IreK-Mediated, Cell Wall-Protective Phosphorylation in Enterococcus faecalis

ABSTRACT Enterococci are opportunistic pathogens and among the leading causes of nosocomial infections. Enterococcus faecalis, the dominant species among infection-derived isolates, has recently been recognized as capable of forming biofilms on abiotic surfaces in vitro as well as on indwelling medical devices. A few bacterial factors known to contribute to biofilm formation in E. faecalis have been characterized. To identify additional factors which may be important to this process, we utilized a Tn917-based insertional mutagenesis strategy to generate a mutant bank in a high-biofilm-forming E. faecalis strain, E99. The resulting mutant bank was screened for mutants exhibiting a significantly reduced ability to form biofilms. One mutant, P101D12, which showed greater than 70% reduction in its ability to form biofilms compared to the wild-type parent, was further characterized. The single Tn917 insertion in P101D12 was mapped to a gene, bee-2, encoding a probable cell wall-anchored protein. Sequence information for the region flanking bee-2 revealed that this gene was a member of a locus (termed the bee locus for biofilm enhancer in enterococcus) comprised of five genes encoding three putative cell wall-anchored proteins and two probable sortases. Contour-clamped homogeneous electric field gel and Southern hybridization analyses suggested that the bee locus is likely harbored on a large conjugative plasmid. Filter mating assays using wild-type E99 or mutant P101D12 as a donor confirmed that the bee locus could transfer conjugally at high frequency to recipient E. faecalis strains. This represents the first instance of the identification of a mobile genetic element conferring biofilm-forming property in E. faecalis.

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Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other IdentifiedEnterococcus faecalisPhage Endolysins

Applied and Environmental Microbiology ◽

10.1128/aem.00555-19 ◽

2019 ◽

Vol 85 (13) ◽

Cited By ~ 7

Author(s):

Hongming Zhang ◽

Bettina A. Buttaro ◽

Derrick E. Fouts ◽

Salar Sanjari ◽

Bradley S. Evans ◽

...

Keyword(s):

Cell Wall ◽

Enterococcus Faecalis ◽

Amino Acid Identity ◽

Lytic Infection ◽

Mass Spectrometry Analysis ◽

Lytic Enzyme ◽

Lytic Enzymes ◽

Content Type ◽

Spectrometry Analysis ◽

Wide Range

ABSTRACTϕEf11 is a temperateSiphoviridaebacteriophage that infects strains ofEnterococcus faecalis. The ϕEf11 genome, encompassing 65 open reading frames (ORFs), is contained within 42,822 bp of DNA. Within this genome, a module of six lysis-related genes was identified. Based upon sequence homology, one of these six genes, ORF28, was predicted to code for anN-acetylmuramoyl-l-alanine amidase endolysin of 46.133 kDa, composed of 421 amino acids. The PCR-amplified ORF28 was cloned and expressed, and the resulting gene product was affinity purified to homogeneity. The purified protein was obtained from a fusion protein that exhibited a molecular mass of 72.5 kDa, consistent with a 46.1-kDa protein combined with a fused 26.5-kDa glutathioneS-transferase tag. It produced rapid, profound lysis inE. faecalispopulations and was active against 73 of 103 (71%)E. faecalisstrains tested. In addition, it caused substantial destruction ofE. faecalisbiofilms. The lysin was quite stable, retaining its activity for three years in refrigerated storage, was stable over a wide range of pHs, and was unaffected by the presence of a reducing agent; however, it was inhibited by increasing concentrations of Ca2+. Liquid chromatography-mass spectrometry analysis ofE. faecaliscell wall digestion products produced by the ORF28 endolysin indicated that the lysin acted as anN-acetylmuramidase, an endo-β-N-acetylglucosaminidase, and an endopeptidase, rather than anN-acetylmuramoyl-l-alanine amidase. The ϕEf11 ORF28 lysin shared 10% to 37% amino acid identity with the lytic enzymes of all other characterizedE. faecalisbacteriophages.IMPORTANCEThe emergence of multidrug-resistant pathogenic microorganisms has brought increasing attention to the urgent need for the development of alternative antimicrobial strategies. One such alternative to conventional antibiotics employs lytic enzymes (endolysins) that are produced by bacteriophages in the course of lytic infection. During lytic infection by a bacteriophage, these enzymes hydrolyze the cell wall peptidoglycan, resulting in the lysis of the host cell. However, external endolysin application can result in lysis from without. In this study, we have cloned, expressed, purified, and characterized an endolysin produced by a bacteriophage infecting strains ofEnterococcus faecalis. The lysin is broadly active against most of the testedE. faecalisstrains and exhibits multifunctional enzymatic specificities that differ from all other characterized endolysins produced byE. faecalisbacteriophages.

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Characterization of Monolaurin Resistance in Enterococcus faecalis

Applied and Environmental Microbiology ◽

10.1128/aem.01013-07 ◽

2007 ◽

Vol 73 (17) ◽

pp. 5507-5515 ◽

Cited By ~ 16

Author(s):

Muriel Dufour ◽

Janet M. Manson ◽

Philip J. Bremer ◽

Jean-Pierre Dufour ◽

Gregory M. Cook ◽

...

Keyword(s):

Cell Wall ◽

Cell Surface ◽

Enterococcus Faecalis ◽

Cell Surface Hydrophobicity ◽

Surface Hydrophobicity ◽

Wall Structure ◽

Transmission Electron ◽

Serious Infections ◽

Autolytic Activity

ABSTRACT There is increasing concern regarding the presence of vancomycin-resistant enterococci in domestically farmed animals, which may act as reservoirs and vehicles of transmission for drug-resistant enterococci to humans, resulting in serious infections. In order to assess the potential for the use of monolaurin as a food preservative, it is important to understand both its target and potential mechanisms of resistance. A Tn917 mutant library of Enterococcus faecalis AR01/DGVS was screened for resistance (MIC, >100 μg/ml) to monolaurin. Three mutants were identified as resistant to monolaurin and were designated DGRM2, DGRM5, and DGRM12. The gene interrupted in all three mutants was identified as traB, which encodes an E. faecalis pheromone shutdown protein and whose complementation in trans restored monolaurin sensitivity in all three mutants. DGRM2 was selected for further characterization. E. faecalis DGRM2 showed increased resistance to gentamicin and chloramphenicol (inhibitors of protein synthesis), while no difference in the MIC was observed with the cell wall-active antibiotics penicillin and vancomycin. E. faecalis AR01/DGVS and DGRM2 were shown to have similar rates (30% cell lysis after 4 h) of cell autolytic activity when activated by monolaurin. Differences in cell surface hydrophobicity were observed between the wild type and the mutant, with the cell surface of the parent strain being significantly more hydrophobic. Analysis of the cell wall structure of DGRM2 by transmission electron microscopy revealed an increase in the apparent cell wall thickness and contraction of its cytoplasm. Taken together, these results suggest that the increased resistance of DGRM2 was due to a change in cell surface hydrophobicity, consequently limiting the diffusion of monolaurin to a potential target in the cytoplasmic membrane and/or cytoplasm of E. faecalis.

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Detection of opsonic antibodies against Enterococcus faecalis cell wall carbohydrates in immune globulin preparations

Infection ◽

10.1007/s15010-014-0628-y ◽

2014 ◽

Vol 42 (4) ◽

pp. 749-755 ◽

Cited By ~ 1

Author(s):

M. Hufnagel ◽

K. Sixel ◽

F. Hammer ◽

A. Kropec ◽

I. G. Sava ◽

...

Keyword(s):

Cell Wall ◽

Enterococcus Faecalis ◽

Immune Globulin ◽

Cell Wall Carbohydrates

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Bactericidal Activity of Gentamicin againstEnterococcus faecalis In Vitro and In Vivo

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.8.2077-2080.2000 ◽

2000 ◽

Vol 44 (8) ◽

pp. 2077-2080 ◽

Cited By ~ 9

Author(s):

Agnès Lefort ◽

Michel Arthur ◽

Louis Garry ◽

Claude Carbon ◽

Patrice Courvalin ◽

...

Keyword(s):

Cell Wall ◽

Enterococcus Faecalis ◽

Bactericidal Activity ◽

Susceptibility Testing ◽

Rabbit Model ◽

Intrinsic Activity ◽

Resistant Mutants ◽

Number Of Bacteria

ABSTRACT The activity of gentamicin at various concentrations against two strains of Enterococcus faecalis was investigated in vitro and in a rabbit model of aortic endocarditis. In vitro, gentamicin at 0.5 to 4 times the MIC failed to reduce the number of bacteria at 24 h. Rabbit or human serum dramatically increased gentamicin activity, leading to a ≥3-log10 CFU/ml decrease in bacterial counts when the drug concentration exceeded the MIC. Susceptibility testing in the presence of serum was predictive of in vivo activity, since gentamicin alone significantly reduced the number of surviving bacteria in the vegetations if the peak-to-MIC ratio was greater than 1. However, gentamicin selected resistant mutants in rabbits. The intrinsic activity of gentamicin should be taken into account in evaluation of combinations of gentamicin and cell wall-active agents against enterococci.

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Cell-Associated Pheromone Peptide (cCF10) Production and Pheromone Inhibition in Enterococcus faecalis

Journal of Bacteriology ◽

10.1128/jb.182.17.4926-4933.2000 ◽

2000 ◽

Vol 182 (17) ◽

pp. 4926-4933 ◽

Cited By ~ 49

Author(s):

B. A. (Leonard) Buttaro ◽

M. H. Antiporta ◽

G. M. Dunny

Keyword(s):

Cell Wall ◽

Protease Inhibitor ◽

Enterococcus Faecalis ◽

Cell Envelope ◽

Conjugative Plasmid ◽

Cell Fractionation ◽

Cell Wall Fraction ◽

Donor Cells ◽

Pheromone Activity ◽

A Cell

ABSTRACT In Enterococcus faecalis, the peptide cCF10 acts as a pheromone, inducing transfer of the conjugative plasmid pCF10 from plasmid-containing donor cells to plasmid-free recipient cells. In these studies, it was found that a substantial amount of cCF10 associates with the envelope of the producing cell. Pheromone activity was detected in both wall and membrane fractions, with the highest activity associated with the wall. Experiments examining the effects of protease inhibitor treatments either prior to or following cell fractionation suggested the presence of a cell envelope-associated pro-cCF10 that can be processed to mature cCF10 by a maturase or protease. A pCF10-encoded membrane protein, PrgY, was shown to prevent self-induction of donor cells by reducing the level of pheromone activity in the cell wall fraction.

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Molecular Basis for Lytic Bacteriophage Resistance in Enterococci

mBio ◽

10.1128/mbio.01304-16 ◽

2016 ◽

Vol 7 (4) ◽

Cited By ~ 39

Author(s):

Breck A. Duerkop ◽

Wenwen Huo ◽

Pooja Bhardwaj ◽

Kelli L. Palmer ◽

Lora V. Hooper

Keyword(s):

Cell Wall ◽

Mouse Model ◽

Enterococcus Faecalis ◽

Molecular Basis ◽

Bacterial Infections ◽

Hypervariable Region ◽

Cell Wall Protein ◽

Phage Resistance ◽

Human Intestine

ABSTRACT The human intestine harbors diverse communities of bacteria and bacteriophages. Given the specificity of phages for their bacterial hosts, there is growing interest in using phage therapies to combat the rising incidence of multidrug-resistant bacterial infections. A significant barrier to such therapies is the rapid development of phage-resistant bacteria, highlighting the need to understand how bacteria acquire phage resistance in vivo . Here we identify novel lytic phages in municipal raw sewage that kill Enterococcus faecalis , a Gram-positive opportunistic pathogen that resides in the human intestine. We show that phage infection of E. faecalis requires a predicted integral membrane protein that we have named PIP EF (for phage infection protein from E. faecalis ). We find that PIP EF is conserved in E. faecalis and harbors a 160-amino-acid hypervariable region that determines phage tropism for distinct enterococcal strains. Finally, we use a gnotobiotic mouse model of in vivo phage predation to show that the sewage phages temporarily reduce E. faecalis colonization of the intestine but that E. faecalis acquires phage resistance through mutations in PIP EF . Our findings define the molecular basis for an evolutionary arms race between E. faecalis and the lytic phages that prey on them. They also suggest approaches for engineering E. faecalis phages that have altered host specificity and that can subvert phage resistance in the host bacteria. IMPORTANCE Bacteriophage therapy has received renewed attention as a potential solution to the rise in antibiotic-resistant bacterial infections. However, bacteria can acquire phage resistance, posing a major barrier to phage therapy. To overcome this problem, it is necessary to understand phage resistance mechanisms in bacteria. We have unraveled one such resistance mechanism in Enterococcus faecalis , a Gram-positive natural resident of the human intestine that has acquired antibiotic resistance and can cause opportunistic infections. We have identified a cell wall protein hypervariable region that specifies phage tropism in E. faecalis . Using a gnotobiotic mouse model of in vivo phage predation, we show that E. faecalis acquires phage resistance through mutations in this cell wall protein. Our findings define the molecular basis for lytic phage resistance in E. faecalis . They also suggest opportunities for engineering E. faecalis phages that circumvent the problem of bacterial phage resistance.

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Enterolysin A, a Cell Wall-Degrading Bacteriocin from Enterococcus faecalis LMG 2333

Applied and Environmental Microbiology ◽

10.1128/aem.69.5.2975-2984.2003 ◽

2003 ◽

Vol 69 (5) ◽

pp. 2975-2984 ◽

Cited By ~ 118

Author(s):

Trine Nilsen ◽

Ingolf F. Nes ◽

Helge Holo

Keyword(s):

Amino Acids ◽

Cell Wall ◽

Amino Acid ◽

Enterococcus Faecalis ◽

Antimicrobial Protein ◽

Terminal Part ◽

Calculated Molecular Weight ◽

Solid Media ◽

Dependent Signal ◽

Modular Structures

ABSTRACT A novel antimicrobial protein, designated enterolysin A, was purified from an Enterococcus faecalis LMG 2333 culture. Enterolysin A inhibits growth of selected enterococci, pediococci, lactococci, and lactobacilli. Antimicrobial activity was initially detected only on solid media, but by growing the bacteria in a fermentor under optimized production conditions (MRS broth with 4% [wt/vol] glucose, pH 6.5, and a temperature between 25 and 35°C), the bacteriocin activity was increased to 5,120 bacteriocin units ml−1. Enterolysin A production was regulated by pH, and activity was first detected in the transition between the logarithmic and stationary growth phases. Killing of sensitive bacteria by enterolysin A showed a dose-response behavior, and the bacteriocin has a bacteriolytic mode of action. Enterolysin A was purified, and the primary structure was determined by combined amino acid and DNA sequencing. This bacteriocin is translated as a 343-amino-acid preprotein with an sec-dependent signal peptide of 27 amino acids, which is followed by a sequence corresponding to the N-terminal part of the purified protein. Mature enterolysin A consists of 316 amino acids and has a calculated molecular weight of 34,501, and the theoretical pI is 9.24. The N terminus of enterolysin A is homologous to the catalytic domains of different cell wall-degrading proteins with modular structures. These include lysostaphin, ALE-1, zoocin A, and LytM, which are all endopeptidases belonging to the M37 protease family. The N-terminal part of enterolysin A is linked by a threonine-proline-rich region to a putative C-terminal recognition domain, which shows significant sequence identity to two bacteriophage lysins.

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