Lack of PNPase activity in Enterococcus faecalis 14 increases the stability of EntDD14 bacteriocin transcripts.

A mutant deficient in polynucleotide phosphorylase (PNPase) activity was previously constructed in Enterococcus faecalis 14; a strain producing a leaderless two-peptide enterocin DD14 (EntDD14). Here, we examined the impact of the absence of PNPase on the expression and synthesis of EntDD14, at the transcriptional and functional levels. As result, EntDD14 synthesis augmented in line with the growth curve, reaching a two- to fourfold increase in the ΔpnpA mutant compared to the E. faecalis 14 wild-type strain (WT). EntDD14 synthesis has reached its highest level after 9 h of growth in both strains. Notably, high expression level of the ddABCDEFGHIJ cluster was registered in ΔpnpA mutant. Transcriptional and in silico analyses support the existence of ddAB and ddCDEFGHIJ independent transcripts, and analysis of the fate of ddAB and ddCDEFGHIJ mRNAs indicated that the differences in mRNA levels and the high EntDD14 activity are likely due to a better stability of the two transcripts in the ΔpnpA mutant, which should result in a higher translation efficiency of the ddAB EntDD14 structural genes and their other protein determinants. Consequently, this study shows a potential link between the mRNA stability and EntDD14 synthesis, secretion and immunity in a genetic background lacking PNPase.

A Review on Enterocin DD14, the Leaderless Two-Peptide Bacteriocin with Multiple Biological Functions and Unusual Transport Pathway.

Enterocin DD14 (EntDD14) is a two-peptide leaderless bacteriocin (LLB) produced by Enterococcus faecalis 14, a human strain isolated from meconium. Studies performed on EntDD14 enabled it to show its activity against Gram-positive bacteria such as Listeria monocytogenes, Clostridium perfringens, Enterococcus faecalis, and Staphylococcus aureus. EntDD14 was also shown to potentiate the activity of different antibiotics such as erythromycin, kanamycin, and methicillin when assessed against methicillin-resistant Staphylococcus aureus (MRSA) in vitro and in vivo in the NMRI-F holoxenic mouse model. Additionally, EntDD14 has an antiviral activity and decreased the secretion of pro-inflammatory IL-6 and IL-8 in inflamed human intestinal Caco-2 cells. The genome of E. faecalis 14 was sequenced and annotated. Molecular tools such as Bagel4 software enabled us to locate a 6.7kb-EntDD14 cluster. Transport of EntDD14 outside of the cytoplasm was shown to be performed synergistically by a channel composed of two pleckstrin-homology-domain-containing proteins, namely DdE/DdF and the ABC transporter DdGHIJ. This latter could also protect the bacteriocinogenic strain against extracellular EntDD14. Here, we focus on academic data and potential therapeutic issues of EntDD14, as a model of two-peptide LLB.

Advances in Characterizing the Transport Systems of and Resistance to EntDD14, A Leaderless Two-Peptide Bacteriocin with Potent Inhibitory Activity.

Enterocin DD14 (EntDD14) is a two-peptide leaderless bacteriocin produced by the Enterococcus faecalis 14 strain previously isolated from meconium. This bacteriocin is mainly active against Gram-positive bacteria. Leaderless bacteriocins do not undergo post-translational modifications and are therefore immediately active after their synthesis. As a result, the cells that produce such bacteriocins have developed means of protection against them which often involve transport systems. In this and our previous work, we constructed different mutants deleted in the genes involved in the transport functions, thus covering all the supposed components of this transport system, using Listeria innocua ATCC 33090 as the indicator strain to assess the activity of externalized EntDD14. We also assessed the self-resistance of the WT and all its engineered derivative mutants against EntDD14, provided extracellularly, in order to evaluate their self-resistance. The results obtained highlight that the ABC transporter constituted by the DdG, H, I, and J proteins contributes to EntDD14 export as well as resistance to an external supply of EntDD14. Our results also have established the essential role of the DdE and DdF proteins as primary transporters dedicated to the externalization of EntDD14. Moreover, the in silico data showed that DdE and DdF appear to assemble in a formation that forms an essential channel for the exit of EntDD14. This channel DdEF may interact with the ABC transporter DdGHIJ in order to control the flow of bacteriocin across the membrane, although the nature of this interaction remains to be elucidated.

The absence of PNPase activity in Enterococcus faecalis results in alterations of the bacterial cell-wall but induces high proteolytic and adhesion activities.

Enterococci are lactic acid bacteria (LAB) used as starters and probiotics, delineating their positive attributes. Nevertheless, enterococci can be culprit for thousands of infectious diseases, including urinary tract infections, bacteremia and endocarditis. Here, we aim to determine the impact of polynucleotide phosphorylase (PNPase) in the biology of Enterococcus faecalis 14; a human isolate from meconium. Thus, a mutant strain deficient in PNPase synthesis, named ΔpnpA mutant, was genetically obtained. After that, a transcriptomic study revealed a set of 244 genes differentially expressed in the ΔpnpA mutant compared with the wild-type strain, when exploiting RNAs extracted from these strains after 3 and 6 h of growth. Differentially expressed genes include those involved in cell wall synthesis, adhesion, biofilm formation, bacterial competence and conjugation, stress response, transport, DNA repair and many other functions related to the primary and secondary metabolism of the bacteria. Moreover, the ΔpnpA mutant showed an altered cell envelope ultrastructure compared with the WT strain, and is also distinguished by a strong adhesion capacity on eukaryotic cell as well as a high proteolytic activity. This study, which combines genetics, physiology and transcriptomics enabled us to show further biological functions that could be directly or indirectly controlled by the PNPase in E. faecalis 14.

Evidence for the Involvement of Pleckstrin Homology Domain-Containing Proteins in the Transport of Enterocin DD14 (EntDD14); a Leaderless Two-Peptide Bacteriocin.

Bacteriocins synthesis is initiated from an inactive precursor, which is composed of an N-terminal leader peptide attached to a C-terminal pro-peptide. However, leaderless bacteriocins (LLB) do not possess this N-terminal leader peptide nor undergo post-translational modifications. These atypical bacteriocins are observed to be immediately active after their translation in the cytoplasm. However, although considered to be simple, the biosynthetic pathway of LLB remains to be fully understood. Enterocin DD14 (EntDD14) is a two-peptide LLB produced by Enterococcus faecalis 14, which is a strain isolated from meconium. In silico analysis of DNA encoding EntDD14 located a cluster of 10 genes ddABCDEFGHIJ, where ddE and ddF encode the peculiar DdE and DdF proteins, carrying pleckstrin homology (PH) domains. These modules are quite common in Eucarya proteins and are known to be involved in intracellular signaling or cytoskeleton organization. To elucidate their role within the EntDD14 genetic determinants, we constructed deletion mutants of the ddE and ddF genes. As a result, the mutants were unable to export EntDD14 outside of the cytoplasm even though there was a clear expression of structural genes ddAB encoding EntDD14, and genes ddHIJ encoding an ABC transporter. Importantly, in these mutant strains (ΔddE and ΔddF), EntDD14 was detected by mass spectrometry in the intracellular soluble fraction exerting, upon its accumulation, a toxic effect on the producing strain as revealed by cell-counting and confocal microscopy analysis. Taken together, these results clearly indicate that PH domain-containing proteins, such as DdE and DdF, are involved in the transport of the leaderless two-peptide EntDD14.

A Leaderless Two-Peptide Bacteriocin, Enterocin DD14, Is Involved in Its Own Self-Immunity: Evidence and Insights.

Enterocin DD14 (EntDD14) is a two-peptide leaderless bacteriocin produced by Enterococcus faecalis 14, a strain previously isolated from meconium. EntDD14 has a strong antibacterial activity against Gram-positive bacteria. Leaderless bacteriocins, unlike bacteriocins with leader peptides, are immediately active after their translation, and a producing strain has then to develop specific mechanisms to protect both intra and extracellular compartments. The in silico analysis of Ent. faecalis 14 genome allowed to locate downstream of structural ddAB genes, 8 other adjacent genes, designed ddCDEFGHIJ, which collectively may form three operons. To gain insights on immunity mechanisms of Ent. faecalis 14, mutant strains knocked out in ddAB genes encoding bacteriocin precursor peptides (Δbac) and/or ABC transporter (ΔddI) of EntDD14 were constructed and characterized. Importantly, Δbac mutant strains, from which structural ddAB genes were deleted, resulted unable to produce EntDD14 and sensitive to exogenous EntDD14 showing their involvement in the Ent. faecalis 14 immunity system. Moreover, the sensitivity of Δbac mutants appeared not to be associated with the down-regulation of ddCDEFGHIJ gene expression since they were similarly expressed in both Δbac and wild-type strains during the log phase while they were found significantly down-regulated in the Δbac mutant strain after 24 h of growth. Data gathered from this study suggest also the implication of the ABC transporter (ddHIJ) in the active export of EntDD14 but ruled-out its involvement in the primary self-immunity system. Interestingly, non-bacteriocin producing Ent. faecalis JH2-2 cells transformed with ddAB, or ddAB plus genes encoding the ABC transporter (ddAB-HIJ) did not produce EntDD14 and remained sensitive to its action. Of note, trans-complementation of the Δbac mutant strain with these constructions allowed to recover the WT phenotype. To the best of our knowledge, this is the first study delineating the role of the intracellular two-peptide leaderless bacteriocins in their self-immunity.

Clusters of Lactobacillus Strains from Vegetal Origins Are Associated with Beneficial Functions: Experimental Data and Statistical Interpretations.

Nine strains of Lactiplantibacillus plantarum and one strain of Lacticaseibacillus paracasei that were recently isolated from prickly pears, fresh figs and blackberries, which are traditionally and largely consumed fruits in Kabylia (north of Algeria), were studied here for their antagonism and antioxidant properties as well as for production of exopolysaccharides. With respect to their inhibitory properties, these strains were tested against three food representative pathogens including Escherichia coli ATCC 8739, Staphylococcus aureus 2S6 and Listeria monocytogenes 162. The antagonism of these pathogens was attributable to lactic acid production, present in the cell free supernatant, at concentrations ranging from 9 to 16.74 g/L. The anti-adhesive properties observed on polystyrene or eukaryotic Caco-2 cells were exerted in a strain dependent-manner. Indeed, the scores obtained ranged from 27% to 75% for S. aureus 2S6, 54% to 95% for L. monocytogenes 162, and 50% to 97% for E. coli ATCC 8739. The co-aggregation of these Lactobacillus strains with the aforementioned target bacteria appeared to be exerted in a strain-dependent manner, with noticeably the upmost rate for Lb. paracasei FB1 on S. aureus 2S6. Interestingly, these novel Lactobacillus strains were able to produce a large amount (315.55 to 483.22 mg/L) of exopolysaccharides, and showed a significant scavenging activity on the 2,2-di-phényl-2-picrylhydrazyle (DPPH) synthetic free radical with rates of 51% to 56%. Of note, the highest antioxidant activity was observed for Lb. paracasei FB1 using the culture supernatants, intact cells or the intracellular extract. The statistical analysis of these data using the principal component analysis (ACP) enabled us to establish three distinct clusters with potential applications as bioprotective and/or probiotic agents, following further evaluation.

Metabolic Shift of an Isogenic Strain of Enterococcus faecalis 14, Deficient in Its Own Bacteriocin Synthesis, as Revealed by a Transcriptomic Analysis.

The production of antimicrobial molecules often involves complex biological pathways. This study aimed at understanding the metabolic and physiological networks of enterocin EntDD14-associated function, in the bacteriocinogenic strain, Enterococcus faecalis 14. A global and comparative transcriptomic study was carried out on E. faecalis 14 and its isogenic mutant Δbac, inactivated in genes coding for EntDD14. The in vitro ability to form biofilm on polystyrene plates was assessed by the crystal violet method, while the cytotoxicity on human colorectal adenocarcinoma Caco-2 cells was determined by the Cell Counting Kit-8. Transcriptomic data revealed that 71 genes were differentially expressed in both strains. As expected, genes coding for EntDD14 were downregulated in the Δbac mutant, whereas the other 69 genes were upregulated. Upregulated genes were associated with phage-related chromosomal islands, biofilm formation capability, resistance to environmental stresses, and metabolic reprogramming. Interestingly, the Δbac mutant showed an improved bacterial growth, a high capacity to form biofilm on inanimate surfaces and a very weak cytotoxicity level. These multiple metabolic rearrangements delineate a new line of defense to counterbalance the loss of EntDD14.

Abundance of Lactobacillus plantarum Strains with Beneficial Attributes in Blackberries (Rubus sp.), Fresh Figs (Ficus carica), and Prickly Pears (Opuntia ficus-indica) Grown and Harvested in Algeria.

This first study performed on traditional fruits consumed in North Africa reveals their richness in microorganisms with beneficial attributes like cholesterol lowering capabilities. Blackberries (Rubus sp.), fresh figs (Ficus carica), and prickly pears (Opuntia ficus-indica) are fruits largely and traditionally consumed in Kabylia, a beautiful northern Algerian region. Here, 85 lactic acid bacteria (LAB)-isolates were isolated and identified by MALDI-TOF mass spectrometry. The identified species belong to Lactobacillus and Leuconostoc genera. These 85 LAB-isolates were then assessed for their capabilities to grow under conditions mimicking the gastrointestinal tract, and the resulting data were statistically treated with principal component analysis (PCA). After which, only 26 LAB-isolates were selected and characterized for their genetic relatedness using random amplified polymorphic DNA (RAPD) method. Following the genetic relatedness assessment, only 10 LAB-strains, among which nine Lactobacillus plantarum and one Lactobacillus paracasei were studied for their pathoproperties and some probiotic features. Interestingly, all of these 10 LAB-strains were devoid of adverse effects, but capable to adhere to human epithelial colorectal adenocarcinoma Caco-2 cells. Of note, these 10 LAB-strains exhibited an important in vitro hypocholesteromia effect, in strain-dependent manner. Moreover, the Lactobacillus strains exhibited a high bile salt hydrolase (BSH) activity which was correlated with expression of bsh2, bsh3 and bsh4 genes.

High rates of CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae in wild boars and Barbary macaques in Algeria.

OBJECTIVES: The present study aimed to screen for the presence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae in wild boars and Barbary macaques in Béjaïa and Jijel, Algeria. METHODS: A total of 216 faecal samples collected between September 2014 and August 2015 were cultured on MacConkey agar supplemented with 1µg/mL ceftazidime. Isolates were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Antimicrobial susceptibility testing was performed by the disk diffusion method, and ESBLs were characterised by PCR and sequencing. Clonal relatedness was studied by multilocus sequence typing (MLST). RESULTS: A total of 47 ESBL-producing isolates were recovered from faecal samples from 40 (44%) of 90 wild boars and 7 (6%) of 126 from Barbary macaques, including 30 Escherichia coli and 17 Klebsiella pneumoniae. Results of PCR and sequencing analysis showed that all of the isolates produced CTX-M-15, and 25 isolates co-produced TEM-1. MLST demonstrated the presence of eight sequence types (STs) among the E. coli isolates (ST617, ST131, ST648, ST405, ST1431, ST1421, ST69 and ST226), whereas only one clone (ST584) was identified for all isolates of K. pneumoniae recovered from wild boars (n=10) and Barbary macaques (n=7). CONCLUSIONS: This is the first report of CTX-M-15-producing E. coli and K. pneumoniae in wild animals from Algeria. The results show that African wildlife can act as a reservoir of the epidemic E. coli clone ST131 producing CTX-M-15, suggesting that this lineage can survive in different ecological niches and adapt to different hosts.

Loss of Antibiotic Tolerance in Sod-Deficient Mutants Is Dependent on the Energy Source and Arginine Catabolism in Enterococci.

UNLABELLED: Enterococci are naturally tolerant to typically bactericidal cell wall-active antibiotics, meaning that their growth is inhibited but they are not killed even when exposed to a high concentration of the drug. The molecular reasons for this extraordinary tolerance are still incompletely understood. Previous work showed that resistance to killing collapsed specifically in mutants affected in superoxide dismutase (Sod) activity, arguing that bactericidal antibiotic treatment led to induction of a superoxide burst. In the present work, we show that loss of antibiotic tolerance in ΔsodA mutants of pathogenic enterococci is dependent on the energy source present during antibiotic treatment. Hexoses induce greater killing than the pentose ribose, and no killing was observed with glycerol as the energy source. These results point to glycolytic reactions as crucial for antibiotic-mediated killing of ΔsodA mutants. A transposon mutant library was constructed in Enterococcus faecalis ΔsodA mutants and screened for restored tolerance of vancomycin. Partially restored tolerance was observed in mutants with transposon integrations into intergenic regions upstream of regulators implicated in arginine catabolism. In these mutants, the arginine deiminase operon was highly upregulated. A model for the action of cell wall-active antibiotics in tolerant and nontolerant bacteria is proposed. IMPORTANCE: Antibiotic tolerance is a serious clinical concern, since tolerant bacteria have considerably increased abilities to resist killing by bactericidal drugs. Using enterococci as models for highly antibiotic-tolerant pathogens, we showed that tolerance of these bacteria is linked to their superoxide dismutase (Sod), arguing that bactericidal antibiotics induce generation of reactive oxygen species inside cells. Wild-type strains are tolerant because they detoxify these deleterious molecules by the activity of Sod, whereas Sod-deficient strains are killed. This study showed that killing depends on the energy source present during treatment and that an increase in arginine catabolism partially restored tolerance of the Sod mutants. These results are used to propose a mode-of-action model of cell wall-active antibiotics in tolerant and nontolerant bacteria.

Analysis of the tolerance of pathogenic enterococci and Staphylococcus aureus to cell wall active antibiotics.

OBJECTIVES: Tolerance refers to the phenomenon that bacteria do not significantly die when exposed to bactericidal antibiotics. Enterococci are known for their high tolerance to these drugs, but the molecular reasons why they resist killing are not understood. In a previous study we showed that the superoxide dismutase (SOD) is implicated in this tolerance. This conclusion was based on the results obtained with one particular strain of Enterococcus faecalis and therefore the objective of the present communication was to analyse whether dependence of tolerance on active SOD is a general phenomenon for enterococci and another Gram-positive pathogen, Staphylococcus aureus. METHODS: Mutants deficient in SOD activity were constructed in pathogenic enterococci. The wild-type sodA gene was cloned into an expression vector and transformed into SOD-deficient strains for complementation with varying levels of SOD activity. Previously constructed SOD-deficient strains of S. aureus were also included in this study. Tolerance to vancomycin and penicillin was then tested. RESULTS: We demonstrated that the dependence on SOD of tolerance to vancomycin and penicillin is a common trait of antibiotic-susceptible pathogenic enterococci. By varying the levels of expression we could also show that tolerance to vancomycin is directly correlated to SOD activity. Interestingly, deletion of the sodA gene in a non-tolerant Enterococcus faecium strain did not further sensitize the mutant to bactericidal antibiotics. Finally, we showed that the SOD enzymes of S. aureus are also implicated in tolerance to vancomycin. CONCLUSION: High tolerance of enterococci to cell wall active antibiotics can be reversed by SOD deficiency.

The lysozyme-induced peptidoglycan N-acetylglucosamine deacetylase PgdA (EF1843) is required for Enterococcus faecalis virulence.

Lysozyme is a key component of the innate immune response in humans that provides a first line of defense against microbes. The bactericidal effect of lysozyme relies both on the cell wall lytic activity of this enzyme and on a cationic antimicrobial peptide activity that leads to membrane permeabilization. Among Gram-positive bacteria, the opportunistic pathogen Enterococcus faecalis has been shown to be extremely resistant to lysozyme. This unusual resistance is explained partly by peptidoglycan O-acetylation, which inhibits the enzymatic activity of lysozyme, and partly by d-alanylation of teichoic acids, which is likely to inhibit binding of lysozyme to the bacterial cell wall. Surprisingly, combined mutations abolishing both peptidoglycan O-acetylation and teichoic acid alanylation are not sufficient to confer lysozyme susceptibility. In this work, we identify another mechanism involved in E. faecalis lysozyme resistance. We show that exposure to lysozyme triggers the expression of EF1843, a protein that is not detected under normal growth conditions. Analysis of peptidoglycan structure from strains with EF1843 loss- and gain-of-function mutations, together with in vitro assays using recombinant protein, showed that EF1843 is a peptidoglycan N-acetylglucosamine deacetylase. EF1843-mediated peptidoglycan deacetylation was shown to contribute to lysozyme resistance by inhibiting both lysozyme enzymatic activity and, to a lesser extent, lysozyme cationic antimicrobial activity. Finally, EF1843 mutation was shown to reduce the ability of E. faecalis to cause lethality in the Galleria mellonella infection model. Taken together, our results reveal that peptidoglycan deacetylation is a component of the arsenal that enables E. faecalis to thrive inside mammalian hosts, as both a commensal and a pathogen.

Aerobic glycerol dissimilation via the Enterococcus faecalis DhaK pathway depends on NADH oxidase and a phosphotransfer reaction from PEP to DhaK via EIIADha.

Two pathways for glycerol dissimilation are present in Enterococcus faecalis. Either glycerol is first phosphorylated by glycerol kinase and then oxidized by glycerol-3-phosphate oxidase with molecular oxygen as the electron acceptor (GlpO/GlpK pathway), or it is first oxidized by glycerol dehydrogenase with NAD(+) as the acceptor of the reduction equivalents and then phosphorylated by dihydroxyacetone kinase (GldA/DhaK pathway). The final end product in both cases is dihydroxyacetone phosphate (DHAP). The genes of the GldA/DhaK pathway are present in a four-gene operon structure encoding GldA, a small hypothetical protein (EF1359), and two subunits of dihydroxyacetone kinase (DhaK and DhaL). We demonstrate in this study that protein EF1359 is part of a phosphorylation cascade which phosphorylates dihydroxyacetone in a phosphoenolpyruvate (PEP)-dependent reaction via EI, HPr, EF1359 and DhaLK. Furthermore we show that aerobic dissimilation of glycerol via the GldA/DhaK pathway is dependent on active NADH oxidase to regenerate NADH in Ent. faecalis. A refined model of the aerobic metabolism of glycerol via the GldA/DhaK pathway is presented.

The prolipoprotein diacylglyceryl transferase (Lgt) of Enterococcus faecalis contributes to virulence.

Enterococcus faecalis is an opportunistic pathogen responsible for nosocomial infections. Lipoproteins in Gram-positive bacteria are translocated across the plasma membrane and anchored by the fatty acid group. They perform critical roles, with some described as virulence determinants. The aim of this study was to explore the roles of E. faecalis lipoproteins in the stress response and virulence. We constructed a mutant affected in the predicted prolipoprotein diacylglyceryl transferase gene lgt, and examined the role of Lgt in membrane anchoring, growth, the stress response and virulence. Inactivation of lgt enhanced growth in a high concentration of Mn(2+) or under oxidative stress in vitro, and significantly decreased virulence.

Galleria mellonella as a model for studying Enterococcus faecium host persistence.

Enterococcus faecium is an opportunistic pathogen responsible for numerous outbreaks worldwide. The basis for the colonization capacities, host persistence and environmental stress response of the hospital-adapted clones emerging from E. faecium are poorly understood. In this study, we propose the use of Galleria mellonella as a simple nonmammalian model to assess E. faecium host persistence. Various strains (n = 10), including hospital-adapted, commensal or animal isolates and a SodA-deficient strain were used to assess the relevance of this model. Compared to Enterococcus faecalis, E. faecium strains do not appear very lethal in a Galleria killing assay. The ability of E. faecium strains to overcome host-immune responses and multiply within the host system was evaluated by monitoring bacterial loads following Galleria infection. Among the E. faecium strains, two hospital-adapted isolates displayed increased colonization ability. In contrast, inactivation of sodA, encoding a putative manganese-dependent superoxide dismutase, significantly reduced survival of E. faecium to Galleria defenses. Galleria appears to be a suitable and convenient surrogate model to study E. faecium survival to host defenses and the role of suspected virulence factors in the colonization process.