Multiple functional domains of Enterococcus faecalis aggregation substance Asc10 contribute to endocarditis virulence

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.

FIG. 1.

NBT vegetation formation, demonstrating interactions between Asc10⁺ E. faecalis and host cells during formation of the septic vegetation in endocarditis. Platelets, fibrin, and other clotting proteins may comprise part of the vegetation, enabling blood-borne E. faecalis to bind to the growing clot.

FIG. 2.

Schematic maps of the Asc10 mutant proteins expressed by the strains used in the rabbit endocarditis model, highlighting the functional domains of the Asc10 protein. (A) Wild-type full-length Asc10 (pCF10). (B) C-terminal domain deletion variant (pCF10-6) with amino acids 688 to 1138 (nucleotides 2064 to 3414) missing. (C) N-terminal aggregation domain deletion variant (pCF10-4) with amino acids 156 to 358 (nucleotides 468 to 1074) deleted. (D) Central aggregation domain insertion variant (pCF10-1) with 31 amino acids inserted at amino acid 546 (nucleotide 1638). Previous studies have determined that amino acid residues 473 to 683 are an essential component of this central aggregation domain. (E) N-terminal single RGDS → RADS substitution variant (pCF10-3). (F) C-terminal single RGDV → RADV substitution variant (pCF10-7). (G) Double RGD → RAD substitution variant (pCF10-2). RGD motifs occur at amino acid residues 606 and 939, as shown in panel A. SS, signal sequence; aa, amino acids. The diagrams are adapted from diagrams of Waters and Dunny (33). Whether expression of each Asc10 variant resulted in visible clumping of the host bacteria is indicated on the right.

FIG. 3.

Asc10 surface expression by the Asc10 mutant strains, as determined by Western blot analysis of purified cell wall fractions. All strains used in Western blot analyses had an OG1SSp background. OG1SSp(pCF10) produced mainly a 150-kDa band, which is larger than the predicted 137-kDa band for Asc10. This may have been due to glycosylation or association of LTA with Asc10. The electrophoretic patterns of the Asc10 derivatives, including the multiple banding patterns, have been observed previously (10, 33). The prgB deletion mutant OG1SSp(pCF10-8) was analyzed on a separate Western blot, and no Asc10 surface expression was detected. The smaller bands observed for pCF10-4, pCF10-5, and pCF10-6 were due to large deletions in the prgB gene.

FIG. 4.

Vegetations on rabbit aortic valves. (A) Aortic valve of a rabbit infected with the central aggregation domain insertion mutant [OG1SSp(pCF10-1)]. The aortic valve is composed of three cup-shaped leaflets, as indicated by the box. The bacterial load of the vegetation removed was 1.50 × 10¹ CFU. The majority of the vegetation is on the right leaflet (indicated by the arrow). (B) Aortic valve of a rabbit infected with an N-terminal single RGDS mutant [OG1SSp(pCF10-3)]. The valves exhibit no apparent vegetations, and the vegetation bacterial load was 5.30 × 10³ CFU. (C) Aortic valve of a rabbit infected with an Asc10⁻ plasmid-free strain (OG1SSp), with one vegetation in the center leaflet (arrow). The vegetation bacterial load was 2.40 × 10⁷ CFU.

FIG. 5.

(A) Plot of vegetation bacterial load (total CFU) versus vegetation weight in a rabbit endocarditis model. E. faecalis cells expressing Asc10 mutant derivatives (2 × 10⁹ CFU) were injected intravenously after catheter placement in the carotid artery for 2 h; the catheter was removed prior to microbe injection. All vegetations on the aortic valve of each rabbit were pooled to obtain the vegetation weight and bacterial load. When no vegetations were found, the valves were removed and plated to determine the valve bacterial load. The low R² value (0.27) indicates that there is no correlation between the sets of values. (B) Arithmetic and geometric mean vegetation bacterial loads in the rabbit endocarditis model, expressed as log₁₀ total CFU. The dashed line indicates the minimum level of detection (1.93 log₁₀ total CFU). *, P ≤ 0.01 for a comparison with the wild-type Asc10⁺ strain.

FIG. 6.

Adherence of mutant Asc10 purified proteins to ECM proteins. The amounts of bound Asc10 proteins were measured by ELISA, and increasing absorbance represented larger amounts of Asc10 protein adhering to the ECM substrate. (A) Asc10 binding to fibrinogen. The N-terminal aggregation domain deletion variant protein (encoded by pCF10-4), but not the wild-type protein, was found to bind fibrinogen. (B) The N-terminal aggregation domain deletion (encoded by pCF10-4) and double aggregation domain (encoded by pCF10-5) variant proteins, but not the wild-type protein, were able to bind vitronectin. (C) The central aggregation domain insertion variant protein (encoded by pCF10-1), but not the wild-type protein, bound von Willebrand factor. The data in each graph are representative of the results of at least three experiments.

FIG. 7.

Proposed functions of Asc10 domains. SS, signal sequence; TNF-α, tumor necrosis factor alpha; IL-1, interleukin-1; IL-6, interleukin-6.