THESEUS1 and RALF34 monitor cell wall integrity

The cell wall is a rigid network being the first barrier between a plant cell and its environment, but at the same time has to be a dynamic network whose cell growth and shape is given by deposition and remodeling of the cell wall. Maintaining cell wall integrity (CWI) is essential for correct plant development and stress response. Members of the family of Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) proteins have been shown to play a role in cell wall homeostasis, mechanoperception, CWI maintenance and growth control. One of the 17 members in Arabidopsis, THESEUS1 (THE1), was identified in a suppressor screen of a cellulose deficient mutant, revealing that the reduction in growth is part of a THE1-mediated compensatory response to cell wall perturbation (Hématy et al., 2007). Interestingly, several CrRLK1L members have been shown to be receptors for Rapid Alkalinisation Factor (RALF) peptides. RALFs are on average 50 amino acids highly basic, cysteine-rich peptides, most of which are predicted to be cleaved from a highly acidic prodomain by a subtilisin protease. Recently, THE1 has been identified as a receptor for RALF34 (Gonneau et al., 2018). However, this peptide might not be the only THE1 ligand, since ralf34 loss-of-function mutants do not phenocopy all aspects of the1 mutants. RALF24 and RALF31 clustered together with RALF34, based on expression values across different tissues. We generated CRISPR/Cas9 mutants on RALF24, RALF31 and RALF34 with the aim to study which THE1 responses depend on these peptides. Our data suggest that RALF34 could not be the ligand for CWI response of THE1. The presence of RALF34 is acting negatively through THE1, inhibiting its response to cell wall damage.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Prevalence and characterization of antibiotic resistant Enterococcus faecalis in French cheeses.

International audiencePrevalence of enterococci and antibiotic resistance profiles of Enterococcus faecalis was analyzed in 126 French cheeses from retail stores. Forty-four percent of pasteurized or thermised-milk cheeses, and up to 92% of raw-milk cheeses contained detectable enterococci. A total of 337 antibiotic resistant enterococci were isolated in 29% and 60% of pasteurized-milk and raw-milk cheeses, respectively. E. faecalis was the predominant antibiotic resistant species recovered (81%), followed by Enterococcus faecium (13%), and Enterococcus durans (6%). The most prevalent antibiotic resistances were tetracycline (Tet) and minocycline (Min), followed by erythromycin (Ery), kanamycin (Kan) and chloramphenicol (Cm). The most common multiple antibiotic resistance phenotype was Cm Ery Kan Min Tet. The occurrence of antibiotic genes, as searched by PCR, was 100 % for aph3'IIIa, 96 % for ermB, 90 % for tetM and 80 % for catA in isolates resistant to Kan, Ery, Tet or Cm, respectively. MLST analysis of 30 multidrug resistant E. faecalis revealed that ST19, CC21, CC25 and CC55 isolates were the most common in cheeses. In conclusion, as in many other European countries, French cheeses do contain enterococci with multiple antibiotics resistances. However, low occurrence of high-level gentamicin resistant or sulfamethoxazole/trimethoprim-resistant enterococci and absence of vancomycin- or ampicillin- resistant enterococci indicate that cheeses cannot be considered as a major reservoir for nosocomial multi-drug resistant enterococci

Role of WAKL receptors in the sensing of PME17 activity

International audienceThe development of bio-control has become a major challenge in agriculture. We have recently characterised that the mutation of the PECTIN METHYL ESTERASE 17 in Arabidopsis ecotype Wassilewskija confers resistance to numerous biotic and abiotic stresses. Our preliminary results suggest that it overproduces phosphorylated polyols potentially derived from the cleavage of membrane lipids and phosphorylated oligosaccharides. In this context, we propose to characterise the role of these newly identified and accumulated carbohydrates in Arabidopsis thaliana mutants able to better adapt to changing environmental conditions. Three axes are developed: (i) a phenotypic characterisation of pme17 in the face of a wide range of stresses, (ii) a detailed characterisation of the carbohydrates produced and the determination of their role in triggering pme17 adaptation mechanisms in the face of these stresses, and (iii) finally the characterisation of a new receptor involved in the regulation of signal transduction. This project should lead to the identification of potential new stimulators of adaptation and plant defences (SDP)

In situ gene expression in cheese matrices: Application to a set of enterococcal genes

International audienceTranscriptional approaches are increasingly used to compare the behaviour of pathogenic and non-pathogenic bacteria in different culture conditions. The purpose of this study was to apply these methods in cheese to better characterize food and clinical Enterococcus faecalis isolates during cheese processing. Because of the complex biochemical composition of the cheese matrix, e.g. the presence of casein and fat, we developed an efficient method to recover total RNA from bacteria in a semi-hard cheese model. To validate the RNA extraction method, we analysed expression of 7 genes from two E. faecalis strains (one clinical and one food isolate) in both cheese and culture medium by semi-quantitative RT-PCR. We then used PCR-based DNA macro-arrays to compare expression of 154 genes from two E. faecalis strains in both cheese and culture medium. The food strain isolated from cheese is transcriptionally active in cheese, as reflected by the higher transcript levels of various genes. Conversely, overall transcript levels of the V583 clinical isolate were lower in cheese, suggesting that the food strain may be more adapted to a dairy environment than the clinical strain. The method described here constitutes a very promising tool for future transcriptomic studies in cheese matrices. Global profiling in foods may prove to be a valid criterion for differentiating food from clinical isolates

The Surface Rhamnopolysaccharide Epa of Enterococcus faecalis Is a Key Determinant of Intestinal Colonization

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

Predicting the influence of climate induced temperature changes on the production of polysaccharides in Arabidopsis seed mucilage

International audienceIn recent years, the climate trend in temperate zones such as Europe and North America has been towards episodes of severe hot weather which are projected to occur more frequently and/or severely over coming decades (IPCC fifth assessment report, 2014). This is predicted to have major effects on ecosystems and the biodiversity of wild species, such as Arabidopsis. Polysaccharides play a key role in plant growth and resistance to stress and how their production will be affected by climate change remains an open question. Mucilage is a hydrogel of polysaccharides formed around imbibed seeds of certain species and is an amenable model system for studying polysaccharide production. We have previously found that natural accessions of Arabidopsis show variation in mucilage production that may be locally adapted to particular climates. To determine how climate change can impact polysaccharide synthesis, we have examined the effect of temperature on the production of seed mucilage polymers. Polysaccharide composition and properties in natural mucilage variants exhibiting divergent traits under standard growth conditions have been compared after seed production at different temperatures. How these are related to the modulation of genes encoding key enzymes in mucilage polysaccharide synthesis will also be presented

Incongruence between the cps type 2 genotype and host-related phenotypes of an Enterococcus faecalis food isolate

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

Rapid alkalinization factor 22 has a structural and signalling role in root hair cell wall assembly

Pressurized cells with strong walls make up the hydrostatic skeleton of plants. Assembly and expansion of such stressed walls depend on a family of secreted RAPID ALKALINIZATION FACTOR (RALF) peptides, which bind both a membrane receptor complex and wall-localized LEUCINE-RICH REPEAT EXTENSIN (LRXs) in a mutually exclusive way. Here we show that, in root hairs, the RALF22 peptide has a dual structural and signalling role in cell expansion. Together with LRX1, it directs the compaction of charged pectin polymers at the root hair tip into periodic circumferential rings. Free RALF22 induces the formation of a complex with LORELEI-LIKE-GPI-ANCHORED PROTEIN 1 and FERONIA, triggering adaptive cellular responses. These findings show how a peptide simultaneously functions as a structural component organizing cell wall architecture and as a feedback signalling molecule that regulates this process depending on its interaction partners. This mechanism may also underlie wall assembly and expansion in other plant cell types.The authors show that RALF22 has a dual role in cell wall assembly in root hairs: as a structural component organizing cell wall architecture and as a feedback signalling molecule that regulates this process depending on its interaction partners

Rapid alkalinization factor 22 has a structural and signalling role in root hair cell wall assembly

Abstract: Pressurized cells with strong walls make up the hydrostatic skeleton of plants. Assembly and expansion of such stressed walls depend on a family of secreted RAPID ALKALINIZATION FACTOR (RALF) peptides, which bind both a membrane receptor complex and wall-localized LEUCINE-RICH REPEAT EXTENSIN (LRXs) in a mutually exclusive way. Here we show that, in root hairs, the RALF22 peptide has a dual structural and signalling role in cell expansion. Together with LRX1, it directs the compaction of charged pectin polymers at the root hair tip into periodic circumferential rings. Free RALF22 induces the formation of a complex with LORELEI-LIKE-GPI-ANCHORED PROTEIN 1 and FERONIA, triggering adaptive cellular responses. These findings show how a peptide simultaneously functions as a structural component organizing cell wall architecture and as a feedback signalling molecule that regulates this process depending on its interaction partners. This mechanism may also underlie wall assembly and expansion in other plant cell types