response and juvenile growth
In order to evaluate the importance of D-Ala-LTA for Drosophila juvenile growth, we  Fig. 7a). These results confirm that LTA are D-alanylated but not WTA as determined above by NMR analysis of purified polymers (Fig. 3). Of note, we hypothesize that the residual D-Ala released for ΔltaS cells might be attached to a glycolipid precursor of LTA as previously observed in Listeria monocytogenes 27 . 280 We then tested the respective TA defective strains for their ability to support Drosophila juvenile growth (Fig. 5a). When associated with germ-free animals, Lp NC8 and ΔtagO strains support optimal Drosophila juvenile growth while Δdltop or ΔltaS strains largely fail to do so despite colonizing well their host's niche ( We had previously established that upon association with Lp, enterocytes from ex-germ free Drosophila sense and signal the presence of Lp cells by at least two independent molecular mechanisms: (i) PGRP-LE-mediated mDAP-PG fragment recognition triggering Imd/Dredd 295 signaling 13 and, (ii) sensing of bacterial cell envelope bearing D-alanylated teichoic acids and signaling by unknown host mechanisms ( Fig. 5b) 14 . Both signals were reported to be important for maximal intestinal peptidase expression and optimal support to growth promotion by Lp ( Fig. 5a,c,d) 14 . To probe the respective contribution of LTA and WTA to this model, we tested the ability of ΔltaS and ΔtagO strains (respectively deprived of LTA or WTA) to support 300 Drosophila growth and intestinal peptidase expression in a Drosophila genetic background (Dredd mutants flies) 28 that blunts the host response to mDAP-PG fragments 29  therefore support the notion that both signals are required for optimal support to larval growth by Lp and that in addition to mDAP-PG fragments, D-Ala-LTAs are likely an additional symbiotic cue sensed by Drosophila enterocytes.

D-Ala-LTAs are direct symbiotic cues supporting Drosophila intestinal response and 315
juvenile growth Next, we tested the hypothesis that D-Ala-LTAs act as direct signals sensed by Drosophila enterocytes. To this end, we purified the major components from the cell envelope of Lp NC8 and ΔdltXABCD strains: WTA, LTA and PG, and tested their ability to rescue Δdltop and ΔltaS mediated larval phenotypes ( Fig. 6a-e). First, germ-free, Δdltop or Lp NC8 -associated animals 320 were treated daily with the purified cell envelope components (WTA, PG and LTA) for 5 days.
On day 6, larvae were harvested and their size measured ( Fig. 6b-e). Daily supplementation with purified WTA or PG either from Lp NC8 or ΔdltXABCD strains did not impact the growth of GF or Δdltop associated larvae (Fig. 6b,c,d). In contrast, the daily supplementation of purified LTA isolated from Lp NC8 (D-Ala-LTAs) to larvae associated with the Δdltop strain shows a 325 growth promoting effect that leads to increased larval size in this condition when compared to the non-supplemented control (Fig. 6d,e). This growth promoting effect was not observed in GF and Lp NC8 associated animals nor when Δdltop associated larvae were supplemented with non-alanylated-LTAs isolated from ΔdltXABCD strain (Fig. 6d,e). We repeated the similar experiment with ΔltaS associated larvae and again observed an improved larval growth upon 330 supplementation with LTA purified from Lp NC8 (D-Ala-LTAs) but not from LTAs purified from ΔdltXABCD strain (ie non-alanylated-LTAs) (Fig. 6e). These results therefore establish that D-Ala-LTAs are necessary and sufficient to restore Drosophila juvenile growth. Previously, we identified through forward genetic screening the pbpX2-dltXABCD operon encoding a bacterial machinery essential to Lp NC8 mediated support to Drosophila larval growth. Here, using complementary structural, biochemical and genetic approaches, we reveal that the protein encoded by the first gene of this operon (now renamed DltE) is not a bona fide PBP as it does not bind Penicillin nor modifies Lp PG. Instead, we establish that DltE is a D-355 alanine esterase acting upon LTA. While studying its physiological function, we discovered that DltE contributes together with the other proteins encoded by the dlt operon (the DltX-A-B-C-D machinery) to the alanylation of LTA on its glycerol residues. The study of DltE's substrates gave insights on the structure and composition of Lp NC8 's TAs, which revealed that only LTA and not WTA are alanylated in this strain. Finally, prompted by this strain specific 360 feature and the importance of the dltEXABCD operon for Lp NC8 -mediated support to Drosophila growth, we identified that LTAs alanylated by the DltE/X/A/B/C/D complex (but not WTAwhich are not alanylated -nor non-alanylated LTA) act as direct symbiotic clues supporting Drosophila intestinal function and juvenile growth.

DltE and the Dlt machinery 365
Regardless of the initial DltE in silico annotation as a PBP (PbpX2), our structural data revealed that the global architecture of its active site cavity is reshaped and different from that of canonical PBP enzymes suggesting an alternative substrate recognition mode and/or a different substrate specificity. Hence, we show that D-Ala LTA and not peptidoglycan is the substrate of DltE. Given its atomic structure and D-Ala esterase activity, DltE may rather relate to 370 Staphylococcus aureus FmtA, a D-amino esterase acting on teichoic acids 30 . FmtA is however different from DltE given that it shows residual PBP activity, is able to interact with ß-lactams and is encoded apart from the dltABCD operon in S. aureus genome. So far, the hypothesis advanced for FmtA biological function concerns the removal of D-Ala from LTAs to make it available to transfer onto WTAs or removal of D-Ala from WTA to reset cell surface charge 375 under particular conditions 30 . Considering that WTA are not alanylated in Lp NC8 , these observations suggest that DltE possesses a distinct function than FmtA. Our work thus paves the way to the characterization of the mechanistical framework in which the interplay between the Dlt machinery and DltE are required for the D-alanylation of LTAs.

Strain specificity in L. plantarum teichoic acids composition and modifications
We analyzed Lp NC8 TAs composition through multidimensional NMR spectroscopy and determined their alanylation levels. Lp NC8 LTAs are, as expected from this strain's genomic information and previous gram-positive bacteria LTA characterization 31,32 , constituted by poly-Glycerol-Phosphate (poly-Gro-P) chains substituted, among others, with Ala. In contrast, but 385 in agreement with the genetic information from Lp NC8 genome, this strain only produces poly-Ribitol-Phosphate (poly-Rbo-P) WTAs given the lack of the tag genes encoding the machinery responsible for the synthesis of poly(Gro-P) chains in its genome 33 . This ability to produce poly(Gro-P) chain is a strain specific feature as the tag gene cluster is only found in 22 out of 54 L. plantarum strains recently studied at the genomic level 34 . However, in the case of the 390 Lp WCFS1 strain, despite its genome carrying the genetic information to synthetize both types of WTA (poly(Gro-P) and poly(Rbo-P)), only poly(Gro-P) WTAs were detected at steady state 33 .
Surprisingly, no Ala substituents were detected in Lp NC8 WTAs. This is an original feature as in other L. plantarum strains such as Lp WCFS1 , WTA appears to be D-alanylated 33  plantarum strains and question about the potential correlation between WTA composition and/or TA D-alanylation patterns and the ability of the strain to benefit Drosophila growth.

Drosophila response to commensals' cell envelop determinants 400
We have previously established that PG fragments 13 and D-Ala esterification to the cell envelope 14 trigger intestinal peptidase expression and support Drosophila juvenile growth.
Here, we report that in addition to PG fragments, D-Ala-LTA from L. plantarum NC8 cell envelop represent a symbiotic cue necessary and sufficient for the ability of Lp NC8 to promote Drosophila growth and upregulation of intestinal digestive peptidases in enterocytes. Given that L. 405 plantarum is a gut luminal microbe and that the Drosophila midgut is lined with both thick chitinous matrix and thin mucus layer, yet permeable to large macromolecules and small nutrient particles 35 , how exactly these macromolecules associated to the bacterial cell membrane reach the enterocytes remains elusive. Similarly to PG fragments, which are shaded in the extracellular media during bacterial cell division, LTA chains may also be shaded after 410 cleavage of the lipid anchor 36 . In addition, our recent findings report Lp NC8 's ability to release microvesicles of multi-nanometers ranges 37  PG fragments are sensed intracellularly in the Drosophila midgut by PGRP-LE which signals the Imd pathway to stimulate intestinal peptidase expression. How D-Ala-LTAs are sensed and signal in enterocytes remain elusive but our results support the notion that another signaling 420 cascade beyond the one engaged by PGRP-LE is leading to D-Ala-LTA mediated intestinal peptidase induction. Taken together, it seems that Drosophila ensures an optimal intestinal response to symbiotic cues by using additive rather than interdependent signals converging locally to intestinal peptidase expression and macroscopically to enhanced juvenile growth.
Given the strong analogies of the impact of L. plantarum strains on Drosophila and Mouse 425 growth 39 and previous work focusing on LTA in host-gram positive bacteria interactions 38,40 , an exciting perspective will be to test the contribution of D-ala-LTA to L. plantarum mediated host growth promotion in other symbiotic models including mammals.

Drosophila diets, stocks and breeding
Drosophila stocks were cultured as described in Erkosar et al., 2015 13 . Briefly, flies were kept at 25°C with 12/12 hours dark/light cycles on a yeast/cornmeal medium containing 50 g/L of inactivated yeast. The low yeast diet was obtained by reducing the amount of inactivated yeast 440 to 7 g/L. Germ-free stocks were established as described in Erkosar et al., 2015 13 . Axenicity was routinely tested by plating serial dilutions of animal lysates on nutrient agar plates.
Drosophila y,w flies were used as the reference strain in this work. The following Drosophila line was also used: y,w,Dredd F64 28 .

Bacterial strains and growth conditions
Strains used in this study are listed in Supplementary Table 3

Knock-in of DltE catalytic dead versions in L. plantarum NC8
L. plantarum NC8 strain carrying a modified version of the dltE gene (dltE S128A ) was built by knocking-in the modified sequence on ΔdltE strain 14 . dltE modified sequence harboring the mutation S128A was obtained by PCR with modified primers (X19). The 5′-and 3′-terminal regions of dltE region together with the entire dltE gene were PCR-amplified with Q5 High-475 Fidelity 2X Master Mix (NEB) from L. plantarum NC8 chromosomal DNA using primers XL17/XL18 and XL19/XL20 (Supplementary Table 4). The 2 fragments were assembled with

Developmental timing determination 500
Axenic adults were placed in breeding cages overnight to lay eggs on sterile poor-yeast diet.
Fresh axenic embryos were collected the next morning and seeded by pools of 40 in tubes containing fly food. A total of 1 × 10 8 CFUs of each strain or PBS was then inoculated homogeneously on the substrate and the eggs and incubated at 25 °C. The emergence of pupae was scored every day until all pupae had emerged. D50 (day when 50% of the pupae emerged) 505 was determined using D50App 14 .

Bacterial loads analysis
To access bacterial CFU in the fly nutritional matrix, microtubes containing food and larvae were inoculated with 1x10 7 CFUs of each strain, independently. Tubes were incubated at 25°C 510 until being processed. Pools of five larvae were deposited in 1.5 ml microtubes containing 0.75-1 mm glass 520 microbeads and 500 µL of PBS.

RNA extraction and RT-qPCR analysis
Axenic y,w and y,w,Dredd embryos were inoculated with 1x10 8  Results are presented as the value of ∆Ct gene /∆Ct rp49 .

Statistical analysis
Data representation and analysis was performed using Graphpad PRISM 8 software (www.graphpad.com). A total of 3 to 5 biological replicates were used for all experiments 535 performed in this study in order to ensure representativity and statistical significance. All samples were included in the analysis. Experiments were done without blinding. Two-sided Mann Whitney's test was applied to perform pairwise statistical analyses between conditions E. coli plasmid construction 540 DNA fragments were amplified by polymerase chain reaction using L. plantarum cDNA as a template and oligonucleotides listed in Supplementary Table 5. The DNA encoding the extracellular domain of wild-type DltE (DltEextra) and the catalytic mutant DltEextra-S128A were cloned into the NcoI and XhoI sites of the pET-28a(+) vector that expresses proteins fused to a C-terminal hexahistidine (His)6 tag (Supplementary Table 6). 545

Protein production and purification
DltEextra and DltEextra-S128A were expressed in E. coli BL21 ( Table 1.

Microscale thermophoresis assays
Protein-ligand interactions were analyzed by microscale thermophoresis (MST) 51  After a short incubation of target-partner complex, the samples were loaded into MST premium glass capillaries and eight measurements were performed (four with target alone and four with target-partner complex) at 22°C. The assays were repeated three times for each binding check 600 experiment. Data analyses were performed using Nanotemper Analysis software provided by the manufacturer.

Preparation of L. plantarum cell walls 635
L. plantarum strains (500 ml) were grown overnight in MRS medium. Cell walls were prepared as described previously 14 , with some modifications. pH of buffer solutions was kept ≤ 6.0 in the different steps to avoid potential D-Ala hydrolysis from teichoic acids. Briefly, bacteria inactivated by heat treatment were boiled in 5% SDS in 50 mM MES buffer pH5.5 for 25 min.

Peptidoglycan purification
Purified cell walls were treated with hydrofluoric acid (48%) for 19h at 0°C to remove WTA and polysaccharides. The remaining insoluble purified PG was washed twice times with 250 655 mM Tris-HCl pH 8.0 and four times with MilliQ H2O and lyophilized.

WTA purification
WTA were extracted from purified cell walls with TCA as described previously 53  LTA was obtained by butanol extraction allowing to keep D-Ala esterified on LTAs as 680 described previously 25,55 . The supernatant was recovered and butanol-1 (1:1 V/V) was added in a glass container. The mixture was stirred for 30 min at room temperature. Insoluble material was discarded after centrifugation at 13.000 g for 20 min in PPCO tubes (Nalgene). The liquid phase was recovered and centrifuged again; the aqueous phase (lower one) containing LTA was recovered. The sample was treated with DNAse II (Sigma D8764) at 50 µg/ml for 2h at 37°C 685 to degrade contaminating DNA and lyophilized. LTA was further purified by hydrophobic interaction chromatography (HIC) with a Hi-prep Octyl FF 16/10 column with an AKTA chromatography system. LTA was dissolved in Buffer A (10 mM ammonium acetate pH 4.7 containing 15% propanol-1). Elution was performed with a gradient of buffer B (10 mM ammonium acetate pH 4.7 containing 60% propanol-1) from 0 to 100% in 1h. Fractions were 690 collected and the presence of LTA was tested by dot-blot. Aliquots were spotted on a PVDF membrane (Immobilon-P transfer membrane, Millipore) and incubated with anti-LTA monoclonal antibody (Origene, AM26274LE-N) at 1/1000 dilution, followed by anti-mouse antibody coupled to horseradish peroxidase (Thermofisher) at 1/2000. Dot-blot was revealed by chemiluminescence detection using Pierce ECL Western blotting substrate (Thermo 695 scientific) and detection with a Chemidoc system (Biorad). Fractions containing LTA were pooled, dialyzed against 10 mM ammonium acetate pH 4.6 with Float-a-Lyzer G2 dialysis devices (cut-off 500-1000 Da) (Spectra/Por (500-1000 Da cut-off), and lyophilized.

PG, WTA and LTA composition analysis and quantification 700
PG was quantified by analysis of muramic acid (Mur) content after acid hydrolysis as described previously 14 . PG (400 µg) was hydrolyzed by 6N HCl for 16 h at 100°C under vacuum. Mur was quantified by high performance anion exchange chromatography coupled with pulse amperometric detection (HPAEC-PAD) with an ICS5000 system (Thermo Scientific) and a Dionex CarboPac PA-1 anion exchange column (4 x 250 mm) (Thermo Scientific) with a guard 705 column. Quantification was made with a standard curve of pure Mur (Sigma-Aldrich) (50 to 1000 pmol).
WTA composition was obtained after hydrolysis with hydrofluoric acid 48% at 0°C for 48h.
LTA composition was obtained after hydrolysis by hydrofluoric acid 48% at 0°C for 48h. After 715 HF evaporation, samples were treated with 4M trifluoroacetic acid (TFA) for 3h at 110°C.
Composition was determined by HPLC analysis with an Aminex HPX-87C column (Biorad) with a Waters 2414 refractive index (RI) detector using alditols (glycerol, ribitol), monosaccharides and phosphate standards. Quantification was performed by comparison with standard amounts of the same compounds. D-Ala esterified to LTA was detected after alkaline 720 hydrolysis using Marfey's reagent derivatization, as described above for whole cells.

NMR analysis
LTA was purified after butanol extraction allowing to keep D-Ala esterified on LTAs as described above 55 . WTA was purified after TCA extraction from cell walls as described above.
pH was kept below 6.0 at all purification steps of both polymers to avoid hydrolysis of the ester 730 bond between D-Ala and the teichoic acid backbone chains. Samples were solubilized in highly enriched deuterated water (99.96% deuterium; EurisoTop, St-Aubin, France) and lyophilized.
This process was repeated twice. Data were recorded on a 9.4-T spectrometer and a 18.8-T spectrometer were 1 H resonated at 400.33 and 800.12 MHz, and 13 C resonated at 100.2 and 200.3 MHz, respectively. All samples were inserted in 3-mm tubes with matching amounts of 735 D2O. Acetone was added as an internal standard, starting from a solution of 2.5 µl of acetone-10 ml of D2O. All pulse sequences were taken from the Bruker library of pulse programs and then optimized for each sample. Spectral widths were 12 and 200 ppm for the proton and carbon observations, respectively. TOCSY was performed with various mixing times of 40 to 120 ms.
Edited 1 H-13 C HSQC spectra were recorded with 1,536 data points for detection and 256 data 740 points for indirect direction.

Test of carboxyesterase activity on purified muropeptide
Disaccharide-depsipentapeptide (GlcNAc-MurNAc-L-Ala-D-Gln-mDAP-L-Ala-L-Lac) was purified from PG of dacA1dacA2 double mutant of L. plantarum NC8 . PG was digested with 745 mutanolysin (Sigma Aldrich) and the resulting soluble muropeptides were reduced by NaBH4 as described previously 24 . The reduced muropeptides were separated by reverse phase ultrahigh-pressure liquid chromatography (RP-UHPLC) with a 1290 chromatography system (Agilent Technologies) and a Zorbax RRHD Eclipse Plus C18 column (100 by 2.1 mm; particle size, 1.8 µm; Agilent Technologies) at 50°C using ammonium phosphate buffer and methanol 750 linear gradient. The eluted muropeptides were detected by UV absorbance at 202 nm. The peak corresponding to the disaccharide-depsipentapeptide was collected. Purified muropeptide was then incubated with 10 µg of purified DltEextra or DacA1 in 50 mM Tris-HCl, 100 mM NaCl buffer overnight at 37°C. The reaction mixtures were analyzed by RP-UHPLC as described above and by LC-MS with an UHPLC instrument (Vanquish Flex, Thermo Scientific) 755 connected to a Q Exactive Focus mass spectrometer (Thermo Scientific). Mass spectra were collected over the range m/z = 380-1400. Data were processed using Xcalibur QualBrowser v2.0 (Thermo Scientific).   ∆dltE and DltE S128A strains and PBS (for the GF condition). Larvae were collected 6 days after 905 association and measured as described in the Methods section. Purple asterisks illustrate statistically significant difference with larval size of Lp NC8 ; ****: p<0,0001. Center values in the graph represent means and error bars represent SD. Representative graph from one out of three independent experiments. c, Day when fifty percent of pupae emerge during a developmental experiment (D50) for GF eggs associated with strains Lp NC8 , ∆dltE and DltE S128A 910 or PBS (for the GF condition). Center values in the graph represent means. Purple asterisks illustrate statistically significant difference with Lp NC8 larval size; ****: p<0,0001. d, Evolution of the number of CFUs on fly food and larvae at days 3, 5, and 7 after inoculation with Lp NC8 , ∆dltE and DltE S128A . Representative graphs from one out of three independent experiments are shown. The purple asterisks represent a statistically significant difference compared with Lp NC8 proteases expression. The green asterisks represent a statistically significant difference compared with the GF condition. NS represents the absence of a statistically significant difference compared 935 to the GF condition or Lp NC8 . **0.001 < P < 0.01; *P < 0.05.   respectively. At day 6 after inoculation, larvae were harvested and measured (see the Methods section for details). Mid-L3 sized-matched larvae were collected for each condition. Their guts were dissected followed by RNA extraction and RT-qPCR targeting proteases expression. b, c, d, Larval longitudinal length after inoculation with strains Lp NC8 , ∆dltop, PBS and purified peptidoglycan (PG) (b) wall teichoic acids (WTA) (c) or lipoteichoic acids (LTA) (d) from 950

Test of D-Ala esterase activity on LTA
Lp NC8 or ∆dltXABCD. Larvae were collected six days after the first association and measured as described in the Methods. The pink asterisks represent a statistically significant difference compared with ∆dltop larval size; violet asterisks represent a statistically significant difference compared with Lp NC8 larval size. NS represents the absence of a statistically significant difference compared to ∆dltop. ****P < 0.0001; **0.001 < P < 0.01. The bars in the graph 955 represent means and s.d. A representative graph from one out of three independent experiments is shown. e, Larval longitudinal length after inoculation with strains Lp NC8 , ∆dltop, ∆ltaS or PBS and purified lipoteichoic acid (LTA) from Lp NC8 or ∆dltXABCD strains. Larvae were collected six days after the first association and measured as described in the Methods. The pink asterisks represent a statistically significant difference compared with ∆dltop larval size; Violet asterisks 960 represent a statistically significant difference compared with Lp NC8 larval size ****P < 0.0001; **0.001 < P < 0.01. The bars in the graph represent means and s.d. A representative graph from one out of three independent experiments is shown. f, g , Mean ± s.d. of 2 -∆Ctgene/∆Ctrp49 ratios for Jon66Cii and Jon65Ai detected in dissected guts of y,w,Dredd size-matched larvae, associated with Lp NC8 , ∆dltop, ∆ltaS or the GF and LTA from Lp NC8 or ∆dltXABCD strains, from 965 five biological replicates. Representative graphs from one out of three independent experiments are shown. The purple asterisks represent a statistically significant difference compared with Lp NC8 protease expression. The pink asterisks represent a statistically significant difference compared with ∆ltaS supplemented with LTA from ∆dltXABCD strain. NS represents the absence of a statistically significant difference compared to ∆ltaS condition. **0.001 < P < 970 0.01; *P < 0.05.