Importance of the collagen adhesin ace in pathogenesis and protection against Enterococcus faecalis experimental endocarditis - PubMed (original) (raw)

Importance of the collagen adhesin ace in pathogenesis and protection against Enterococcus faecalis experimental endocarditis

Kavindra V Singh et al. PLoS Pathog. 2010.

Erratum in

Abstract

Ace is an adhesin to collagen from Enterococcus faecalis expressed conditionally after growth in serum or in the presence of collagen. Here, we generated an ace deletion mutant and showed that it was significantly attenuated versus wild-type OG1RF in a mixed infection rat endocarditis model (P<0.0001), while no differences were observed in a peritonitis model. Complemented OG1RFDeltaace (pAT392::ace) enhanced early (4 h) heart valve colonization versus OG1RFDeltaace (pAT392) (P = 0.0418), suggesting that Ace expression is important for early attachment. By flow cytometry using specific anti-recombinant Ace (rAce) immunoglobulins (Igs), we showed in vivo expression of Ace by OG1RF cells obtained directly from infected vegetations, consistent with our previous finding of anti-Ace antibodies in E. faecalis endocarditis patient sera. Finally, rats actively immunized against rAce were less susceptible to infection by OG1RF than non-immunized (P = 0.0004) or sham-immunized (P = 0.0475) by CFU counts. Similarly, animals given specific anti-rAce Igs were less likely to develop E. faecalis endocarditis (P = 0.0001) and showed fewer CFU in vegetations (P = 0.0146). In conclusion, we have shown for the first time that Ace is involved in pathogenesis of, and is useful for protection against, E. faecalis experimental endocarditis.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Flow cytometry analysis of cell surface expression of Ace by E. faecalis OG1RF, its isogenic ace deletion mutant and its in trans complemented ace deletion mutant.

(A) Comparison of the effect of growth for 10 h under different conditions on expression levels of Ace in OG1RF using both pre-immune Igs (PI) and anti-rAce Igs. (B) Analysis of Ace expression by the OG1RF ace deletion mutant and the effect of its in trans complementation. OG1RFΔ_ace_, ace deletion mutant; OG1RFΔ_ace_ (pAT392::ace), complemented ace deletion mutant; OG1RFΔ_ace_ (pAT392), ace deletion mutant with the empty vector. Reactivity to affinity-purified specific anti-rAce Igs is shown for each isogenic strain. Bacteria were analyzed using side scatter as the threshold for detection. Binding by specific anti-rAce Igs is indicated as log fluorescence intensity on the X-axis. For each histogram, 50,000 events of bacterium-sized particles were counted.

Figure 2

Figure 2. Adherence of E. faecalis OG1RF and its derivatives to immobilized collagens.

(A) Adherence to collagen type I (CI). (B) Adherence to collagen type IV (CIV). (C) Adherence to fibrinogen (Fg). Mean % of cells adhering ± SD from two independent experiments representing 12 wells/sample are shown.

Figure 3

Figure 3. Flow cytometry analysis of Ace surface expression by E. faecalis OG1RF cells derived from vegetations of rat experimental infective endocarditis.

(A) Surface expression of Ace by bacterium sized particles derived from vegetations. Vegetations were produced as described in methods, and some rats were injected (i.v.) with BHI-grown OG1RF. Processed sterile and infected vegetations were incubated with specific anti-Ace A-domain Igs or Igs purified from antiserum raised against heat-killed E. faecalis strain HH22-whole-cells (anti-Efs), followed by incubation with R-phycoerythrin-conjugated antibody. Specific binding by anti-Ace or anti-Efs antibodies is indicated as log fluorescence intensity on the X_-_axis. Each histogram represents 5,000 (non-infected vegetations) to 25,000 (infected vegetations) events of bacterium-sized particles. (B) For comparison, OG1RF cells grown in vitro in BHI at 37°C (inoculum used for infection) and 46°C (the growth condition that exhibited most in vitro Ace expression) as well as in BHIS at 37°C, stained with pre-immune Igs and anti-Ace and the same batch of R-phycoerythrin-conjugated secondary antibody, are shown in panel B.

Figure 4

Figure 4. E. faecalis OG1RF and OG1RFΔ_ace_ (TX5467) in a competition (mixed infection) assay in the rat endocarditis model.

Percentages of OG1RF and OG1RFΔ_ace_ present in inocula and recovered from vegetations 72 h post infection of 12 rats are shown. Horizontal bars represent the means (P<0.0001 by paired t test) for percentages of total bacteria in the vegetations of OG1RF versus OG1RFΔ_ace_. Empty circles and empty triangles represent percentages of OG1RF and OG1RFΔ_ace_ in inocula, respectively, while solid circles and solid triangles represent percentages of OG1RF and OG1RFΔ_ace_ in vegetations, respectively.

Figure 5

Figure 5. Complemented OG1RFΔ_ace_ (pAT392::ace) [TX5647] and OG1RFΔ_ace_ (pAT392) [TX5648] in early (mono-infection) colonization of aortic valves in the rat model.

In the panel on the left, empty circles and empty triangles represent OG1RFΔ_ace_ (pAT392::ace) and OG1RFΔ_ace_ (pAT392) inocula, respectively. In the panel on the right, solid circles and solid triangles represent OG1RFΔ_ace_ (pAT392::ace) and OG1RFΔ_ace_ (pAT392) in vegetations, respectively. Data are expressed as log10 CFU/gm recovered from the vegetations 4 h post infection of 12 rats (OG1RFΔ_ace_ (pAT392::ace) and 11 rats (OG1RFΔ_ace_ (pAT392), respectively. Horizontal bars represent the geometric mean titers. Significantly enhanced (by a mean ± SD increase of 1.4±0.6 log10 CFU/gm) vegetation titer by OG1RFΔ_ace_ (pAT392::ace) versus OG1RFΔ_ace_ (pAT392) (P = 0.0417) by unpaired t test for mean log10CFU/gm is shown.

Figure 6

Figure 6. Comparison of serum anti-Ace titers in immunized and non-immunized rats.

Rats were immunized and boosted twice with 100 μg rAce. Antibody levels were measured by ELISA. Mean serum anti-Ace titers were plotted for each antibody dilution tested.

Figure 7

Figure 7. rAce active-immunization in rat endocarditis model.

(A) BHI-grown OG1RF. In the panel on the left, empty circles and empty triangles represent OG1RF used for non-immunized and rAce active-immunized rats, respectively. In the panel on the right, solid circles and solid triangles represent OG1RF recovered from the vegetations, 48 h post infection. Horizontal bars represent the geometric means. Significantly fewer rats were infected by OG1RF in rAce active-immunized (5/16) versus non-immunized (16/16) (P = 0.0001 by Fisher's exact test). Vegetations showed 4.2±1.0 log10 more OG1RF CFU/gm (mean ± SD) from non-immunized versus rAce active-immunized rats (P = 0.0004, by unpaired t test). (B) BHIS-grown OG1RF. In the panel on the left, empty circles, empty triangles and empty diamonds represent OG1RF used for non-immunized, rAce active-immunized and sham-immunized rats, respectively. In the panel on the right, solid circles, solid triangles and solid diamonds represent OG1RF recovered from the vegetations, 48 h post infection. Horizontal bars represent the geometric means. Significantly reduced vegetation bacterial counts of OG1RF in rAce active-immunized (n = 18), versus non-immunized (n = 10) (P = 0.0231) and versus sham-immunized (n = 33) (P = 0.0475) by unpaired t test are shown. Rats (non-immunized) showed a mean ± SD increase of 3.2±1.3 log10 OG1RF CFU/gm from vegetations versus rAce active-immunized rats while rats (FCA-FICA immunized) showed a mean ± SD increase of 2.0±1.0 log10 OG1RF CFU/gm from vegetations versus rAce active-immunized rats.

Figure 8

Figure 8. Passive immunization (anti-rAce Ig versus PI Ig) in rat endocarditis model.

In the panel on the left, empty circles and empty triangles represent OG1RF inocula for pre-immune (PI) Ig treated and affinity purified specific anti-rAce Ig treated rats, respectively. In the panel on the right, solid circles and solid triangles represent OG1RF recovered from rat vegetations 24 h post infection. Horizontal bars represent the geometric means. Significantly fewer rats were infected by OG1RF in rAce Ig (2 mg/kg) (2/10) versus PI Ig (2 mg/kg) (5/6) treated rats (P = 0.0001 by Fisher's exact test). Rats (PI Ig treated) showed a mean ± SD increase of 3.8±1.4 log10 OG1RF CFU/gm from vegetations versus anti-rAce Ig treated rats (P = 0.0146 by unpaired t test).

Figure 9

Figure 9. Kaplan-Meier survival plots of wild-type OG1RF and the ace mutant in the mouse peritonitis model.

(A) Survival plots of OG1RF and OG1RFΔ_ace_ using 109 inocula (B) Survival plots of OG1RF and OG1RFΔ_ace_ using 108 inocula. Six mice were tested with each inoculum of each of the strains shown.

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