scholarly journals Putative Surface Proteins Encoded within a Novel Transferable Locus Confer a High-Biofilm Phenotype to Enterococcus faecalis

2006 ◽  
Vol 188 (6) ◽  
pp. 2063-2072 ◽  
Author(s):  
Preeti M. Tendolkar ◽  
Arto S. Baghdayan ◽  
Nathan Shankar
Keyword(s):  
Cell Wall ◽  
Enterococcus Faecalis ◽  
Insertional Mutagenesis ◽  
Surface Proteins ◽  
Conjugative Plasmid ◽  
Sequence Information ◽  
Wild Type ◽  
Abiotic Surfaces ◽  
Mutant Bank

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.

scholarly journals A genomic and proteomic analysis of surface proteins in bovine-adapted lineages of Staphylococcus aureus

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Shauna D. Drumm ◽  
Rebecca Owens ◽  
Jennifer Mitchell ◽  
Orla M. Keane
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Surface Proteins ◽  
Host Cells ◽  
Mass Spectrometry Analysis ◽  
In Vitro Assays ◽  
Immune Recognition ◽  
Independent Manner ◽  
Genes Encoding

In Ireland, Staphylococcus aureus is the most common cause of intramammary infection (IMI) in cattle with the bovine-adapted lineages CC151 and CC97 most commonly found. Surface proteins play a major role in establishing and maintaining the infection. A previous study revealed that a strain from the CC151 lineage showed significant decay in genes encoding predicted surface proteins. Twenty-three S. aureus strains, twelve belonging to CC151 and eleven belonging to CC97, isolated from clinical IMI, were sequenced and genes encoding cell wall anchored (CWA) proteins predicted. Analysis showed that a minority of genes encoding putative CWA proteins were intact in the CC151 strains compared to CC97. Of the 26 known CWA proteins in S. aureus, the CC151 strains only encoded 10 intact genes while CC97 encoded on average 18 genes. Also within the CC97 lineage, the repertoire of genes varied depending on individual strains, with strains encoding between 17-20 intact genes. Although CC151 is reported to internalize within bovine host cells, it does so in a fibronectin-binding protein (FnBPA and FnBPB) independent manner. In-vitro assays were performed and results showed that strains from CC151, and surprisingly also CC97, weakly bound bovine fibronectin and that the FnBPs were poorly expressed in both these lineages. Mass spectrometry analysis of cell wall extracts revealed that SdrE and AdsA were the most highly expressed CWA proteins in both lineages. These results demonstrate significant differences between CC151 and CC97 in their repertoire of genes encoding CWA proteins, which may impact immune recognition of these strains and their interactions with host cells.

scholarly journals Recovery of Fusarium oxysporum Fo47 Mutants Affected in Their Biocontrol Activity After Transposition of the Fot1 Element

2002 ◽  
Vol 92 (9) ◽  
pp. 936-945 ◽  
Author(s):  
Sophie Trouvelot ◽  
Chantal Olivain ◽  
Ghislaine Recorbet ◽  
Quirico Migheli ◽  
Claude Alabouvette
Keyword(s):  
Fusarium Oxysporum ◽  
Insertional Mutagenesis ◽  
Wild Type Strain ◽  
Growth Habit ◽  
Antagonistic Activity ◽  
Phenotypic Characterization ◽  
Wild Type ◽  
Biocontrol Activity

To investigate the biocontrol mechanisms by which the antagonistic Fusarium oxysporum strain Fo47 is active against Fusarium wilt, a Fot1 transposon-mediated insertional mutagenesis approach was adopted to generate mutants affected in their antagonistic activity. Ninety strains in which an active Fot1 copy had transposed were identified with a phenotypic assay for excision and tested for their biocontrol activity against F. oxysporum f. sp. lini on flax in greenhouse experiments. Sixteen strains were affected in their capacity to protect flax plants, either positively (more antagonistic than Fo47) or negatively (less antagonistic). The molecular characterization of these mutants confirms the excision of Fot1 and its reinsertion in most of the cases. Moreover, we demonstrate that other transposable elements such as Fot2, impala, and Hop have no transposition activity in the mutant genomes. The phenotypic characterization of these mutants shows that they are affected neither in their in vitro growth habit nor in their competitiveness in soil compared with wild-type strain Fo47. These results show that mutants are not impaired in their saprophytic phase and suggest that the altered biocontrol phenotype should likely be expressed during the interaction with the host plant.

scholarly journals Antibodies to a Surface-Exposed, N-terminal Domain of Aggregation Substance Are Not Protective in the Rabbit Model of Enterococcus faecalis Infective Endocarditis

2001 ◽  
Vol 69 (5) ◽  
pp. 3305-3314 ◽  
Author(s):  
John K. McCormick ◽  
Helmut Hirt ◽  
Christopher M. Waters ◽  
Timothy J. Tripp ◽  
Gary M. Dunny ◽  
...  
Keyword(s):  
Infective Endocarditis ◽  
Enterococcus Faecalis ◽  
Rabbit Model ◽  
Surface Protein ◽  
Conjugative Plasmid ◽  
Aggregation Substance ◽  
Exposed Region ◽  
Correct Sequence

ABSTRACT The aggregation substance (AS) surface protein fromEnterococcus faecalis has been implicated as an important virulence factor for the development of infective endocarditis. To evaluate the role of antibodies specific for Asc10 (the AS protein from the conjugative plasmid pCF10) in protective immunity to infective endocarditis, an N-terminal region of Asc10 lacking the signal peptide and predicted to be surface exposed (amino acids 44 to 331; AS44–331) was cloned with a C-terminal histidine tag translational fusion and expressed fromEscherichia coli. N-terminal amino acid sequencing of the purified protein revealed the correct sequence, and rabbit polyclonal antisera raised against AS44–331 reacted specifically to Asc10 expressed from E. faecalis OG1SSp, but not to other proteins as judged by Western blot analysis. Using these antisera, flow cytometry analysis demonstrated that antibodies to AS44–331 bound to a surface-exposed region of Asc10. Furthermore, antibodies specific for AS44–331were opsonic for E. faecalis expressing Asc10 in vitro but not for cells that did not express Asc10. New Zealand White rabbits immunized with AS44–331 were challenged intravenously withE. faecalis cells constitutively expressing Asc10 in the rabbit model of experimental endocarditis. Highly immune animals did not show significant differences in clearance of organisms from the blood or spleen or in formation of vegetations on the aortic valve, in comparison with nonimmune animals. Although in vivo expression of Asc10 was demonstrated by immunohistochemistry, these experiments provide evidence that immunity to Asc10 does not play a role in protection from experimental infective endocarditis due toE. faecalis and may have important implications for the development of immunological approaches to combat enterococcal endocarditis.

scholarly journals Use of Recombinase-BasedIn VivoExpression Technology To Characterize Enterococcus faecalis Gene Expression during Infection IdentifiesIn Vivo-Expressed Antisense RNAs and Implicates the Protease Eep in Pathogenesis

2011 ◽  
Vol 80 (2) ◽  
pp. 539-549 ◽  
Author(s):  
Kristi L. Frank ◽  
Aaron M. T. Barnes ◽  
Suzanne M. Grindle ◽  
Dawn A. Manias ◽  
Patrick M. Schlievert ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Rabbit Model ◽  
Microscopic Analysis ◽  
Mammalian Host ◽  
Wild Type ◽  
Antisense Transcripts ◽  
Content Type ◽  
Antisense Rnas

ABSTRACTEnterococcus faecalisis a member of the mammalian gastrointestinal microflora that has become a leading cause of nosocomial infections over the past several decades.E. faecalismust be able to adapt its physiology based on its surroundings in order to thrive in a mammalian host as both a commensal and a pathogen. We employed recombinase-basedin vivoexpression technology (RIVET) to identify promoters on theE. faecalisOG1RF chromosome that were specifically activated during the course of infection in a rabbit subdermal abscess model. The RIVET screen identified 249 putativein vivo-activated loci, over one-third of which are predicted to generate antisense transcripts. Three predicted antisense transcripts were detected inin vitro- andin vivo-grown cells, providing the first evidence ofin vivo-expressed antisense RNAs inE. faecalis. Deletions in thein vivo-activated genes that encode glutamate 5-kinase (proB[EF0038]), the transcriptional regulator EbrA (ebrA[EF1809]), and the membrane metalloprotease Eep (eep[EF2380]) did not hinder biofilm formation inin vitroassays. In a rabbit model of endocarditis, the ΔebrAstrain was fully virulent, the ΔproBstrain was slightly attenuated, and the Δeepstrain was severely attenuated. The Δeepvirulence defect could be complemented by the expression of the wild-type gene intrans. Microscopic analysis of early Δeepbiofilms revealed an abundance of small cellular aggregates that were not observed in wild-type biofilms. This work illustrates the use of a RIVET screen to provide information about the temporal activation of genes during infection, resulting in the identification and confirmation of a new virulence determinant in an important pathogen.

scholarly journals Enterococcus faecalis 6-Phosphogluconolactonase Is Required for Both Commensal and Pathogenic Interactions with Manduca sexta

2014 ◽  
Vol 83 (1) ◽  
pp. 396-404 ◽  
Author(s):  
Jonathan F. Holt ◽  
Megan R. Kiedrowski ◽  
Kristi L. Frank ◽  
Jing Du ◽  
Changhui Guan ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Manduca Sexta ◽  
Wild Type ◽  
Genetic Determinants ◽  
Content Type ◽  
Polystyrene Plate ◽  
Insect Gut ◽  
Insight Into

Enterococcus faecalisis a commensal and pathogen of humans and insects. InManduca sexta,E. faecalisis an infrequent member of the commensal gut community, but its translocation to the hemocoel results in a commensal-to-pathogen switch. To investigateE. faecalisfactors required for commensalism, we identifiedE. faecalisgenes that are upregulated in the gut ofM. sextausing recombinase-basedin vivoexpression technology (RIVET). The RIVET screen produced 113 clones, from which we identified 50 genes that are more highly expressed in the insect gut than in culture. The most frequently recovered gene was locus OG1RF_11582, which encodes a 6-phosphogluconolactonase that we designatedpglA. ApglAdeletion mutant was impaired in both pathogenesis and gut persistence inM. sextaand produced enhanced biofilms compared with the wild type in anin vitropolystyrene plate assay. Mutation of four other genes identified by RIVET did not affect persistence in caterpillar guts but led to impaired pathogenesis. This is the first identification of genetic determinants forE. faecaliscommensal and pathogenic interactions withM. sexta. Bacterial factors identified in this model system may provide insight into colonization or persistence in other host-associated microbial communities and represent potential targets for interventions to preventE. faecalisinfections.

scholarly journals The Enterococcal Surface Protein, Esp, Is Involved in Enterococcus faecalis Biofilm Formation

2001 ◽  
Vol 67 (10) ◽  
pp. 4538-4545 ◽  
Author(s):  
Alejandro Toledo-Arana ◽  
Jaione Valle ◽  
Cristina Solano ◽  
Marı́a Jesús Arrizubieta ◽  
Carme Cucarella ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Biofilm Formation ◽  
Insertional Mutagenesis ◽  
Surface Protein ◽  
Structural Similarity ◽  
Abiotic Surfaces ◽  
Polyvinyl Chloride Plastic ◽  
Primary Attachment ◽  
Encoding Gene ◽  
The Relationship

ABSTRACT The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalisisolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on theE. faecalis isolate, insertional mutagenesis ofesp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboringesp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.

scholarly journals Bactericidal Activity of Gentamicin againstEnterococcus faecalis In Vitro and In Vivo

2000 ◽  
Vol 44 (8) ◽  
pp. 2077-2080 ◽  
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.

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

2000 ◽  
Vol 182 (17) ◽  
pp. 4926-4933 ◽  
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.

scholarly journals Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori

2007 ◽  
Vol 189 (24) ◽  
pp. 8914-8921 ◽  
Author(s):  
Anna Skoglund ◽  
Britta Björkholm ◽  
Christina Nilsson ◽  
Anders F. Andersson ◽  
Cecilia Jernberg ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Restriction Enzyme ◽  
Dna Methyltransferase ◽  
Insertional Mutagenesis ◽  
Phase Variation ◽  
Restriction Enzyme Digestion ◽  
Wild Type ◽  
Gene Mutant ◽  
H Pylori

ABSTRACT A large number of genes encoding restriction-modification (R-M) systems are found in the genome of the human pathogen Helicobacter pylori. R-M genes comprise approximately 10% of the strain-specific genes, but the relevance of having such an abundance of these genes is not clear. The type II methyltransferase (MTase) M.HpyAIV, which recognizes GANTC sites, was present in 60% of the H. pylori strains analyzed, whereof 69% were resistant to restriction enzyme digestion, which indicated the presence of an active MTase. H. pylori strains with an inactive M.HpyAIV phenotype contained deletions in regions of homopolymers within the gene, which resulted in premature translational stops, suggesting that M.HpyAIV may be subjected to phase variation by a slipped-strand mechanism. An M.HpyAIV gene mutant was constructed by insertional mutagenesis, and this mutant showed the same viability and ability to induce interleukin-8 in epithelial cells as the wild type in vitro but had, as expected, lost the ability to protect its self-DNA from digestion by a cognate restriction enzyme. The M.HpyAIV from H. pylori strain 26695 was overexpressed in Escherichia coli, and the protein was purified and was able to bind to DNA and protect GANTC sites from digestion in vitro. A bioinformatic analysis of the number of GANTC sites located in predicted regulatory regions of H. pylori strains 26695 and J99 resulted in a number of candidate genes. katA, a selected candidate gene, was further analyzed by quantitative real-time reverse transcription-PCR and shown to be significantly down-regulated in the M.HpyAIV gene mutant compared to the wild-type strain. This demonstrates the influence of M.HpyAIV methylation in gene expression.

scholarly journals Conjugative delivery of CRISPR-Cas9 for the selective depletion of antibiotic-resistant enterococci

2019 ◽  
Author(s):  
Marinelle Rodrigues ◽  
Sara W. McBride ◽  
Karthik Hullahalli ◽  
Kelli L. Palmer ◽  
Breck A. Duerkop
Keyword(s):  
Antibiotic Resistance ◽  
Enterococcus Faecalis ◽  
Resistance Genes ◽  
Bacterial Infections ◽  
Antibiotic Resistance Genes ◽  
Multidrug Resistant ◽  
Conjugative Plasmid ◽  
Antibiotic Resistant ◽  
Resistance Determinants

AbstractThe innovation of new therapies to combat multidrug-resistant (MDR) bacteria is being outpaced by the continued rise of MDR bacterial infections. Of particular concern are hospital-acquired infections (HAIs) recalcitrant to antibiotic therapies. The Gram-positive intestinal pathobiontEnterococcus faecalisis associated with HAIs and some strains are MDR. Therefore, novel strategies to controlE. faecalispopulations are needed. We previously characterized anE. faecalisType II CRISPR-Cas system and demonstrated its utility in the sequence-specific removal of antibiotic resistance determinants. Here we present work describing the adaption of this CRISPR-Cas system into a constitutively expressed module encoded on a pheromone-responsive conjugative plasmid that efficiently transfers toE. faecalisfor the selective removal of antibiotic resistance genes. Usingin vitrocompetition assays, we show that these CRISPR-Cas-encoding delivery plasmids, or CRISPR-Cas antimicrobials, can reduce the occurrence of antibiotic resistance in enterococcal populations in a sequence-specific manner. Furthermore, we demonstrate that deployment of CRISPR-Cas antimicrobials in the murine intestine reduces the occurrence of antibiotic-resistantE. faecalisby several orders of magnitude. Finally, we show thatE. faecalisdonor strains harboring CRISPR-Cas antimicrobials are immune to uptake of antibiotic resistance determinantsin vivo. Our results demonstrate that conjugative delivery of CRISPR-Cas antimicrobials may be adaptable for future deployment from probiotic bacteria for exact targeting of defined MDR bacteria or for precision engineering of polymicrobial communities in the mammalian intestine.ImportanceCRISPR-Cas nucleic acid targeting systems hold promise for the amelioration of multidrug-resistant enterococci, yet the utility of such tools in the context of the intestinal environment where enterococci reside is understudied. We describe the development of a CRISPR-Cas antimicrobial, deployed on a conjugative plasmid, for the targeted removal of antibiotic resistance genes from intestinalEnterococcus faecalis. We demonstrate that CRISPR-Cas targeting reduces antibiotic resistance ofE. faecalisby several orders of magnitude in the intestine. Although barriers exist that influence the penetrance of the conjugative CRISPR-Cas antimicrobial among target recipientE. faecaliscells, the removal of antibiotic resistance genes inE. faecalisupon uptake of the CRISPR-Cas antimicrobial is absolute. In addition, cells that obtain the CRISPR-Cas antimicrobial are immunized against the acquisition of new antibiotic resistance genes. This study suggests a potential path toward plasmid based CRISPR-Cas therapies in the intestine.

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