Acceleration of Enterococcus faecalis biofilm formation by aggregation substance expression in an ex vivo model of cardiac valve colonization

Abstract

Infectious endocarditis involves formation of a microbial biofilm in vivo. Enterococcus faecalis Aggregation Substance (Asc10) protein enhances the severity of experimental endocarditis, where it has been implicated in formation of large vegetations and in microbial persistence during infection. In the current study, we developed an ex vivo porcine heart valve adherence model to study the initial interactions between Asc10(+) and Asc10(-)E. faecalis and valve tissue, and to examine formation of E. faecalis biofilms on a relevant tissue surface. Scanning electron microscopy of the infected valve tissue provided evidence for biofilm formation, including growing masses of bacterial cells and the increasing presence of exopolymeric matrix over time; accumulation of adherent biofilm populations on the cardiac valve surfaces during the first 2-4 h of incubation was over 10-fold higher than was observed on abiotic membranes incubated in the same culture medium. Asc10 expression accelerated biofilm formation via aggregation between E. faecalis cells; the results also suggested that in vivo adherence to host tissue and biofilm development by E. faecalis can proceed by Asc10-dependent or Asc10-independent pathways. Mutations in either of two Asc10 subdomains previously implicated in endocarditis virulence reduced levels of adherent bacterial populations in the ex vivo system. Interference with the molecular interactions involved in adherence and initiation of biofilm development in vivo with specific inhibitory compounds could lead to more effective treatment of infectious endocarditis.

Figure 1. Asc10 variants used in this study; prgB mutant constructs were generated in the native context of pCF10.

Wild-type Asc10 protein shown in shaded areas. Derivatives of pCF10 are shown in different regions; brackets denote altered regions of protein. pCF10-1, 31-aa insertion in central aggregation domain at aa 546; the central aggregation domain was postulated to fall between aa 473–683 in previous studies , but it is possible that it covers a larger region. pCF10-2, RGD motifs changed to RAD (the more N-terminal RGDS motif falls at aa 606, RGDV at aa 939). pCF10-4, deletion of N-terminal lipoteichoic acid (LTA) binding aggregation domain (aa 156–358); pCF10-5, combined mutations in both aggregation domains; pCF10-6, C-terminal domain deletion (aa 688–1138). pCF10-8 is not shown here, but this mutant derivative is contains an in-frame deletion of the prgB gene, which retains only the first three and last three codons.

Figure 2. Increase in Asc10⁺ E. faecalis aggregate size on valve tissue over time.

Scanning electron micrographs of E. faecalis Asc10⁺ OG1SSp (pCF10) incubated with heart valve segments for 0.5 h (A), 2 h (B), and 4 h (C), showing noticeable enlargement of bacterial aggregates over time, an observation compatible with biofilm formation. Part A also highlights preferential adherence of E. faecalis to areas of noticeable tissue damage, as opposed to areas where the tissue appears more intact (lower right of photograph). An uninfected valve is shown in part D, where the tissue is mostly intact, compared to the other panels in the presence of bacteria. Scale bars: A, 1 µm; B, 2 µm; C, 4 µm; D, 1 µm.

Figure 3. Asc10⁺ OG1SSp (pCF10) colonizes valve tissue more heavily than an Asc10⁻ prgB deletion mutant.

(A, B, C) Valve tissue infected with Asc10⁺ OG1SSp (pCF10) (panel A, B) and Asc10⁻ prgB deletion mutant strains (OG1SSp [pCF10-8]; C), as analyzed by scanning electron microscopy. Note the density of Asc10⁺ OG1SSp (pCF10) cells colonizing the valve (A), though not all areas of the valve were colonized as heavily, as shown in part B. In contrast, the prgB deletion mutant bound in single cells or short chains; all images were taken at 4 h post-infection. Scale bar: A, B, C  = 3 µm. (D) Porcine aortic, tricuspid, and mitral valves were infected with E. faecalis strains carrying wild-type pCF10 and the prgB deletion derivative pCF10-8 for 0.5, 1.0, 2.0 and 4.0 h. Valves were washed and homogenized, and adherent bacteria were quantified by plating onto agar. The data shown are a compilation of at least three experiments, each with valve sections from a different heart.

Figure 4. Comparison of porcine heart valve tissue and cellulose membrane adherence by Asc10⁺ OG1SSp (pCF10) and a prgB deletion mutant (pCF10-8).

Both strains are demonstrated to bind to valve tissue at a much higher density than to an abiotic surface such as the cellulose membrane. * indicates p-value ≤0.02, with respect to valve bacterial load.

Figure 5. Greater accumulation of exopolymeric matrix on Asc10⁺ E. faecalis biofilms, in comparison to Asc10⁻ biofilms.

Scanning electron micrographs of E. faecalis Asc10⁺ OG1SSp (pCF10) incubated 0.5 h (A) and 2 h (B) showing typical comparatively smooth appearance of the bacterial surface at early incubation times (A) compared to the fibrillar strands that became more evident over time (B). Use of a fixative containing alcian blue on the 4 h Asc10⁺ OG1SSp (pCF10)-infected valve tissue reveals the presence of an interwoven matrix covering most of the bacterial cells, which are seen as raised areas under the matrix (C). At higher magnification (D), bacterial cells can be observed clearly under the matrix, which appears more fibrillar in this view. Arrows in parts C and D indicate cells partially covered by matrix material. (E, F) Alcian blue-fixed valve sections colonized with the prgB deletion mutant (pCF10-8) revealed less matrix material as compared to the Asc10⁺ strain, in addition to a marked decrease in bacterial cells adherent to the valve tissue. In part F, the matrix material coats an Asc10⁻ E. faecalis cell, with areas of attachment to the valve tissue. Scale bars: A = 0.3 µm, B = 0.5 µm, C = 3.0 µm, D = 1.0 µm, E = 4.0 µm, F = 1.0 µm.

Figure 6. Alteration of the aggregation domain mutants in Asc10 lowers the ability of E. faecalis to bind to valve tissue.

(A) Double aggregation domain mutant (OG1SSp [pCF10-5]) with N-terminal aggregation domain deletion and central aggregation 31-aa insertion. The mutant is unable to bind as well as Asc10⁺ OG1SSp (pCF10). (B) Single aggregation domain mutants bind as well as Asc10⁺ OG1SSp (pCF10) initially, but over time the gap between the Asc10⁺ OG1SSp (pCF10) and mutant grows increasingly. The data shown are a compilation of at least three experiments. * denotes p≤0.02, with respect to Asc10⁺ OG1SSp (pCF10) valve bacterial loads.

Figure 7. Effects of mutations altering Asc10 subdomains on adherence and biofilm development.

(A) At initial timepoints, the double RGD mutant (OG1SSp [pCF10-2]) binds as well or better than Asc10⁺ OG1SSp (pCF10); no deficiency in binding to porcine valve tissue was detected until 4 h. (B) Deletion of the C-terminal domain of Asc10 does not significantly affect the ability of E. faecalis to bind to valve tissue. The data shown are a compilation of at least three experiments. * denotes p≤0.02, with respect to Asc10⁺ OG1SSp (pCF10) valve bacterial loads.

Figure 8. Asc10-independent and -dependent biofilm formation models.

E. faecalis cell attachment to the valve surface can be mediated by chromosomally-encoded adhesins (A), or by Asc10 (B). In the absence of Asc10, accumulation of biofilm mass takes place as individual E. faecalis cells bind to the valve tissue (C). Asc10 expression allows for bacterial aggregation, and thus accelerated growth of biofilm mass through adherence of aggregates to the surface.