scholarly journals Multiple Functional Domains of Enterococcus faecalis Aggregation Substance Asc10 Contribute to Endocarditis Virulence

2008 ◽  
Vol 77 (1) ◽  
pp. 539-548 ◽  
Author(s):  
Olivia N. Chuang ◽  
Patrick M. Schlievert ◽  
Carol L. Wells ◽  
Dawn A. Manias ◽  
Timothy J. Tripp ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Bacterial Load ◽  
Rabbit Model ◽  
Surface Proteins ◽  
Functional Domains ◽  
Mammalian Host ◽  
Bacterial Cells ◽  
Wild Type ◽  
Aggregation Substance

ABSTRACT Aggregation substance proteins encoded by sex pheromone plasmids increase the virulence of Enterococcus faecalis in experimental pathogenesis models, including infectious endocarditis models. These large surface proteins may contain multiple functional domains involved in various interactions with other bacterial cells and with the mammalian host. Aggregation substance Asc10, encoded by plasmid pCF10, is induced during growth in the mammalian bloodstream, and pCF10 carriage gives E. faecalis a significant selective advantage in this environment. We employed a rabbit model to investigate the role of various functional domains of Asc10 in endocarditis. The data suggested that the bacterial load of the infected tissue was the best indicator of virulence. Isogenic strains carrying either no plasmid, wild-type pCF10, a pCF10 derivative with an in-frame deletion of the prgB gene encoding Asc10, or pCF10 derivatives expressing other alleles of prgB were examined in this model. Previously identified aggregation domains contributed to the virulence associated with the wild-type protein, and a strain expressing an Asc10 derivative in which glycine residues in two RGD motifs were changed to alanine residues showed the greatest reduction in virulence. Remarkably, this strain and the strain carrying the pCF10 derivative with the in-frame deletion of prgB were both significantly less virulent than an isogenic plasmid-free strain. The data demonstrate that multiple functional domains are important in Asc10-mediated interactions with the host during the course of experimental endocarditis and that in the absence of a functional prgB gene, pCF10 carriage is actually disadvantageous in vivo.

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 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 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 Leishmania mexicana Mutants Lacking Glycosylphosphatidylinositol (GPI):Protein Transamidase Provide Insights into the Biosynthesis and Functions of GPI-anchored Proteins

2000 ◽  
Vol 11 (4) ◽  
pp. 1183-1195 ◽  
Author(s):  
James D. Hilley ◽  
Jody L. Zawadzki ◽  
Malcolm J. McConville ◽  
Graham H. Coombs ◽  
Jeremy C. Mottram
Keyword(s):  
Normal Growth ◽  
Surface Proteins ◽  
Host Cells ◽  
Mammalian Host ◽  
Putative Protein ◽  
Major Class ◽  
Major Surface Proteins ◽  
Major Surface

The major surface proteins of the parasitic protozoonLeishmania mexicana are anchored to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. We have cloned the L. mexicana GPI8 gene that encodes the catalytic component of the GPI:protein transamidase complex that adds GPI anchors to nascent cell surface proteins in the endoplasmic reticulum. Mutants lacking GPI8 (ΔGPI8) do not express detectable levels of GPI-anchored proteins and accumulate two putative protein–anchor precursors. However, the synthesis and cellular levels of other non–protein-linked GPIs, including lipophosphoglycan and a major class of free GPIs, are not affected in the ΔGPI8 mutant. Significantly, the ΔGPI8 mutant displays normal growth in liquid culture, is capable of differentiating into replicating amastigotes within macrophages in vitro, and is infective to mice. These data suggest that GPI-anchored surface proteins are not essential to L. mexicana for its entry into and survival within mammalian host cells in vitro or in vivo and provide further support for the notion that free GPIs are essential for parasite growth.

Identification of secreted and surface proteins from Enterococcus faecalis

2009 ◽  
Vol 55 (8) ◽  
pp. 967-974 ◽  
Author(s):  
Abdellah Benachour ◽  
Thierry Morin ◽  
Laurent Hébert ◽  
Aurélie Budin-Verneuil ◽  
André Le Jeune ◽  
...  
Keyword(s):  
Cell Surface ◽  
Enterococcus Faecalis ◽  
Virulence Factors ◽  
Gel Electrophoresis ◽  
Ion Trap ◽  
Surface Proteins ◽  
Two Dimensional Gel Electrophoresis ◽  
Proteomic Approach ◽  
Associated Proteins

Secreted and surface proteins of bacteria are key molecules that interface the cell with the environment. Some of them, corresponding to virulence factors, have already been described for Enterococcus faecalis , the predominant species involved in enterococcal nosocomial infections. In a global proteomic approach, the identification of the most abundant secreted and surface-associated proteins of E. faecalis JH2-2 strain was carried out. These proteins were separated by gel electrophoresis or directly subjected to in vivo trypsinolysis and then analyzed by liquid chromatography – electrospray ion trap tandem mass spectrometry. Putative functions were assigned by homology to the translated genomic database of E. faecalis. A total of 44 proteins were identified, eight secreted proteins from the supernatant culture and 38 cell surface proteins from two-dimensional gel electrophoresis and in vivo trypsinolysis among which two are common to the two groups. Their sequences analysis revealed that 35 of the 44 proteins harbour characteristic features (signal peptide or transmembrane domains) consistent with an extracellular localization. This study may be considered as an important step to encourage proteomic-based investigations of E. faecalis cell surface associated proteins that could lead to the discovery of virulence factors and to the development of new therapeutic tools.

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 Enterococcal Aggregation Substance and Binding Substance Are Not Major Contributors to Urinary Tract Colonization by Enterococcus faecalis in a Mouse Model of Ascending Unobstructed Urinary Tract Infection

2004 ◽  
Vol 72 (4) ◽  
pp. 2445-2448 ◽  
Author(s):  
James R. Johnson ◽  
Connie Clabots ◽  
Helmut Hirt ◽  
Christopher Waters ◽  
Gary Dunny
Keyword(s):  
Urinary Tract ◽  
Enterococcus Faecalis ◽  
Recombinant Strain ◽  
Plasmid Segregation ◽  
Cognate Receptor ◽  
Aggregation Substance ◽  
Binding Substance ◽  
Lower Urinary

ABSTRACT Isogenic Enterococcus faecalis strains that differ in their expression of aggregation substance (AS) and its cognate receptor, enterococcal binding substance (EBS), were compared for urovirulence in mice. Strain OG1SSp/pCF500 (inducible AS+, constitutive EBS+) failed to outcompete isogenic derivative INY3000 (AS− EBS−) in the urine, bladders, or kidneys of mice harvested at 48 h postinoculation. Neither mouse nor human urine induced AS expression by OG1SSp/pCF500. Recombinant strain OG1SSp/pINY1801 (constitutive AS+, EBS+) exhibited plasmid segregation that was as extensive in vivo as in vitro. These data suggest that AS and EBS do not contribute to upper or lower urinary tract colonization by E. faecalis and that growth in urine does not induce AS expression by strains carrying plasmids in the pCF10 family.

scholarly journals The Electricidal Effect Is Active in an Experimental Model of Staphylococcus epidermidis Chronic Foreign Body Osteomyelitis

2009 ◽  
Vol 53 (10) ◽  
pp. 4064-4068 ◽  
Author(s):  
Jose L. Del Pozo ◽  
Mark S. Rouse ◽  
Gorane Euba ◽  
Cheol-In Kang ◽  
Jayawant N. Mandrekar ◽  
...  
Keyword(s):  
Foreign Body ◽  
Staphylococcus Epidermidis ◽  
Bacterial Load ◽  
Rabbit Model ◽  
Electrical Current ◽  
Bacterial Biofilms ◽  
Control Group ◽  
Doxycycline Treatment ◽  
Continuous Delivery

ABSTRACT Treatment with low-amperage (200 μA) electrical current was compared to intravenous doxycycline treatment or no treatment in a rabbit model of Staphylococcus epidermidis chronic foreign body osteomyelitis to determine if the electricidal effect is active in vivo. A stainless steel implant and 104 CFU of planktonic S. epidermidis were placed into the medullary cavity of the tibia. Four weeks later, rabbits were assigned to one of three groups with treatment administered for 21 days. The groups included those receiving no treatment (n = 10), intravenous doxycycline (n = 14; 8 mg/kg of body weight three times per day), and electrical current (n = 15; 200 μA continuous delivery). Following treatment, rabbits were sacrificed and the tibias quantitatively cultured. Bacterial load was significantly reduced in the doxycycline (median, 2.55 [range, 0.50 to 6.13] log10 CFU/g of bone) and electrical-current (median, 1.09 [range, 0.50 to 2.99] log10 CFU/g of bone) groups, compared to the level for the control group (median, 4.16 [range, 3.70 to 5.66] log10 CFU/g of bone) (P < 0.0001). Moreover, treatment with electrical current was statistically significantly more efficacious (P = 0.035) than doxycycline treatment. The electricidal effect (the bactericidal activity of low-amperage electrical current against bacterial biofilms) is active in vivo in the treatment of experimental S. epidermidis chronic foreign body osteomyelitis.

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 Interaction of Pneumococcal Histidine Triad Proteins with Human Complement

2010 ◽  
Vol 78 (5) ◽  
pp. 2089-2098 ◽  
Author(s):  
Merit Melin ◽  
Emmanuel Di Paolo ◽  
Leena Tikkanen ◽  
Hanna Jarva ◽  
Cecile Neyt ◽  
...  
Keyword(s):  
Factor H ◽  
Surface Proteins ◽  
Bacterial Cells ◽  
Wild Type ◽  
Protein Antigens ◽  
Pneumococcal Infections ◽  
Relative Contribution ◽  
Bacterial Lysates ◽  
Affect Factor ◽  
Complement Deposition

ABSTRACT The pneumococcal histidine triad (Pht) proteins PhtA, PhtB, PhtD, and PhtE form a group of conserved pneumococcal surface proteins. Humans produce antibodies to Pht proteins upon exposure to pneumococcus, and immunization of mice has provided protective immunity against sepsis and pneumonia and reduced nasopharyngeal colonization. Pht proteins are candidates for inclusion in multicomponent pneumococcal protein vaccines. Their biological function in pneumococcal infections is not clear, but a role in complement inhibition has been suggested. We measured complement deposition on wild-type and Pht mutant strains in four genetic backgrounds: Streptococcus pneumoniae D39 (serotype 2) and R36A (unencapsulated derivative of D39) and strains of serotypes 3, 4, and 19F. PspA and PspC single and double mutants were compared to the wild-type and Pht-deficient D39 strains. Factor H binding was measured to bacterial cells, lysates, and protein antigens. Deletion of all four Pht proteins (Pht−) resulted in increased C3 deposition on the serotype 4 strain but not on the other strains. Pht antigens did not bind factor H, and deletion of Pht proteins did not affect factor H binding by bacterial lysates. The Pht− mutant serotype 4 strain bound slightly less factor H than the wild-type strain when binding was measured by flow cytometry. Pht proteins may play a role in immune evasion, but the mechanism of function is unlikely to be mediated by factor H binding. The relative contribution of Pht proteins to the inhibition of complement deposition is likely to be affected by the presence of other pneumococcal proteins and to depend on the genetic background.

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