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The intestinal microbiota is known to influence postnatal growth. We previously found that a strain of Lactiplantibacillus plantarum (strain Lp WJL ) buffers the adverse effects of chronic undernutrition on the growth of juvenile germ-free mice. Here, we report that Lp WJL sustains the postnatal growth of malnourished conventional animals and supports both insulin-like growth factor–1 (IGF-1) and insulin production and activity. We have identified cell walls isolated from Lp WJL , as well as muramyl dipeptide and mifamurtide, as sufficient cues to stimulate animal growth despite undernutrition. Further, we found that NOD2 is necessary in intestinal epithelial cells for Lp WJL -mediated IGF-1 production and for postnatal growth promotion in malnourished conventional animals. These findings indicate that, coupled with renutrition, bacteria cell walls or purified NOD2 ligands have the potential to alleviate stunting.

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.

Teichoic acids (TA) are crucial for the homeostasis of the bacterial cell wall as well as their developmental behavior and interplay with the environment. TA can be decorated by different modifications, modulating thus their biochemical properties. One major modification consists in the esterification of TA by d-alanine, a process known as d-alanylation. TA d-alanylation is performed by the Dlt pathway, which starts in the cytoplasm and continues extracellularly after d-Ala transportation through the membrane. In this study, we combined structural biology and in vivo approaches to dissect the cytoplasmic steps of this pathway in Lactiplantibacillus plantarum, a bacterial species conferring health benefits to its animal host. After establishing that AcpS, DltB, DltC1 and DltA are required for the promotion of Drosophila juvenile growth under chronic undernutrition, we solved their crystal structure and/or used NMR and molecular modeling to study their interactions. Our work demonstrates that the suite of interactions between these proteins is ordered with a conserved surface of DltC1 docking sequentially AcpS, DltA and eventually DltB. Altogether, we conclude that DltC1 acts as an interaction hub for all the successive cytoplasmic steps of the TA d-alanylation pathway.

E. faecalis strain VE14089 was derived from V583 cured of its plasmids. Although VE14089 had no major DNA rearrangements, it presented significant growth and host adaptation differences from the reference strain V583 of our collection. To construct a strain with better fitness, we sequenced the genome of VE14089, identified single nucleotide polymorphisms (SNPs), and repaired the genes that could account for these changes. Using this reference-derivative strain, we provide a novel genetic system to understand the role of the variable region of epa in the enterococcal lifestyle., Commensal and generally harmless in healthy individuals, Enterococcus faecalis causes opportunistic infections in immunocompromised patients. Plasmid-cured E. faecalis strain VE14089, derived from sequenced reference strain V583, is widely used for functional studies due to its improved genetic amenability. Although strain VE14089 has no major DNA rearrangements, with the exception of an ∼20-kb integrated region of pTEF1 plasmid, the strain presented significant growth differences from the V583 reference strain of our collection (renamed VE14002). In the present study, genome sequencing of strain VE14089 identified additional point mutations. Excision of the integrated pTEF1 plasmid region and sequential restoration of wild-type alleles showing nonsilent mutations were performed to obtain the VE18379 reference-derivative strain. Recovery of the growth ability of the restored VE18379 strain at a level similar to that seen with the reference strain points to GreA and Spx as bacterial fitness determinants. Virulence potential in Galleria mellonella and intestinal colonization in mouse demonstrated host adaptation of the VE18379 strain equivalent to VE14002 host adaptation. We further demonstrated that deletion of the 16.8-kb variable region of the epa locus recapitulates the key role of Epa decoration in host adaptation, providing a genetic system to study the role of specific epa-variable regions in host adaptation independently of other genetic variations. IMPORTANCE E. faecalis strain VE14089 was derived from V583 cured of its plasmids. Although VE14089 had no major DNA rearrangements, it presented significant growth and host adaptation differences from the reference strain V583 of our collection. To construct a strain with better fitness, we sequenced the genome of VE14089, identified single nucleotide polymorphisms (SNPs), and repaired the genes that could account for these changes. Using this reference-derivative strain, we provide a novel genetic system to understand the role of the variable region of epa in the enterococcal lifestyle.

The AI-2 i nterspecies quorum-sensing molecule is produced by the LuxS enzyme and has been ascribed a role in virulence in several bacteria. The nosocomial pathogenEnterococcus faecalisinhabits several different environments where multispecies communities are established. However, despite the presence of aluxSgene in this pathogen, its role inE. faecalispathogenesis has never been assessed. In the present work, we deleted theluxSgene from the vancomycin-resistant clinical isolateE. faecalisV583 and demonstrated the lack of AI-2 production by the mutant strain. Using microarrays and externally added (S)-4,5-dihydroxy-2,3-pentanedione we showed that AI-2 is not sensed byE. faecalisas a canonical quorum-sensing molecule and that theluxSmutation caused pleiotropic effects in gene expression, which could not be complemented by extracellularly added AI-2. These global differences in gene expression affected several gene functional roles, mainly those enrolled in metabolism and transport. Metabolic phenotypi ng of theluxSmutant, using Biolog plates, showed differences in utilization of galactose. AI-2 production by LuxS was shown to be irrelevant for some phenotypes related to the pathogenic potential ofE. faecalisnamely biofilm formation, adhesion to Caco-2 cells, resistance to oxidative stress and survival inside J-774 macrophages. However, theluxSmutant was attenuated when tested in theDrosophilaseptic injury model, as its deletion led to delayed fly death. Overall our findings show that differential gene expression related to theluxSmutation cannot be ascribed to quorum-sensing. Moreover, the role of LuxS appears to be limited to metabolism.

SummaryThe microbial environment influence animal physiology. However, the underlying molecular mechanisms of such functional interactions are largely undefined. Previously, we showed that upon chronic undernutrition, strains of Lactobacillus plantarum, a dominant commensal partner of Drosophila, promote host juvenile growth and maturation partly via enhanced expression of intestinal peptidases. By screening a transposon insertion library of Lactobacillus plantarum in gnotobiotic Drosophila larvae, we identify a bacterial cell wall modifying machinery encoded by the pbpX2-dltXABCD operon that is critical to enhance host digestive capabilities and promote growth and maturation. Deletion of this operon leads to bacterial cell wall alteration with a complete loss of teichoic acids D-alanylation. We thus conclude that teichoic acids modifications participate in commensal-host interactions and specifically, D-alanine esterification of teichoic acids contributes to optimal L. plantarum mediated intestinal peptidase expression and Drosophila juvenile growth upon chronic undernutrition.Highlights- LpNC8 mutant library screening identifies genes affecting Drosophila growth promotion.- pbpX2-dlt operon is required for D-alanylation of teichoic acids and Drosophila growth.- Deleting the pbpX2-dlt operon alters host intestinal peptidase expression.- Peptidoglycan and pbpX2-dlt dependent signals are required for LpNC8 mediated growth promotion.eTOC blurbAnimals establish interactions with their microbial communities that shape many aspects of their physiology including juvenile growth. However, the underlying molecular mechanisms are largely undefined. Matos et al. reveal that bacterial teichoic acids modifications contribute to host juvenile growth promotion.

The microbial environment influences animal physiology. However, the underlying molecular mechanisms of such functional interactions are largely undefined. Previously, we showed that during chronic undernutrition, strains of Lactobacillus plantarum, a major commensal partner of Drosophila, promote host juvenile growth and maturation partly through enhanced expression of intestinal peptidases. By screening a transposon insertion library of Lactobacillus plantarum in gnotobiotic Drosophila larvae, we identify a bacterial cell-wall-modifying machinery encoded by the pbpX2-dlt operon that is critical to enhance host digestive capabilities and promote animal growth and maturation. Deletion of this operon leads to bacterial cell wall alteration with a complete loss of d-alanylation of teichoic acids. We show that L. plantarum cell walls bearing d-alanylated teichoic acids are directly sensed by Drosophila enterocytes to ensure optimal intestinal peptidase expression and activity, juvenile growth and maturation during chronic undernutrition. We thus conclude that besides peptidoglycan, teichoic acid modifications participate in the host–commensal bacteria molecular dialogue occurring in the intestine. Under nutritional limitation, modification of the Lactobacillus plantarum cell wall by d-alanylation of teichoic acids is important for host intestinal peptidase expression and consequently growth of the Drosophila host, providing further insights into host–commensal interactions.

We discovered a chromosomal locus containing 2 toxin-antitoxin modules (TAs) with an antisense transcriptional organization in the E. faecalis clinical isolate V583. These TAs are homologous to the type I txpA-ratA system and the type II mazEF, respectively. We have shown that the putative MazF is toxic for E. coli and triggers RNA degradation, and its cognate antitoxin MazE counteracts toxicity. The second module, adjacent to mazEF, expresses a toxin predicted to belong to the TxpA type I family found in Firmicutes, and the antisense RNA antidote, RatA. Genomic analysis indicates that the cis-association of mazEF and txpA-ratA modules has been favored during evolution, suggesting a selective advantage for this TA organization in the E. faecalis species. We showed regulatory interplays between the 2 modules, involving transcription control and RNA stability. Remarkably, our data reveal that MazE and MazEF have a dual transcriptional activity: they act as autorepressors and activate ratA transcription, most likely in a direct manner. RatA controls txpA RNA levels through stability. Our data suggest a pivotal role of MazEF in the coordinated expression of mazEF and txpA-ratA modules in V583. To our knowledge, this is the first report describing a crosstalk between type I and II TAs.

Enterococcus faecalis is a commensal bacterium of the human intestine and a major opportunistic pathogen in immunocompromised and elderly patients. The pathogenesis of E. faecalis infection relies in part on its capacity to colonize the gut. Following disruption of intestinal homeostasis, E. faecalis can overgrow, cross the intestinal barrier, and enter the lymph and bloodstream. To identify and characterize E. faecalis genes that are key to intestinal colonization, our strategy consisted in screening mutants for the following phenotypes related to intestinal lifestyle: antibiotic resistance, overgrowth, and competition against microbiota. From the identified colonization genes, epaX encodes a glycosyltransferase located in a variable region of the enterococcal polysaccharide antigen (epa) locus. We demonstrated that EpaX acts on sugar composition, promoting resistance to bile salts and cell wall integrity. Given that EpaX is enriched in hospital-adapted isolates, this study points to the importance of the epa variability as a key determinant for enterococcal intestinal colonization.

Polylysogeny is frequently considered to be the result of an adaptive evolutionary process in which prophages confer fitness and/or virulence factors, thus making them important for evolution of both bacterial populations and infectious diseases. The Enterococcus faecalis V583 isolate belongs to the high-risk clonal complex 2 that is particularly well adapted to the hospital environment. Its genome carries 7 prophage-like elements (V583-pp1 to -pp7), one of which is ubiquitous in the species. In this study, we investigated the activity of the V583 prophages and their contribution to E. faecalis biological traits. We systematically analyzed the ability of each prophage to excise from the bacterial chromosome, to replicate and to package its DNA. We also created a set of E. faecalis isogenic strains that lack from one to all six non-ubiquitous prophages by mimicking natural excision. Our work reveals that prophages of E. faecalis V583 excise from the bacterial chromosome in the presence of a fluoroquinolone, and are able to produce active phage progeny. Intricate interactions between V583 prophages were also unveiled: i) pp7, coined EfCIV583 for E. faecalis chromosomal island of V583, hijacks capsids from helper phage 1, leading to the formation of distinct virions, and ii) pp1, pp3 and pp5 inhibit excision of pp4 and pp6. The hijacking exerted by EfCIV583 on helper phage 1 capsids is the first example of molecular piracy in Gram positive bacteria other than staphylococci. Furthermore, prophages encoding platelet-binding-like proteins were found to be involved in adhesion to human platelets, considered as a first step towards the development of infective endocarditis. Our findings reveal not only a role of E. faecalis V583 prophages in pathogenicity, but also provide an explanation for the correlation between antibiotic usage and E. faecalis success as a nosocomial pathogen, as fluoriquinolone may provoke release of prophages and promote gene dissemination among isolates., Author Summary Enterococcus faecalis is a member of the core-microbiome of the human gastrointestinal tract. In the last decades however, this bacterial species has emerged as a major cause of hospital-acquired infections worldwide. Some isolates are particularly adapted to the hospital environment, and this adaptation was recently linked with enrichment in mobile genetic elements including prophages, which are chromosomal integrated genomes of bacterial viruses. We characterized the biological prophage activity in an E. faecalis strain of clinical origin that harbors 7 prophages. Six active prophages exhibit intricate interactions, one of which is involved in a molecular piracy phenomenon. We also established, for the first time, a direct correlation between prophage and adhesion to human platelets, an initial step towards infective endocarditis. Finally, we showed that fluoroquinolone increases prophage activity and can thus contribute to horizontal gene spreading. Overall, we provide evidence that prophages are key players in E. faecalis evolution towards pathogenicity.

Enterococcus faecalis is a nosocomial opportunistic pathogen, but is also found in fermented food products where it plays a fundamental role in the fermentation process. Previously, we have described the nonstarter E. faecalis cheese isolate QA29b as harboring virulence genes and proven to be virulent in Galleria mellonella virulence model. In this study, we further characterized this food strain concerning traits relevant for the host-pathogen relationship. QA29b was found to belong to sequence type (ST) 72, a common ST among food isolates, and thus we consider it as a good representative of food E. faecalis strains. It demonstrated high ability to form biofilms, to adhere to epithelial cells and was readily eliminated by J774.A1 macrophage cells. Despite carrying the cps locus associated with the capsular polysaccharide CPS 2 type, cps genes were not expressed, likely due to an IS6770 inserted in the cpsC-cpsK promoter region. This work constitutes the first study of traits important for interaction, colonization and infection in the host performed on a good representative of E. faecalis food isolates. Reported results stress the need for a reliable serotyping assay of E. faecalis, as cps genotyping may not be reliable. Overall, QA29b characterization shows that despite its virulence potential in an insect model, this food strain is readily eliminated by mammalian macrophages. Thus, fine tuned approaches combining cellular and mammalian models are needed to address and elucidate the multifactorial aspect of virulence potential associated with food isolates. (C) 2012 Elsevier B.V. All rights reserved.

Bacitracin is used both in clinical practice and as an animal growth promoter in some countries. Recent reports revealed the dissemination of high-level bacitracin resistance (HLBR) in the genus Enterococcus. The bcrABD operon confers HLBR in an Enterococcus faecalis strain, and the presence of the regulator BcrR was stated as essential for this phenotype. Our objectives were to investigate the spread of the bcrABDR genes in the genus Enterococcus and to evaluate the role of this genotype in the HLBR phenotype. Fifty-eight enterococcal isolates from different environments and species were screened for the presence of the bcrABDR genes, and minimum inhibitory concentrations for bacitracin were determined. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to study the expression of bacitracin resistance genes in strains with HLBR following growth in the presence and absence of the antibiotic. The bcrABDR genotype was found associated with E. faecalis strains from clinical settings bearing a HLBR phenotype. The most disseminated mechanism of bacitracin resistance was the BcrAB ABC transporter, encoded by the bcrAB genes. This transporter, which is constitutively expressed in the absence of bcrR, was sufficient to confer HLBR. Our findings stress the need for further evaluation of bacitracin resistance in this genus.

Metazoans establish with microorganisms complex interactions for their mutual benefits. Drosophila, which has already proven useful host model to study several aspects of innate immunity and host-bacteria pathogenic associations has become a powerful model to dissect the mechanisms behind mutualistic host-microbe interactions. Drosophila microbiota is composed of simple and aerotolerant bacterial communities mostly composed of Lactobacillaceae and Acetobactereaceae. Drosophila mono- or poly-associated with lactobacilli strains constitutes a powerful model to dissect the complex interplay between lactobacilli and host biologic traits. Thanks to the genetic tractability of both Drosophila and lactobacilli this association model offers a great opportunity to reveal the underlying molecular mechanisms. Here, we review our current knowledge about how the Drosophila model is helping our understanding of how lactobacilli shapes host biology.