Primers used in this study.

<p>^<i>a</i>Introduced restriction sites are underlined.</p

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

<p>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 (<i>P</i> = 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 (<i>P</i> = 0.0146 by unpaired <i>t</i> test).</p

Adherence of <i>E. faecalis</i> OG1RF and its derivatives to immobilized collagens.

<p>(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.</p

<i>E. faecalis</i> OG1RF and OG1RFΔ<i>ace</i> (TX5467) in a competition (mixed infection) assay in the rat endocarditis model.

<p>Percentages of OG1RF and OG1RFΔ<i>ace</i> present in inocula and recovered from vegetations 72 h post infection of 12 rats are shown. Horizontal bars represent the means (<i>P</i><0.0001 by paired <i>t</i> test) for percentages of total bacteria in the vegetations of OG1RF versus OG1RFΔ<i>ace</i>. Empty circles and empty triangles represent percentages of OG1RF and OG1RFΔ<i>ace</i> in inocula, respectively, while solid circles and solid triangles represent percentages of OG1RF and OG1RFΔ<i>ace</i> in vegetations, respectively.</p

Bacterial strains and plasmids used in this study.

<p><i>^a</i>Chl, chloramphenicol; Fus, fusidic acid; Gen, gentamicin; Kan, kanamycin; Rif, rifampicin; and ts, temperature-sensitive. Superscript “s” designates sensitivity and “r” designates resistance; “r” is defined for enterococci as MIC >500 for Gen and >2000 for Kan.</p

Complemented OG1RFΔ<i>ace</i> (pAT392::<i>ace</i>) [TX5647] and OG1RFΔ<i>ace</i> (pAT392) [TX5648] in early (mono-infection) colonization of aortic valves in the rat model.

<p>In the panel on the left, empty circles and empty triangles represent OG1RFΔ<i>ace</i> (pAT392::<i>ace</i>) and OG1RFΔ<i>ace</i> (pAT392) inocula, respectively. In the panel on the right, solid circles and solid triangles represent OG1RFΔ<i>ace</i> (pAT392::<i>ace</i>) and OG1RFΔ<i>ace</i> (pAT392) in vegetations, respectively. Data are expressed as log₁₀ CFU/gm recovered from the vegetations 4 h post infection of 12 rats (OG1RFΔ<i>ace</i> (pAT392::<i>ace</i>) and 11 rats (OG1RFΔ<i>ace</i> (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<i>ace</i> (pAT392::<i>ace</i>) versus OG1RFΔ<i>ace</i> (pAT392) (<i>P</i> = 0.0417) by unpaired <i>t</i> test for mean log₁₀CFU/gm is shown.</p

Flow cytometry analysis of cell surface expression of Ace by <i>E. faecalis</i> OG1RF, its isogenic <i>ace</i> deletion mutant and its <i>in trans</i> complemented <i>ace</i> deletion mutant.

<p>(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 <i>ace</i> deletion mutant and the effect of its <i>in trans</i> complementation. OG1RFΔ<i>ace</i>, <i>ace</i> deletion mutant; OG1RFΔ<i>ace</i> (pAT392::<i>ace</i>), complemented <i>ace</i> deletion mutant; OG1RFΔ<i>ace</i> (pAT392), <i>ace</i> 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.</p

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

<p>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.</p

rAce active-immunization in rat endocarditis model.

<p>(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) (<i>P</i> = 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 (<i>P</i> = 0.0004, by unpaired <i>t</i> 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) (<i>P</i> = 0.0231) and versus sham-immunized (n = 33) (<i>P</i> = 0.0475) by unpaired <i>t</i> 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.</p

Kaplan-Meier survival plots of wild-type OG1RF and the <i>ace</i> mutant in the mouse peritonitis model.

<p>(A) Survival plots of OG1RF and OG1RFΔ<i>ace</i> using 10⁹ inocula (B) Survival plots of OG1RF and OG1RFΔ<i>ace</i> using 10⁸ inocula. Six mice were tested with each inoculum of each of the strains shown.</p