Dag Anders Brede

Comparative genomic analysis of pathogenic and probiotic Enterococcus faecalis isolates, and their transcriptional responses to growth in human urine.

Urinary tract infection (UTI) is the most common infection caused by enterococci, and Enterococcus faecalis accounts for the majority of enterococcal infections. Although a number of virulence related traits have been established, no comprehensive genomic or transcriptomic studies have been conducted to investigate how to distinguish pathogenic from non-pathogenic E. faecalis in their ability to cause UTI. In order to identify potential genetic traits or gene regulatory features that distinguish pathogenic from non-pathogenic E. faecalis with respect to UTI, we have performed comparative genomic analysis, and investigated growth capacity and transcriptome profiling in human urine in vitro. Six strains of different origins were cultivated and all grew readily in human urine. The three strains chosen for transcriptional analysis showed an overall similar response with respect to energy and nitrogen metabolism, stress mechanism, cell envelope modifications, and trace metal acquisition. Our results suggest that citrate and aspartate are significant for growth of E. faecalis in human urine, and manganese appear to be a limiting factor. The majority of virulence factors were either not differentially regulated or down-regulated. Notably, a significant up-regulation of genes involved in biofilm formation was observed. Strains from different origins have similar capacity to grow in human urine. The overall similar transcriptional responses between the two pathogenic and the probiotic strain suggest that the pathogenic potential of a certain E. faecalis strain may to a great extent be determined by presence of fitness and virulence factors, rather than the level of expression of such traits.

Characterization of Garvicin ML, a Novel Circular Bacteriocin Produced by Lactococcus garvieae DCC43, Isolated from Mallard Ducks (Anas platyrhynchos)

ABSTRACT Lactococcus garvieae DCC43 produces a bacteriocin, garvicin ML (GarML), with a molecular mass of 6,004.2 Da. Data from de novo amino acid sequencing by tandem mass spectrometry and nucleotide sequencing by reverse genetics suggested that the bacteriocin is synthesized as a 63-amino-acid precursor with a 3-amino-acid leader peptide that is removed by cleavage. Subsequently, a covalent linkage between the N and C termini forms the mature version of this novel 60-amino-acid circular bacteriocin.

Comparative genomics of Enterococcus faecalis from healthy Norwegian infants

BackgroundEnterococcus faecalis, traditionally considered a harmless commensal of the intestinal tract, is now ranked among the leading causes of nosocomial infections. In an attempt to gain insight into the genetic make-up of commensal E. faecalis, we have studied genomic variation in a collection of community-derived E. faecalis isolated from the feces of Norwegian infants.ResultsThe E. faecalis isolates were first sequence typed by multilocus sequence typing (MLST) and characterized with respect to antibiotic resistance and properties associated with virulence. A subset of the isolates was compared to the vancomycin resistant strain E. faecalis V583 (V583) by whole genome microarray comparison (comparative genomic hybridization (CGH)). Several of the putative enterococcal virulence factors were found to be highly prevalent among the commensal baby isolates. The genomic variation as observed by CGH was less between isolates displaying the same MLST sequence type than between isolates belonging to different evolutionary lineages.ConclusionThe variations in gene content observed among the investigated commensal E. faecalis is comparable to the genetic variation previously reported among strains of various origins thought to be representative of the major E. faecalis lineages. Previous MLST analysis of E. faecalis have identified so-called high-risk enterococcal clonal complexes (HiRECC), defined as genetically distinct subpopulations, epidemiologically associated with enterococcal infections. The observed correlation between CGH and MLST presented here, may offer a method for the identification of lineage-specific genes, and may therefore add clues on how to distinguish pathogenic from commensal E. faecalis. In this work, information on the core genome of E. faecalis is also substantially extended.

The Transcriptome of the Nosocomial Pathogen Enterococcus faecalis V583 Reveals Adaptive Responses to Growth in Blood

Background Enterococcus faecalis plays a dual role in human ecology, predominantly existing as a commensal in the alimentary canal, but also as an opportunistic pathogen that frequently causes nosocomial infections like bacteremia. A number of virulence factors that contribute to the pathogenic potential of E. faecalis have been established. However, the process in which E. faecalis gains access to the bloodstream and establishes a persistent infection is not well understood. Methodology/Principal Findings To enhance our understanding of how this commensal bacterium adapts during a bloodstream infection and to examine the interplay between genes we designed an in vitro experiment using genome-wide microarrays to investigate what effects the presence of and growth in blood have on the transcriptome of E. faecalis strain V583. We showed that growth in both 2xYT supplemented with 10% blood and in 100% blood had a great impact on the transcription of many genes in the V583 genome. We identified several immediate changes signifying cellular processes that might contribute to adaptation and growth in blood. These include modulation of membrane fatty acid composition, oxidative and lytic stress protection, acquisition of new available substrates, transport functions including heme/iron transporters and genes associated with virulence in E. faecalis. Conclusions/Significance The results presented here reveal that cultivation of E. faecalis in blood in vitro has a profound impact on its transcriptome, which includes a number of virulence traits. Observed regulation of genes and pathways revealed new insight into physiological features and metabolic capacities which enable E. faecalis to adapt and grow in blood. A number of the regulated genes might potentially be useful candidates for development of new therapeutic approaches for treatment of E. faecalis infections.

Molecular and Genetic Characterization of a Novel Bacteriocin Locus in Enterococcus avium Isolates from Infants

ABSTRACT Enterococci are among the most common human intestinal lactic acid bacteria, and they are known to produce bacteriocins. In this study, fecal enterococci were isolated from infants and screened for bacteriocin production. Bacteriocin-producing Enterococcus avium isolates were obtained, and a new pediocin-like bacteriocin was purified and characterized. This bacteriocin, termed avicin A, was found to be produced by isolates from two healthy infants. It was purified to homogeneity from culture supernatant by ion-exchange and reversed-phase chromatography, and part of its amino acid sequence was obtained. The sequence of a 7-kb DNA fragment of a bacteriocin locus was determined by PCR and DNA sequencing. The bacteriocin locus was organized into four operon-like structures consisting of (i) the structural genes encoding avicin A and its immunity protein, (ii) a divergicin-like bacteriocin (avicin B) gene, (iii) an ABC bacteriocin transporter gene and two regulatory genes (histamine protein kinase- and response regulator-encoding genes), and (iv) induction peptide pheromone- and transport accessory protein-encoding genes. It was shown that the production of avicin A was regulated by the peptide pheromone-inducible regulatory system. Avicin A shows very high levels of similarity to mundticin KS and enterocin CRL35. This bacteriocin showed strong antimicrobial activity against many species of Gram-positive bacteria, including the food-borne pathogen Listeria monocytogenes. The avicin A locus is the first bacteriocin locus identified in E. avium to be characterized at the molecular level.

The Maltose ABC Transporter in Lactococcus lactis Facilitates High-Level Sensitivity to the Circular Bacteriocin Garvicin ML

ABSTRACT We generated and characterized a series of spontaneous mutants of Lactococcus lactis IL1403 with average 6- to 11-fold-lowered sensitivities to the circular bacteriocin garvicin ML (GarML). Carbohydrate fermentation assays highlighted changes in carbohydrate metabolism, specifically loss of the ability to metabolize starch and maltose, in these mutants. PCR and sequencing showed that a 13.5-kb chromosomal deletion encompassing 12 open reading frames, mainly involved in starch and maltose utilization, had spontaneously occurred in the GarML-resistant mutants. Growth experiments revealed a correlation between sensitivity to GarML and carbon catabolite repression (CCR); i.e., sensitivity to GarML increased significantly when wild-type cells were grown on maltose and galactose as sole carbohydrates, an effect which was alleviated by the presence of glucose. Among the genes deleted in the mutants were malEFG, which encode a CCR-regulated membrane-bound maltose ABC transporter. The complementation of mutants with these three genes recovered normal sensitivity to the bacteriocin, suggesting an essential role of the maltose ABC transporter in the antimicrobial activity of GarML. This notion was supported by the fact that the level of sensitivity to GarML was dose dependent, increasing with higher expression levels of malEFG over a 50-fold range. To our knowledge, this is the first time a specific protein complex has been demonstrated to be involved in sensitivity to a circular bacteriocin.

Comparative genomic analysis reveals significant enrichment of mobile genetic elements and genes encoding surface structure-proteins in hospital-associated clonal complex 2 Enterococcus faecalis

BackgroundEnterococci rank among the leading causes of nosocomial infections. The failure to identify pathogen-specific genes in Enterococcus faecalis has led to a hypothesis where the virulence of different strains may be linked to strain-specific genes, and where the combined endeavor of the different gene-sets result in the ability to cause infection. Population structure studies by multilocus sequence typing have defined distinct clonal complexes (CC) of E. faecalis enriched in hospitalized patients (CC2, CC9, CC28 and CC40).ResultsIn the present study, we have used a comparative genomic approach to investigate gene content in 63 E. faecalis strains, with a special focus on CC2. Statistical analysis using Fishers exact test revealed 252 significantly enriched genes among CC2-strains. The majority of these genes were located within the previously defined mobile elements phage03 (n = 51), efaB5 (n = 34) and a vanB associated genomic island (n = 55). Moreover, a CC2-enriched genomic islet (EF3217 to -27), encoding a putative phage related element within the V583 genome, was identified. From the draft genomes of CC2-strains HH22 and TX0104, we also identified a CC2-enriched non-V583 locus associated with the E. faecalis pathogenicity island (PAI). Interestingly, surface related structures (including MSCRAMMs, internalin-like and WxL protein-coding genes) implicated in virulence were significantly overrepresented (9.1%; p = 0.036, Fishers exact test) among the CC2-enriched genes.ConclusionIn conclusion, we have identified a set of genes with potential roles in adaptation or persistence in the hospital environment, and that might contribute to the ability of CC2 E. faecalis isolates to cause disease.

An antimicrobial peptide is produced by extracellular processing of a protein from Propionibacterium jensenii.

A protease-activated antimicrobial peptide (PAMP) and its inactive precursor were purified from the culture supernatant of Propionibacterium jensenii LMG 3032 and characterized at the molecular level. PAMP is a 64-amino-acid cationic peptide of 6,383 Da with physicochemical features similar to those of bacteriocins from gram-positive bacteria. This peptide displayed bactericidal activity against several propionibacteria and lactobacilli. DNA sequencing indicated that the PAMP-encoding gene (pamA) is translated as a proprotein of 198 amino acids with an N-terminal signal peptide of 27 amino acids and that PAMP constitutes the C-terminal part of this precursor. The amino acid sequence of pro-PAMP showed no similarity to those of other known proteins. By using activity tests and mass spectrometry, we showed that PAMP was formed upon protease treatment of the precursor protein. The propionibacteria produced the PAMP precursor constitutively during growth up to a level of approximately 4 mg/liter, but the producing bacteria were unable to activate the precursor. The requirement for an external protease represents a novel strategy for generating antimicrobial peptides.

Circular Bacteriocins: Biosynthesis and Mode of Action

ABSTRACT Circular bacteriocins are a group of N-to-C-terminally linked antimicrobial peptides, produced by Gram-positive bacteria of the phylum Firmicutes. Circular bacteriocins generally exhibit broad-spectrum antimicrobial activity, including against common food-borne pathogens, such as Clostridium and Listeria spp. These peptides are further known for their high pH and thermal stability, as well as for resistance to many proteolytic enzymes, properties which make this group of bacteriocins highly promising for potential industrial applications and their biosynthesis of particular interest as a possible model system for the synthesis of highly stable bioactive peptides. In this review, we summarize the current knowledge on this group of bacteriocins, with emphasis on the recent progress in understanding circular bacteriocin genetics, biosynthesis, and mode of action; in addition, we highlight the current challenges and future perspectives for the application of these peptides.

Complete genome sequence of the commensal Enterococcus faecalis 62 isolated from a healthy Norwegian infant.

The genome of Enterococcus faecalis 62, a commensal isolate from a healthy Norwegian infant, revealed multiple adaptive traits to the gastrointestinal tract (GIT) environment and the milk-containing diet of breast-fed infants. Adaptation to a commensal existence was emphasized by lactose and other carbohydrate metabolism genes within genomic islands, accompanied by the absence of virulence traits.