Effect of Lactobacillus salivarius Bacteriocin Abp118 on the Mouse and Pig Intestinal Microbiota

Lactobacilli are Gram-positive bacteria that are a subdominant element in the human gastrointestinal microbiota, and which are commonly used in the food industry. Some lactobacilli are considered probiotic, and have been associated with health benefits. However, there is very little culture-independent information on how consumed probiotic microorganisms might affect the entire intestinal microbiota. We therefore studied the impact of the administration of Lactobacillus salivarius UCC118, a microorganism well characterized for its probiotic properties, on the composition of the intestinal microbiota in two model animals. UCC118 has anti-infective activity due to production of the bacteriocin Abp118, a broad-spectrum class IIb bacteriocin, which we hypothesized could impact the microbiota. Mice and pigs were administered wild-type (WT) L. salivarius UCC118 cells, or a mutant lacking bacteriocin production. The microbiota composition was determined by pyrosequencing of 16S rRNA gene amplicons from faeces. The data show that L. salivarius UCC118 administration had no significant effect on proportions of major phyla comprising the mouse microbiota, whether the strain was producing bacteriocin or not. However, L. salivarius UCC118 WT administration led to a significant decrease in Spirochaetes levels, the third major phylum in the untreated pig microbiota. In both pigs and mice, L. salivarius UCC118 administration had an effect on Firmicutes genus members. This effect was not observed when the mutant strain was administered, and was thus associated with bacteriocin production. Surprisingly, in both models, L. salivarius UCC118 administration and production of Abp118 had an effect on Gram-negative microorganisms, even though Abp118 is normally not active in vitro against this group of microorganisms. Thus L. salivarius UCC118 administration has a significant but subtle impact on mouse and pig microbiota, by a mechanism that seems at least partially bacteriocin-dependent

Identification et caractérisation d'Ers, régulateur transcriptionnel impliqué dans la virulence d'Enterocccus faecalis

Ers a été identifié comme un régulateur transcriptionnel chez Enterococcus faecalis, une bactérie lactique responsable d infections nosocomiales. Cette protéine fait partie de la branche PrfA des régulateurs de la famille Crp/Fnr et serait, chez E. faecalis, l homologue de PrfA, régulateur majeur de la virulence chez Listeria monocytogenes. Elle possède en effet des similitudes structurales avec les protéines de cette famille et en particulier avec PrfA. De plus, on retrouve une séquence similaire de la boîte PrfA dans la région promotrice d ers. Celle-ci a été montrée comme étant incluse dans le segment d ADN reconnu par Ers. D autre part, cette protéine est impliquée dans la survie de la bactérie au stress oxydatif et dans les macrophages. Un mutant deltaers est, de plus, moins virulent que la souche sauvage dans un modèle murin de péritonite. Ers joue également un rôle dans le métabolisme de l arginine, du citrate et du glycérol. Les membres actuellement connus du régulon Ers sont les gènes ef0082 (codant un transporteur du glycérol), arcABC (codant des enzymes du métabolisme de l arginine), ef1459 (codant une protéine hypothétique), citF (codant la sous-unité alpha de la citrate lyase), ace (codant une adhésine impliquée dans la virulence) et enfin glpKOF (codant des protéines jouant un rôle dans le catabolisme du glycérol). L ensemble de ces résultats révèle qu Ers est un régulateur pléiotrope impliqué dans la virulence et le métabolisme cellulaire chez E. faecalis.Ers is a transcriptional regulator of Enterococcus faecalis, a lactic acid bacterium more and more involved in hospital acquired infection. It is part of the PrfA branch of the Crp/Fnr family regulators and may exert a PrfA-like activity in E. faecalis. This protein shares strong structural homologies with proteins of this family including PrfA, the major regulator of virulence in Listeria monocytogenes. Moreover, a sequence similar to the PrfA-box is found in the promoter regions of ers and it has been shown to be included in the DNA segment recognized by Ers. Furthermore, this protein is important for the survival of this opportunistic pathogen towards oxidative stress and within macrophages. In addition, in a mouse peritonitis model of virulence, the deltaers mutant appeared significantly less lethal than the JH2-2 wild type strain. Ers is also involved in the arginine, citrate and glycerol metabolism. At the present time, the Ers regulon is composed of ef0082 encoding a glycerol transporter, arcABC, encoding enzymes of the arginine deiminase system, ef1459 encoding a hypothetical protein, citF, encoding the alpha subunit of the citrate lyase involved in the citrate catabolism, ace, an adhesin involved in virulence and finally glpKOF, encoding two glycerol degradation enzymes, GlpK and GlpO, and a glycerol uptake facilitator, GplF. Taken together, these results indicate that the regulator Ers has a pleiotropic effect, especially in the cellular metabolism and in the virulence of E. faecalisCAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF

Ers Controls Glycerol Metabolism in Enterococcus faecalis

International audienceErs is a pleiotropic transcriptional regulator in Enterococcus faecalis, an opportunistic bacterium. The authors have already shown that this protein is important for survival against oxidative stress and within macrophages as well as for survival of mice, and that Ers also is involved in the regulation of citrate and arginine metabolisms. The current study highlights the involvement of Ers also in the regulation of glycerol metabolism. The results suggest that EF0082, a known member of the Ers regulon encoding a major facilitator family transporter, may be able to transport glycerol. Moreover, the study demonstrates that Ers acts as a positive regulator of the glpKOF operon encoding glycerol kinase, glycerol-3-phosphate oxidase, and glycerol transport facilitator proteins

Enterococcus faecalis Maltodextrin Gene Regulation by Combined Action of Maltose Gene Regulator MalR and Pleiotropic Regulator CcpA

International audienceEnterococcus faecalis represents a leading cause of hospital-acquired infections worldwide. Several studies highlighted the importance of carbohydrate metabolism in the infection process of this bacterium. The genes required for maltodextrin metabolism are particularly induced during mouse infection and, therefore, should play an important role for pathogenesis. Since no data were hitherto available concerning the regulation of expression of the maltodextrin operons, we have conducted experiments to study the underlying mechanisms

Identification of the general stress stimulon related to colonization in Enterococcus faecalis

International audienceEnterococcus faecalis has to cope with major stress conditions during colonization. To understand the effects of stress encountered during infection, the present study assessed the transcriptomic response of the bacteria facing exposure to serum, urine, bile salts, acid pH, or oxidative stress. Compared to non-stressed culture, 30% of the E. faecalis genes were differentially expressed. The transcriptome analysis reveals common but also specific responses, depending on stresses encountered: thus, urine exposure has the most important impact, and the highest number of genes with modified expression is involved in transport and metabolism. The results also pinpoint many stress-related sRNA or intergenic regions not yet characterized. This study identified the general stress stimulon related to infection: when the commensal bacterium initiates its response to stress related to infection, it increases its ability to survive to rough conditions for colonization, rather than promoting expression of virulence factors, and becomes this opportunistic pathogen that thrives in hospital settings

Ers a Crp/Fnr-like transcriptional regulator of Enterococcus faecalis

International audienceErs has been identified in a recent study as a protein involved in the pathogenesis and the stress response of the lactic acid bacterium Enterococcus faecalis, an opportunistic pathogen. In the E. faecalis sequenced genome, Ers is annotated as a transcriptional regulator member of the Crp/Fnr family. This protein has been shown to be involved in the oxidative stress response as well as in the survival within macrophages. In the present study, we sum up the characteristics of Ers and provide further evidence that this protein is a member of the PrfA branch of this regulator family. These features emphasize the importance of studying Ers since PrfA is the major regulator of virulence in Listeria monocytogenes. Ers shares common Crp/Fnr family characteristics, including a HTH motif, a cyclic nucleotide binding domain and conserved amino acid residues. Furthermore, a “PrfA-box-like” sequence has been identified in the ers promoter region. A similar sequence is present in the ef0082 promoter, a gene known to be a member of the Ers regulon. Moreover, ers shares the same genetic neighborhood as other PrfA-like proteins, present in Gram positive bacteria. Lastly, by comparison with PrfA, we have identified an amino acid substitution in the Ers sequence. Such a substitution could imply that Ers is in a constitutively active form

Characterization of the Ers Regulon of Enterococcus faecalis▿

Ers has been qualified as the PrfA-like transcriptional regulator of Enterococcus faecalis. In a previous study we reported that Ers is important for the survival within macrophages of this opportunist pathogenic bacterium. In the present work we have used proteomic and microarray expression profiling of E. faecalis JH2-2 and an ers-deleted mutant (Δers mutant) strains to define the Ers regulon. In addition to EF_0082 (encoding a putative facilitator family transporter), already known to be under Ers regulation, three genes or operons displayed a significant decrease (confirmed by reverse transcription quantitative PCR) in expression in the Δers mutant. The first locus corresponds to three genes: arcA, arcB, and arcC1 (arcABC). These genes are members of the ADI operon, encoding enzymes of the arginine deiminase system. The second is the EF_1459 gene, which encodes a hypothetical protein and is located within a putative phage genetic element. Lastly, Ef_3319 is annotated as the alpha subunit of the citrate lyase encoded by citF. citF is a member of a putative 12-gene operon involved in citrate catabolism. Moreover, the promoter sequence, similar to the “PrfA box” and found in the promoter regions of ers and EF_0082, has been shown to be included in the DNA segment recognized by Ers. Phenotypic analysis of the Δers mutant strain revealed a growth defect when cultured with arginine or citrate as the energy source; this was not seen for the wild type. As expected, similar results were obtained with mutants in which arcA and citF were inactivated. In addition, in the mouse peritonitis model of virulence, the Δers mutant appeared significantly less lethal than the JH2-2 wild-type strain. Taken together, these results indicate that the regulator Ers has a pleiotropic effect, especially in the cellular metabolism and virulence of E. faecalis

ace, Which Encodes an Adhesin in Enterococcus faecalis, Is Regulated by Ers and Is Involved in Virulence▿

Enterococcus faecalis is an opportunistic pathogen that causes numerous infectious diseases in humans and is a major agent of nosocomial infections. In this work, we showed that the recently identified transcriptional regulator Ers (PrfA like), known to be involved in the cellular metabolism and the virulence of E. faecalis, acts as a repressor of ace, which encodes a collagen-binding protein. We characterized the promoter region of ace, and transcriptional analysis by reverse transcription-quantitative PCR and mobility shift protein-DNA binding assays revealed that Ers directly regulates the expression of ace. Transcription of ace appeared to be induced by the presence of bile salts, probably via the deregulation of ers. Moreover, with an ace deletion mutant and the complemented strain and by using an insect (Galleria mellonella) virulence model, as well as in vivo-in vitro murine macrophage models, we demonstrated for the first time that Ace can be considered a virulence factor for E. faecalis. Furthermore, animal experiments revealed that Ace is also involved in urinary tract infection by E. faecalis

Enzymes required for maltodextrin catabolism in Enterococcus faecalis exhibit novel activities

Maltose and maltodextrins are formed during the degradation of starch or glycogen. Maltodextrins are composed of a mixture of maltooligosaccharides formed by α-1,4- but also some α-1,6-linked glucosyl residues. The α-1,6-linked glucosyl residues are derived from branching points in the polysaccharides. In Enterococcus faecalis, maltotriose is mainly transported and phosphorylated by a phosphoenolpyruvate:carbohydrate phosphotransferase system. The formed maltotriose-6α-phosphate is intracellularly dephosphorylated by a specific phosphatase, MapP. In contrast, maltotetraose and longer maltooligosaccharides up to maltoheptaose are taken up without phosphorylation via the ATP binding cassette transporter MdxEFG-MsmX. We show that the maltose-producing maltodextrin hydrolase MmdH (GenBank accession no. EFT41964) in strain JH2-2 catalyzes the first catabolic step of α-1,4-linked maltooligosaccharides. The purified enzyme converts even-numbered α-1,4-linked maltooligosaccharides (maltotetraose, etc.) into maltose and odd-numbered (maltotriose, etc.) into maltose and glucose. Inactivation of mmdH therefore prevents the growth of E. faecalis on maltooligosaccharides ranging from maltotriose to maltoheptaose. Surprisingly, MmdH also functions as a maltogenic α-1,6-glucosidase, because it converts the maltotriose isomer isopanose into maltose and glucose. In addition, E. faecalis contains a glucose-producing α-1,6- specific maltodextrin hydrolase (GenBank accession no. EFT41963, renamed GmdH). This enzyme converts panose, another maltotriose isomer, into glucose and maltose. A gmdH mutant had therefore lost the capacity to grow on panose. The genes mmdH and gmdH are organized in an operon together with GenBank accession no. EFT41962 (renamed mmgT). Purified MmgT transfers glucosyl residues from one α-1,4-linked maltooligosaccharide molecule to another. For example, it catalyzes the disproportionation of maltotriose by transferring a glucosyl residue to another maltotriose molecule, thereby forming maltotetraose and maltose together with a small amount of maltopentaose.Fil: Joyet, Philippe. Université Paris-Saclay; FranciaFil: Mokhtari, Abdelhamid. Université Paris-Saclay; Francia. 8 May 1945 University; ArgeliaFil: Riboulet-Bisson, Eliette. Normandie University; FranciaFil: Blancato, Victor Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Espariz, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Magni, Christian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Hartke, Axel. Normandie University; FranciaFil: Deutscher, Josef. Université Paris-Saclay; Francia. Centre National de la Recherche Scientifique; FranciaFil: Sauvageot, Nicolas. Normandie University; Franci