Aggregation Substance proteins encoded by sex pheromone plasmids increase
virulence of Enterococcus faecalis in experimental pathogenesis models, including
infectious endocarditis. 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 the 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 vegetation was the best indicator of
virulence. Previously identified aggregation domains contributed to the virulence
associated with the wild-type protein, and a strain expressing an Asc10 derivative where
glycine residues in two RGD motifs were changed to alanines 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. In addition, mutants carrying Tn917 insertions in the
prgB gene demonstrated that secreted N-terminal Asc10 fragments possess activity
promoting endocarditis virulence. 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.
Since Asc10 is important as a virulence factor in E. faecalis endocarditis
pathogenesis, developing immunization approaches against this surface protein will be useful in combating endocarditis disease. Use of Fab fragment antibodies against
Asc10 was found to decrease vegetation size and bacterial load in the rabbit
endocarditis model. In addition, microarray and histological studies revealed two routes
of infection in vegetation formation; one in the absence of Asc10, characterized by a
robust inflammatory response, and the second in which the presence of Asc10 dampens
this response, possibly impeding the influx of immune cells into the vegetation.
We also employed an ex vivo porcine heart valve adherence model to study the initial
interactions between Asc10+ E. faecalis and valve tissue, and to examine formation of
biofilms. We found that the aggregation domains contribute most to Asc10-mediated E.
faecalis valve adherence, whereas the RGD motifs have importance in later stages of
valve colonization. Again, an N-terminal Asc10 fragment expressed from a prgB
Tn917 insertion mutant mediated adherence of E. faecalis cells, emphasizing the
importance of the aggregation domains in valve attachment. Most of the Asc10 mutants
examined showed some defects in valve adherence at 4 h, corroborating results from
our rabbit endocarditis model, and implying that Asc10 contributes mainly to
persistence of E. faecalis during endocarditis infection. Extracellular matrix (ECM)
protein studies to determine the eukaryotic Asc10 ligand in valve tissue found that fulllength
Asc10 protein did not mediate E. faecalis binding to vitronectin, fibronectin,
fibrinogen, von Willebrand factor, heparan sulfate, or chondroitin sulfate. In scanning
electron microscopy analysis of the infected valve tissue, we found evidence of biofilm
formation, including growing aggregates of bacteria, and the increasing presence of
exopolymeric matrix over time. Additionally, E. faecalis cells preferentially bound to
damaged tissue, though it was difficult to determine whether the bacteria caused the
damage, or if it was due to deterioration of the tissue over time. This porcine heart
valve tissue colonization model will serve as a useful tool in future studies of biofilm
University of Minnesota Ph.D. dissertation. August 2009. Major: Microbiology, Immunology and Cancer Biology. Advisor: Gary M. Dunny. 1 computer file (PDF); xvi, 119 pages, appendix pages 113-119.
Chuang-Smith, Olivia Newton.
Enterococcus faecalis aggregation substance (Asc10) as liaison between bacterium and heart valve in endocarditis..
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