Importance of the Collagen Adhesin Ace in Pathogenesis and Protection against Enterococcus faecalis Experimental Endocarditis

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 OG1RFΔace (pAT392::ace) enhanced early (4 h) heart valve colonization versus OG1RFΔace (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.


Introduction
Enterococci are gram-positive cocci of intestinal origin first reported as a cause of infective endocarditis (IE) in 1899 [1]. They were recognized as the 3 rd most common cause of IE as early as the 1920's, and have remained the 3 rd most common cause of community onset IE since then with Enterococcus faecalis accounting for .90% of isolates from enterococcal IE when identified to the species level [1,2,3,4,5]. Over the past 20 years, enterococci have also become the 2 nd -3 rd most common organisms isolated from nosocomial (healthcare-associated) infections including UTIs, bacteremia, intraabdominal and wound infections, endocarditis, sepsis in neonates, among others [1,3]. Indeed, among causes of endocarditis, enterococci (predominantly E. faecalis) have been variably reported as the #1 and #2 cause [6,7]. Since healthcareassociated infections, particularly those caused by antibiotic resistant bacteria, result in enormous increases in hospital stays and costs, enterococci clearly represent an important drain on healthcare dollars. In one study, the attributable mortality of enterococcal bacteremia was 31% [8], emphasizing the clinical, not just the financial, seriousness of these infections.
The first step in infective endocarditis is vascular tissue colonization, which can be mediated by cell-wall anchored adhesins such as MSCRAMMs (for microbial surface components recognizing adhesive matrix molecules) [9] of gram-positive bacteria. Our previous in silico analyses of the E. faecalis genome identified a family of genes encoding MSCRAMM-like proteins containing one or more regions of ca. 150 aa segments with deviant Ig-like fold(s), characteristic of the Staphylococcus aureus MSCRAMMs [10]. One of these, called Ace (for Adhesin to collagen of E. faecalis), has been studied in detail. Genetic and biochemical analyses showed that Ace mediates adherence of E. faecalis cells to bovine and rat collagen type I (CI), human collagen type IV (CIV), and mouse laminin [11,12,13], as well as human dentin [14].
Crystal structure analysis of the ligand-binding segment of Ace showed that the Ace A domain is composed of two sub-domains, N1 and N2, each adopting an Ig-like fold [15]. Subsequent point and truncation mutation analyses suggested that Ace binds to collagen by a mechanism called the ''Collagen Hug'' [15], a variant of the ''Dock, Lock and Latch'' ligand-binding mechanism shown for Staphylococcus epidermidis fibrinogen (Fg) adhesin SdrG [16,17]. The ace gene is ubiquitous [18] in E. faecalis and conserved among diverse isolates albeit with at least four variants due to variation in the number of repeats of the B domain [19]. Conditional in vitro production of Ace (i.e., markedly enhanced production after growth at 46uC, growth in brain-heart infusion plus 40% serum (BHIS) or growth in the presence of collagen versus growth in BHI broth at 37uC) by different strains correlates with conditional adherence of these E. faecalis strains to collagens and laminin [19,20]. Most sera from patients with E. faecalis IE show reactivity with rAce, indicating that different strains express Ace during human infection and that it is antigenic in vivo [19].
Furthermore, anti-Ace antibodies (affinity purified from human serum or animals immunized with rAce) were shown to inhibit in vitro adherence of E. faecalis strains to collagen and laminin [11,19]. In a recent study, anti-Ace40 (ligand-binding A-domain of Ace) monoclonal antibodies were shown to completely inhibit binding of Ace40 to human CI and collagen type VI and inhibited binding of Ace-coated fluorescent beads to epithelial cell lines, thus suggesting Ace as a potential therapeutic target antigen against E. faecalis infections [21].
In the present study, we have studied the role of Ace in the pathogenesis of E. faecalis endocarditis by generating an ace deletion mutant in E. faecalis strain OG1RF, by complementing this mutant (OG1RFDace), by comparing these isogenic strains with OG1RF for adherence to various extracellular matrix (ECM) proteins and for their ability to infect aortic valves in a rat endocarditis model. Finally, we also determined the importance of Ace as a protective antigen against experimental endocarditis in a rat model by using active and passive immunization.

Characterization of the Dace mutant and complementation construct
Our previous disruption mutant of ace was found to be unstable in vivo (see below). We therefore constructed an allelic replacement ace deletion mutant of OG1RF (TX5467, OG1RFDace::cat; resistant to chloramphenicol 10 mg/ml). Deletion of ace from OG1RF was verified by sequencing confirming the correct deletion of ace from 223 to + 2200 (including the RBS, complete ace gene, and 34 bp downstream of ace), and by pulsed field gel electrophoresis (PFGE) and hybridizations (Table 1). Growth (OD 600 ) of the Dace mutant was similar to wild-type (WT) OG1RF

Author Summary
Enterococcus faecalis was recognized as a common cause of infective endocarditis (IE) by the early 1900s. It is still third in community-onset IE, but is the second most common cause of hospital-associated IE. Complications due to E. faecalis IE include congestive heart failure, septic emboli and death and current management involves a combination of antimicrobials, often with surgery. Emergence of antimicrobial resistance has created the need for alternative strategies (such as immunoprophylaxis) that target in vivo expressed virulence-associated surface proteins. One such E. faecalis protein is Ace, which is antigenic during human IE and mediates attachment of E. faecalis cells to host extracellular matrix proteins collagen and laminin. Using a rat model, we now show that ace contributes to E. faecalis IE pathogenesis and demonstrate that Ace is expressed at high levels during IE even though produced at low levels under laboratory conditions; both active and passive immunization based on the collagen-binding domain of Ace conferred significant protection against IE. These observations, along with data that human antibodies against Ace inhibit collagen adherence of E. faecalis, indicate that Ace is an important virulence-associated factor and a promising target for prophylactic and possibly therapeutic strategies against E. faecalis IE. in BHI (data not shown). We have previously shown, using western blotting and RT-PCR, that ace is expressed at higher levels when grown in BHIS at 37uC or in BHI at 46uC [11] than in BHI at 37uC. Here, we assessed surface localization of Ace in OG1RF at 10 h using flow cytometry analyses with affinity-purified anti-rAce Igs. The mean fluorescence intensity levels for different culture conditions increased progressively with cells grown in BHI at 37uC, BHIS at 37uC and BHI at 46uC ( Figure 1A), consistent with our previous western and immunofluorescence microscopy data [11,20]. The percentages (%) of Ace-expressing cells in BHIS cultures of OG1RF, OG1RFDace, OG1RFDace (pAT392) (empty vector control), and OG1RFDace (pAT392::ace) (complementation) were .70%, ,5%, ,5%, and .90%, respectively, demonstrating the inability of OG1RFDace to produce Ace and the efficient complementation of OG1RFDace by pAT392::ace. In these experiments, pAT392-containing strains were grown without added gentamicin, the same conditions that we used for preparing inocula for the rat endocarditis experiments. When BHIS was supplemented with gentamicin, expression of Ace increased to .95% of cells in OG1RFDace (pAT392::ace) ( Figure 1B), likely due to improved plasmid stability (see below). OG1RF and its isogenic Dace mutant as well as the complementation constructs were tested for their ability to adhere to immobilized ECM proteins and BSA. Consistent with our previous demonstration of adherence of OG1RF to CI, CIV and Fg after growth in BHIS at 37uC and to CI and CIV after growth in BHI at 46uC (but not in BHI [11] at 37uC), we observed here that OG1RF adhered to CI and CIV when grown in BHIS at 37uC, unlike OG1RFDace which showed markedly reduced adherence to CI (from ,36 to 15%) (Figure 2A), and CIV (43 to 3%) ( Figure 2B), but no change in adherence to Fg (( Figure 2C). This corroborates our earlier data with a mutant with an insertional disruption of ace [11,20]. Introduction of the ace gene in trans into OG1RFDace resulted in even greater adherence to collagens than WT (.1.5-fold higher), whereas OG1RFDace electroporated with pAT392 retained its reduced adherence phenotype ( Figure 2); these results are consistent with Ace expression data from flow cytometry analysis.

In vivo surface expression of Ace
To determine if Ace is produced during infection, we performed flow cytometry analyses on extracts directly processed from IE vegetations infected with OG1RF grown in BHI at 37uC. Forward and side scatter pattern analyses of particles from processed vegetations and comparisons with those from in vitro grown OG1RF cells indicated that most of the gated particles detected by flow cytometry are likely OG1RF bacterial cells, thus confirming the removal of the majority of host tissue particles from the vegetations during the processing steps described in methods. Sterile processed vegetations from non-infected rats probed with anti-rAce-specific Igs (negative control) showed labeling of a minor fraction (,3%) of bacterium-sized particles ( Figure 3A), while processed vegetations from OG1RF infected rats probed with Igs from an antiserum raised against formalin-killed E. faecalis strain HH22-whole-cells (positive control) bound 85% of bacterium-sized particles, further indicating that the majority of these particles were E. faecalis cells ( Figure 3A). Affinity-purified anti-rAce-specific Igs bound to ,40 to 45% of bacterium-sized particles from different rat endocarditis vegetations infected with OG1RF ( Figure 3), demonstrating that Ace is actively expressed in host vegetations during IE.
In vivo testing of WT OG1RF and ace mutants in a mixed infection competition assay Although our initial mixed-infection competition experiments showed a clear advantage for the WT over an ace disruption mutant TX5256 [11,20] to develop IE in rat model (data not shown), subsequent experiments identified instability of this single cross-over ace disruption mutant during in vivo growth. Hence, we generated an OG1RFDace mutant for further in vivo testing.
In an initial mono-infection experiment (n = 2) with our ace deletion mutant (OG1RFDace), TX5467, we first determined the expression of cat (encoding chloramphenicol acetyl transferase) in OG1RFDace, which carries this chloramphenicol resistance marker gene in place of ace, by analyzing individual colonies for chloramphenicol resistance and by high stringency hybridization using intragenic cat and ace DNA probes. We found that ,10% of the colonies recovered from vegetations were chloramphenicol (10 mg/ml) susceptible even though they were cat probe positive and ace probe negative, indicating that, although the cat gene was present, silencing of its expression had occurred in these colonies. Previously, it has been shown that some antibiotic resistance genes of Escherichia coli are silenced in vivo; specifically, expression of an intact antibiotic resistance gene was switched off during the course of gut colonization in pigs, a phenomenon suggested to be helpful for bacterial fitness [22]. Therefore, for our mixed infection animal experiments, all the results reported here are based on high (B) Analysis of Ace expression by the OG1RF ace deletion mutant and the effect of its in trans complementation. OG1RFDace, ace deletion mutant; OG1RFDace (pAT392::ace), complemented ace deletion mutant; OG1RFDace (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. doi:10.1371/journal.ppat.1000716.g001 stringency hybridization with ace and cat probes using ,200 CFUs/rat vegetation for all the rats used. Of interest, we also tested for chloramphenicol resistance but did not observe further cat silencing.
In the mixed infection competition assay, all 12 rats were infected with an approximately 1:1 mixture (as predicted by OD 600 ) of BHI-grown OG1RF (determined geometric mean (GM) CFU 3.8610 7 /rat, representing 47% of the inoculum): OG1RF-Dace (GM CFU 4.4610 7 /rat, representing 53% of the inoculum) ( Figure 4). Bacterial CFUs from vegetations on aortic valves were recovered at ,72 h from all 12 rats and are shown in Figure 4. The mean percentage (%) of OG1RF in the total CFU of bacteria recovered was 81.5% versus 18.5% for OG1RFDace (P,0.0001), thus demonstrating a clear advantage of OG1RF versus OG1RFDace at 72 h for heart valve colonization in rats. The mean virulence index or competitive index [23,24], which is a sensitive measure of the relative degree of virulence attenuation of a particular mutant in a mixed infection with the WT strain, was calculated using the equation shown in Materials and Methods. The mean virulence index of the ace mutant relative to WT in vegetations was 0.077; this indicates that ace has an important role in this endovascular infection.
In vivo testing of the complemented OG1RFDace (pAT392::ace) construct and OG1RFDace (pAT392) in the mono-infection model In initial mono-infection experiments with complementation constructs and testing 24 h after inoculation, we observed loss of the plasmid from cells recovered from vegetations, with 94%-98% loss from OG1RFDace (pAT392::ace) (7 rats) and 14%-100% loss from OG1RFDace (pAT392) (8 rats) (data not shown). We also tried growing both constructs in BHIS supplemented with gentamicin for the preparation of inocula for infection, but in vivo loss of the plasmid still occurred 24 h after inoculation. To minimize in vivo growth time and to determine the role of Ace in the early stage of valve colonization in rats, we tested both OG1RFDace (pAT392::ace) and OG1RFDace (pAT392) in the rat model 4 h after inoculation. Two independent mono-infection experiments were done and the combined results are shown in Figure 5. Rats inoculated with OG1RFDace (pAT392::ace) (n = 12) showed 1.460.6 log 10 more CFU/gm than OG1RFDace (pAT392) (n = 11) (P = 0.0417) ( Figure 5), thus demonstrating that Ace has a significant role in early colonization of heart valves in E. faecalis rat IE. Reduced time in vivo also resulted in much less loss of the plasmid from each construct. In the case of OG1RFDace (pAT392::ace), gentamicin susceptible colonies were recovered in only 2/12 rats (2/2 colonies from one and 3/3 colonies from the other were gentamicin susceptible), while with OG1RFDace (pAT392), 3-100% of colonies (among 8-48 tested) recovered from 5/11 rats were gentamicin susceptible. These results corroborated the above described complementation of ace surface expression ( Figure 1B) and restoration of in vitro adherence of OG1RFDace (pAT392::ace) to CI and CIV to similar levels as observed for OG1RF.

Active immunization with rAce and in vivo protection against E. faecalis experimental IE in rats
Since Ace was found to be an important virulence factor in rat experimental IE, an Ace-specific immune response might hinder the development of IE. To study this, rats were vaccinated s.c. thrice with 99% pure 100 mg rAce or PBS or Freund's complete adjuvant -Freund's incomplete adjuvant (FCA -FICA) and were challenged with 10 7 to 10 9 CFU of E. faecalis OG1RF per rat (see methods). Comparison of anti-Ace antibody levels of 10 immunized and three non-immunized animals by ELISA showed that all 10 immunized rats tested had high levels of anti-Ace titers (1: .50,000), whereas no anti-Ace antibodies were detected in any of the three control rats ( Figure 6). Sixteen of 16 no-treatment control rats (100%) developed E. faecalis endocarditis after challenge with 10 times the ID 50 of BHI-grown OG1RF compared with 5 of 14 rats (35%) in the rAce active-immunization group (P = 0.0001) ( Figure 7A). The no-treatment control rats showed a mean of 4.261.0 log 10 more CFU/gm than the rAce active-immunized (P = 0.0004) in vegetations recovered from heart valves. In an independent experiment, in order to mimic in vivo growth conditions more closely and because we had found that Ace is expressed at higher levels by OG1RF when grown in BHIS [11], we used BHIS-grown OG1RF for the preparation of inocula. rAce (n = 10) rats were inoculated with a higher inoculum of 1.4610 9 CFU/rat (,100 times the ID 50 ), while non-immunized controls (n = 18) were inoculated with 3.1610 8 -1.1610 9 CFU/ rat. Fifteen of 18 non-immunized control animal (83%) developed E. faecalis endocarditis compared with 5 of 10 rats (50%) in the rAce-immunized group with a mean 6 standard deviation (SD) increase of 3.261.3 log 10 CFU (P = 0.0231) in the non-immunized group ( Figure 7B), showing significantly reduced bacterial titers in rAce-immunized group even when given a higher inoculum. Thirty-three sham (FCA -FICA)-immunized control group rats inoculated with 1.2-3.8610 8 CFU/rat also showed significantly higher bacterial counts with a mean 6 SD increase of 2.061.0 log 10 CFU/gm (P = 0.0475) versus the rAce active-immunized treatment group (n = 10) ( Figure 7B). These results demonstrate reproducibility of in vivo protection by active immunization with rAce against E. faecalis experimental endocarditis in rats using two different growth conditions to prepare the inocula.
In vivo protection against E. faecalis experimental endocarditis in rats using anti-rAce Igs for passive immunization Five of 6 (83%) of rats administered purified control Igs (2 mg/ kg) from pre-immune serum 1 h prior to inoculation of OG1RF developed E. faecalis IE compared with 2/10 rats (20%) of the group given anti-rAce Igs (2 mg/kg) affinity-purified from immune serum (P = 0.0001) (Figure 8). Mean bacterial titers recovered from control rat aortic vegetations showed 3.861.4 log 10 more CFU/ gm than the group given anti-rAce Igs (P = 0.0146) ( Figure 8). Thus, these results corroborated the protection results seen above in active immunization using rAce against E. faecalis IE in rats.

Mouse peritonitis model
In this in vivo model, both OG1RF and OG1RFDace caused animal mortality at similar rates with all the inocula tested (data with two inocula were shown in Figure 9) showing that OG1RFDace was not attenuated versus OG1RF.

Discussion
Infective endocarditis, which affects the endothelial lining of the heart, is among the most severe of the wide range of enterococcal  infections encountered in humans, presenting a major therapeutic challenge and resulting in considerable mortality even when treated with antibiotics [5,7,8,25]. Development of endocarditis can be initiated by injury to the valvular endothelium, which disrupts the normal valve structure and exposes underlying tissues, including ECM material. Deposition of host proteins, such as fibrin, as well as platelets at the site of injury then leads to the formation of a sterile thrombotic vegetation. This endovascular lesion may become colonized by circulating bacteria, leading to the growth of an infected vegetation. Valvular and aortic tissues are rich in collagen [26], and collagen is also found in sterile vegetations [26]. Previous studies have demonstrated that Ace plays a major role in the in vitro adherence of E. faecalis isolates to immobilized collagen [11,12,13]. Therefore, we reasoned that collagen could serve as a potential adhesion target for enterococci during bacteremia and that Ace could mediate bacterial attachment to these collagen-containing sites. To date, no studies have demonstrated a role for Ace in endocarditis and only very recently has a report appeared showing that Ace is important in a murine urinary tract infection model [27]. However, our previous demonstration of the role of host-derived cues (i.e., using moieties typically encountered in the host, such as serum or collagen [19,20]), for induction of both Ace expression and adherence of E. faecalis cells to collagen, suggested that both of these phenotypes are elicited by close association of this organism with a mammalian host/tissue. Moreover, we found that 90% of patients with prior E. faecalis endocarditis have Ace-specific antibodies in their sera, implying that Ace is expressed in vivo during the infection and that it is immunogenic [19]. For these reasons, we chose an experimental IE model in this study to explore the role of Ace in E. faecalis pathogenesis.
We first looked for direct evidence showing that Ace is expressed during E. faecalis infection. E. faecalis OG1RF cells recovered directly from infected vegetations showed surface expression of Ace with a much higher (,36) mean fluorescence intensity compared to cells grown in vitro in BHI at 37 uC, indicating that the host environment in vegetations, similar to collagen and serum [19,20], can induce production of Ace and its localization on the cell surface.
As anticipated, in vitro ECM protein adherence results with OG1RFDace corroborated our previous results with an insertionally inactivated ace [11,20]. Collagen adherence of the ace deletion mutant was restored by complementation in trans and the adherence of the complemented strain was 1.5-fold above the level of the WT parent strain, likely due to the higher number of Ace molecules displayed on the surface of the complemented strain, as determined by flow cytometry.  Deletion of ace resulted in significant attenuation in the ability of the mutated E. faecalis OG1RF strain to compete successfully with its isogenic WT parent in infection of vegetations in a mixedinoculum rat IE model. To the best of our knowledge, this is the first demonstration that Ace contributes to E. faecalis virulence in endocarditis. When we complemented the ace mutant in trans, significantly more colonization of heart valves was observed at 4 h after infection by this strain than by an isogenic strain containing an empty vector. Thus, these results confirmed our 72 h results with the ace deletion mutant and, furthermore, provide evidence that Ace plays an important role during the initial attachment and colonization stage of IE development, possibly by mediating adherence of E. faecalis cells to exposed collagen at the site of endovascular injury. This is consistent with the high surface expression levels of Ace in the complemented strain shown by our flow cytometry analysis. While stably maintained by the majority of E. faecalis cells during early colonization (4 h), the high instability of the complementation vector after extended growth in vegetations (94-98% of cells had lost the plasmid by 24 h) reduced its effect at later stages of endocarditis. The residual ability of OG1RFDace to cause endocarditis in some rats indicates that Ace is not absolutely required for E. faecalis to cause endocarditis; this is in agreement with published studies that showed a role for additional factors in causing E. faecalis IE [28,29,30]. While the precise mechanism of action of Ace for initiating, maintaining and/or propagating IE has yet to be elucidated, we infer that the difference in virulence of OG1RFDace may be due to its reduced ability to adhere to collagen. However, we cannot exclude the possibility of another ligand or another function of this protein.
Interestingly, deletion of ace did not result in observable effects in the mouse peritonitis model in terms of either time to death or total mortality, suggesting that ace is not important for this infection or that the direct administration of a large inoculum of bacteria into the peritoneal cavity may bypass an early infection stage where Ace might be involved. These results also indicate that deletion of ace did not affect growth or survival of OG1RF in vivo in general, consistent with the similar growth rate and viability of the ace deletion mutant and WT when grown in vitro.
We have recently shown that Acm, a collagen adhesin from E. faecium, is an important factor for endocarditis caused by that species [23]; this is similar to a previous observation with Cna of S. aureus [31], which is also involved in other infections, such as septic arthritis [32]. These MSCRAMMs share a large degree of sequence conservation in their collagen-binding domains; similar proteins are present in several other species of gram-positive pathogens, such as Streptococcus equi [33], Arcanobacterium pyogenes [34], Bacillus anthracis [35] and Streptococcus gallolyticus [36], and they possibly share a similar collagen-binding mechanism, called the ''Collagen Hug'' that has been characterized for Cna and Ace [15,37]. Therefore, it seems plausible that this family of proteins has been preserved or acquired across different gram-positive species/genera as a generalized mechanism to provide a binding function, although the ligand in the ecological niches where enterococci are found in nature and the purpose for these adhesins is not known. Recently, we described Ebp pili as another important factor for E. faecalis endocarditis [29], as well as urinary tract infections and biofilm formation [29,38], a further indication of the significance of surface proteins of the MSCRAMM family for E. faecalis pathogenesis.
Our results with active immunization of rats using the collagenbinding domain of Ace showed that only 25% of immunized rats developed endocarditis, while the infection rate in the untreated group was 100%. Protection was also evident when bacterial counts were evaluated. Consistent with these results, prophylactic treatment of rats with affinity-purified anti-Ace antibodies raised against the collagen-binding domain of Ace significantly reduced bacterial numbers in vegetations, demonstrating that passive transfer of Ace-specific antibodies confers significant protection against E. faecalis IE in rat. The differences in pre-infection procedures between the active-and passive-immunized groups preclude direct comparison of results from these two methods. Based on the results presented in this study, it seems likely that these preventive strategies specifically target the initial attachment and colonization stage of endocarditis by blocking collagen adherence of E. faecalis cells. Consistent with this hypothesis, we have previously shown that Ace-specific polyclonal antibodies purified from immunized rabbits or from humans with a prior E. faecalis endocarditis infection were effective in inhibiting adherence of Ace-expressing E. faecalis isolates to collagen [11,19]. Furthermore, a recent study that generated monoclonal antibodies against rAce showed that some of the mAbs completely inhibited binding of rAce to collagen and Ace-coated fluorescent beads to epithelial cell lines [21].The ace gene is ubiquitously present among isolates of E. faecalis and its encoded amino acid sequence, especially within the collagen-binding domain, is highly conserved [19]. Therefore, targeting ace could potentially offer protective immunization against a large spectrum of genetically diverse E. faecalis isolates, an advantage over other virulence-associated factors, such as aggregation substance, hemolysin and gelatinase, which were found to be produced by ,45% of endocarditis isolates [39] and for which protective efficacy has not been shown [40]. So far, only one E. faecalis antigen, the capsular polysaccharide, has shown promise as a potential vaccine candidate, as passive and active immunization against it lowered bacterial counts in kidneys, spleens and livers in a mouse i.v. infection model [41]. To our knowledge, our study is the first report of an immunization strategy that reduces E. faecalis colonization of aortic valves and shows protection against the development of E. faecalis endocarditis, thus, suggesting Ace as a promising alternative target for prophylaxis of E. faecalis endocarditis in high risk patients. However, the ability of OG1RF to cause IE in some of the rAce-immunized rats and also in some anti-Ace antibody-treated rats indicates that targeting multiple MSCRAMMs may be necessary for a robust protection of E. faecalis IE. Consistent with this, a recent study [42] showed full vaccine protection against abscess formation or lethal challenge with S. aureus strains when a combination of four MSCRAMM antigens were used versus a moderate reduction in bacterial load when used as individual vaccine antigens.
In summary, we have demonstrated here that i) deletion of the ace gene resulted in significant attenuation of the ability of E. faecalis to colonize aortic valves and cause endocarditis in an experimental rat IE model, coinciding (ii) with reduced in vitro adherence by the ace deletion mutant to collagen types I and IV; we have also shown that (iii) Ace is actively expressed within host vegetations during endocarditis and that (iv) both active and passive immunization against the collagen-binding domain of Ace conferred significant protection against endocarditis and reduced the numbers of bacteria found in vegetations. Taken together, 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 6 SD increase of 3.861.4 log 10 OG1RF CFU/gm from vegetations versus anti-rAce Ig treated rats (P = 0.0146 by unpaired t test). doi:10.1371/journal.ppat.1000716.g008 these results demonstrate that Ace is an important virulenceassociated factor and a likely target for prophylactic and therapeutic strategies against E. faecalis endocarditis. Since Acelike proteins are widespread among streptococci and staphylococci, future cross-protection studies may reveal novel opportunities for the development of vaccines or immunotherapeutics that may be useful for the prevention and treatment of gram-positive infective endocarditis.

Ethics statement
The rat endocarditis model and surgical procedures were performed in accordance with the institutional policies and the guidelines stipulated by the animal welfare committee, University of Texas Health Science Center at Houston (AWC, UTHSC). This study was reviewed and approved by the University Institutional Review Board (AWC approval # HSC-AWC-08-067).
Bacterial strains, plasmids, materials, standard molecular techniques, and growth conditions E. coli and E. faecalis strains and all plasmids used in this study are listed in Table 1. All constructs were given TX numbers and plasmids from these constructs were assigned respective pTEX numbers (Table 1). E. coli strains were grown in Luria-Bertani media (Difco Laboratories, Detroit, Mich.). Enterococci were grown either in BHI, BHIS, Todd-Hewitt (TH) broth/agar (Difco Laboratories) or Enterococcosel TM Agar (EA) (Becton Dickinson) at 37uC, unless a different growth temperature is specified. The following antibiotic concentrations were used with E. faecalis: chloramphenicol 10 mg/ml, kanamycin 2000 mg/ml, rifampicin 100 mg/ml and gentamicin 125 mg/ml. With E. coli, the concentrations used were chloramphenicol 10 mg/ml, kanamycin 50 mg/ml, and gentamicin 25 mg/ml. Resistance of enterococci to gentamicin and kanamycin was defined as MICs .500 and .2000 mg/ml, respectively [18].

Materials
All antibiotics were obtained from Sigma (St. Louis, Mo.). Tran 35 S-label and bovine serum albumin (BSA) were purchased from MP Biomedicals Inc. (Irvine, Calif.). C I and CIV were from Sigma and Fg was from Enzyme Research Laboratories (South Bend, Ind.). Oligonucleotide primers were purchased from Invitrogen (Carlsbad, Calif.) or IDT (Coralville, Iowa) or Sigma and their sequences are provided in Table 2. Restriction enzymes and DNA modification enzymes were mostly from Invitrogen and New England BioLabs, Inc. (Beverly, Mass.). All other chemicals used in the investigation were of molecular biology grade.

Standard molecular techniques
Chromosomal DNA from E. faecalis isolates was prepared following the hexadecyltrimethyl ammonium bromide method described earlier [43]. Plasmid DNA was isolated from E. coli using the Wizard Plus SV minipreps DNA purification system (Promega Corporation, Madison, Wis.) and, from E. faecalis, by a previously described method [44]. General recombinant DNA techniques such as ligation and agarose gel electrophoresis were performed using standard methods [45]. When necessary, DNA fragments were purified with low melting temperature agarose gel followed by purification using QIAquick-gel extraction kit (Qiagen Inc., Valencia, Calif.). PCR reactions were performed with a Perkin-Elmer GeneAmp PCR system 9700 using the optimized buffer B (1 6 buffer: 60 mM Tris-HCl [pH 8.5], 15 mM ammonium sulfate and 2 mM MgCl 2 ) obtained from Invitrogen. PCRgenerated fragments were purified using the Wizard PCR DNA Cleanup System (Promega Corporation). Recombinant plasmids were generated in E. coli DH5a or XL1-blue. Electroporation of E. coli and E. faecalis was carried out using a Gene Pulser (Bio-RAD Laboratories, Richmond, Calif.) as described previously [46,47]. Agarose plugs containing genomic DNA were digested with SmaI and PFGE was performed using a previously described method [1]. Southern blotting was performed using Hybond-N + nylon membrane and 0.4 N sodium hydroxide solution. Preparation of colony lysate blots was described elsewhere [48]. The RadPrime DNA Labeling System (Invitrogen) was used for labeling DNA probes with [a-32 P] dCTP (GE Healthcare, Piscataway, N.J.) and hybridizations were carried out using high stringency conditions [48,49]. DNA sequencing reactions were performed by the Taq dye-deoxy terminator method and an automated ABI Prism sequencer (Applied Biosystems, Foster city, Calif.).

Construction of an ace deletion mutant in OG1RF and its complementation
An E. faecalis ace mutant (OG1RFDace::cat) was constructed by allelic replacement using pTEX5500ts as described earlier for E. faecium [50]. We used a replacement strategy in this study to facilitate distinguishing between WT and OG1RFDace during in vivo animal experiments with mixed cultures; bioinformatics and mRNA analyses of the ace locus predicts the absence of a polar effect of ace deletion by the cat replacement (unpublished data). E. faecalis OG1RFDace was constructed by allelic replacement using pTEX5500ts as described earlier for E. faecium [50]. A 1027-bp DNA fragment (AceDelUp) encompassing the region upstream of ace was amplified from OG1RF genomic DNA template using primers AceDelF1 and AceDelR1 (Table 2), digested with BamHI and HindIII, and ligated with similarly digested pTEX5500ts. Similarly, a 989-bp DNA fragment (AcedelDn) encompassing the region downstream of ace was amplified from the same genomic DNA template using primers AceDelF2 and AceDelR2 ( Table 2). The AceDelDn PCR product digested with PstI and EcoRI was ligated to similarly digested pTEX5500ts::AceDelUp and was then transformed into E. coli DH5a to obtain TX5428. The plasmid from this construct, pTEX5428 (pTEX5500ts::AceDelUp+Ace-DelDn), was introduced into electrocompetent cells of OG1RF and cells were then plated on gentamicin plates at the permissive temperature (28uC). A single gentamicin and chloramphenicol resistant colony from these plates was grown overnight at 42uC, then plated on chloramphenicol plates and incubated at 37uC. After confirming the specific single crossover integration (OG1R-FaceUp::pTEX5428) by PCR (with primer sets AceUpF1 and CmR as well as AceDnR1 and CmF), one of the integrants was picked, grown for eight overnight serial passages at 42uC, and then plated on BHI to select for plasmid excision by double crossover recombination. The colonies from these BHI plates were then replica plated to chloramphenicol plates and gentamicin plates to identify colonies that retained the cat gene but not the vector.
To complement OG1RFDace in trans, an ,2 kb fragment containing the ace open reading frame plus its ribosome-binding site (amplified using primers aceComF1 and aceComR1; Table 2) was cloned under the control of the P2 promoter of the shuttle vector, pAT392 [51]. This in vitro-ligated construct for complementation (designated as pTEX5646) was transformed into E. coli XL1-Blue to obtain TX5646 and was then introduced into electrocompetent cells of TX5467 to obtain TX5647 (OG1RFDace (pAT392::ace). Surface expression of Ace in OG1RFDace (pA-T392::ace) was determined by flow cytometry (see below).

Growth curves
Overnight cultures were inoculated into BHI broth at a dilution of 1:100. The cultures were then grown at 37uC with shaking in an orbital shaker and aliquots were removed hourly from 0 to 12 h and at 24 h, for determining the absorbance at 600 nm (OD 600 ) with a spectrophotometer.

In vitro adherence assay
Adherence of E. faecalis to CI, CIV, Fg and BSA was determined in four independent experiments using Tran 35 S-labeled bacteria by a previously described assay [11].
Expression and purification of (His) 6

tagged Ace A domain
Construction of the recombinant plasmid pTEX5254 (complete ace A domain of OG1RF cloned into pBAD/HisA expression vector) was described previously [11]. Expression cultures of TX5254 were induced with arabinose and the N-terminally His 6 tagged proteins were purified using nickel affinity chromatography and anion exchange chromatography, as described previously [11,52]. Protein concentrations were determined by absorption spectroscopy at 280 nm using calculated molar absorption coefficient values [53].

Ace specific polyclonal antibodies
Expression and purification of (His) 6 -tagged recombinant Ace A domain was done using a previously described construct and methods [11]. Goat polyclonal serum against recombinant rAce A domain (rAce) was generated by Bethyl Laboratories (Montgomery, TX). Ace A-domain specific antibodies were eluted from rAceA coupled to cyanogen bromide-activated Sepharose 4B, according to the manufacturer's protocol (Amersham Biosciences, Piscataway, N.J.). The antibodies were concentrated by ultrafiltration with a 10,000-Da molecular-weight-cutoff filter (Millipore, Bedford, Mass.), dialyzed against PBS and concentrations were determined by absorption spectroscopy.

Protein extraction and Western blotting
Surface protein extracts from E. faecalis isolates were prepared using mutanolysin (Sigma) as described earlier [11]. Protein extracts were electrophoresed in 4-12% NuPAGE Bis-Tris gels (Invitrogen) under reducing conditions in MOPS buffer, and transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were then incubated with either affinity-purified anti-Ace A-domain specific immunoglobulins (Igs) [11] or pre-immune rabbit serum Igs followed by horseradish peroxidase-conjugated anti-goat antibodies. The blots were then developed with Supersignal West Pico Chemiluminescent substrate (PIERCE, Rockford, Ill.). Purified recombinant Ace A-domain was used as a positive control.

Flow cytometry
Bacteria. Surface expression of Ace on OG1RF or OG1RFDace (pAT392::ace) cells was detected by flow cytometry using affinity purified Ace A-domain (ligand-binding domain)specific antibodies, as described earlier [54]. Bacteria grown for 10 h in appropriate conditions were probed with pre-immune or affinity purified anti-rAceA specific-antibodies followed by donkey anti-goat IgG F(ab') 2 fragment conjugated with R-phycoerythrin (Jackson Immunoresearch Laboratory, West Grove, Pa.). The cells were fixed in paraformaldehyde and analyzed with a Coulter EPICS XL AB6064 flow cytometer (Beckman Coulter, Fullerton, Calif.) and System II software.

Endocarditis
vegetations. Sterile vegetations were produced in 11 rats, as described below, and nine rats were injected (i.v.) with OG1RF. Vegetations harvested after 48 h from the nine infected and two non-infected rat heart valves were processed to remove host tissue debris, using a previously described method [23]. Processed samples in groups of three infected vegetations were mixed and then divided into aliquots for labeling with a) Igs purified from antiserum raised against formalin-killed E. faecalis HH22-whole-cells (positive control) and b) affinity-purified anti-rAce-specific Igs. To assess possible crossreactivity of anti-rAce Igs with host tissue, the non-infected vegetation sample was probed with affinity-purified anti-rAcespecific Igs. Forward scatter (for analysis of particle sizes in the sample) and side scatter (for analysis of cell granularity or internal complexity) of vegetation processed cells were analyzed and compared with the in vitro grown E. faecalis OG1RF cells.

Antibody titers
Anti-Ace antibody titers in rat sera were determined by ELISA as described by [19] with some modifications. Briefly, 96-well plates (Immulon 4HBX, Thermo Fisher Scientific, Waltham, Mass.) were coated with 1 mg of rAce in 0.05 M carbonatebicarbonate buffer, pH 9.6. Rat sera were tested in duplicate with serial dilutions from 1:100 to 1:240,800, followed by detection with peroxidase-conjugated anti-rat secondary antibody (Jackson Im-munoResearch Laboratory, West Grove, Pa.) and TMB peroxidase substrate (Bethyl Laboratories, Montgomery, Tex.). The reaction was stopped with 2 M H 2 SO 4 . Antibody titers were expressed as the highest serum dilution with an A 450nm $0.10 at 3 min after addition of the substrate [55].
Testing the effect of E. faecalis OG1RF, ace mutants, complemented mutants and immunizations in experimental endocarditis Aortic valve endocarditis was produced in rats by following previously published methods [23,30,55,56,57,58]. In brief, for induction of endocarditis, white Sprague-Dawley rats (,200 gm) were used. The animals were anesthetized with isoflurane for placement of intravascular catheters. The right carotid artery was exposed and a sterile polyethylene catheter was inserted through a small incision and advanced to ,4 cm into the left ventricle. Proper positioning was assured by sensing resistance and vigorous pulsation of the line.
Mixed infection competition assay using OG1RF and ace mutants. For testing OG1RF and OG1RFDace in a mixed infection competition assay, bacteria (,1:1 by OD) were inoculated via the catheter 20 min after catheter placement; the catheter was heat-sealed and left in place during the course of the experiment and the skin was closed with sutures. Bacterial geometric mean (GM) CFUs determined for OG1RF and OG1RFDace from the inocula were 3.8610 7 /rat and 4.4610 7 / rat, respectively. Animals were sacrificed 72 h after infection. Hearts were aseptically removed and aortic valves were examined. The platelet-fibrin vegetations formed on aortic valves were excised, weighed, homogenized in 1 ml of saline and dilutions were plated onto BHI and EA media. Ninety-six colonies/rat were picked into microtiter plate wells containing BHI, grown for 4-6 h and then replica plated onto EA and BHI supplemented with chloramphenicol 10 mg/ml, or BHI supplemented with kanamycin 2000 mg/ml in the case of the ace disruption mutant, to verify the phenotypic markers of OG1RF and OG1RFDace. DNA lysates from colonies obtained from the OG1RFDace plus OG1RF mixed infection were hybridized under high stringency conditions [48], using intragenic DNA probes of ace and cat in order to generate the percentage (%) of OG1RF and OG1RFDace colonies of the recovered bacteria from vegetations. Rats with sterile cultures of undiluted vegetation homogenates (,1 ml) were considered to have had no induction of endocarditis. Data were expressed as percentages (%) of WT and mutant per vegetation. For vegetations showing only OG1RF colonies, for example, when 96/96 colonies were cat negative and ace positive and 0/96 colonies were cat positive and ace negative, the proportion of OG1RFDace in the vegetation was assigned the value of 1/97 (,1%) (i.e., assuming the next colony picked would have been a mutant).

Mono-infection testing using complemented OG1RFDace
(pAT392::ace) and OG1RFDace (pAT392). For testing of OG1RFDace (pAT392::ace) versus OG1RFDace (pAT392) in the mono-infection model, rats were inoculated (i.v.) 24 h postcatheterization and were sacrificed at 4 h or 24 h post infection. Due to the instability of pAT392 seen in in vivo experiments, inocula were then grown in the presence of gentamicin (125 mg/ ml) and animals were sacrificed at 24 h post infection. To minimize the loss of pAT392 over time and to determine the role of Ace in early colonization of aortic valves by OG1RFDace (pAT392::ace) versus OG1RFDace (pAT392), we also sacrificed a group of 11 to 12 animals administered with bacteria grown in BHIS at 4 h post inoculation. Rats with sterile cultures of undiluted vegetation homogenates were considered to have had no induction of endocarditis. In order to determine the in vivo stability of plasmid pAT392 in complemented OG1RFDace (pAT392::ace) and OG1RFDace (pAT392), ,95 random colonies recovered from vegetations were picked into microtiter wells and were replica plated onto BHI supplemented with gentamicin 125 mg/ml versus BHI to differentiate between gentamicin resistant (the marker of pAT392) [51] and gentamicin susceptible colonies.
Estimation of WT OG1RF ID 50 in rat IE model. ID 50 of OG1RF was determined for BHI and BHIS grown cultures. Twenty one rats were inoculated (i.v.) with either BHI or BHIS grown OG1RF in a range of 1.6610 4 -2.6610 8 and 7.2610 5 -1.1610 9 CFU/ rat, respectively. The ID 50 was determined by the method of Reed and Muench [59].
Immunizations. For active immunization, animals were divided into three groups: (a) rAce active-immunized, (b) nonimmunized and (c) FCA -FICA sham-immunized. Immunizations were done following previously published methods [55,56]. In brief, animals in group (a) received an initial dose of 100 mg of rAce in FCA (week 1) followed by a second and third dose of 100 mg of rAce in FICA at a two weeks interval. Animals in group (c) received FCA (week 1) followed by a second and third dose of FICA at a two weeks interval. Surgeries and catheter placement were done as described above for testing the ace mutant. Twentyfour hours post catheterization, animals were inoculated (i.v.) using BHI-or BHIS-grown OG1RF and were sacrificed at 48 h post infection. Three independent experiments were done and results were combined.
For the passive immunization group, animals were injected (i.v.) with 2 mg/kg of affinity purified Ace Igs purified from immunized goat serum 24 h post-catheterization and 1 h prior to bacterial inoculation; controls received 2 mg/kg of purified Igs from preimmune goat serum. Animals were sacrificed at 24 h post infection. Two independent experiments were done and results were combined.
Post euthanasia procedures. Hearts were aseptically removed from all euthanized animals. The vegetations on aortic valves were excised, weighed, homogenized in 1 ml of saline and dilutions were plated onto BHI, EA and BHI supplemented with rifampicin 100 mg/ml for bacterial CFU recovery. Rats with sterile cultures of undiluted vegetation homogenates were considered to have had no induction of endocarditis.

PFGE
Randomly picked colonies recovered from vegetations of infected rats were also tested by PFGE to reconfirm the infecting organism [49]. In brief, agarose plugs containing genomic DNA were digested with SmaI and PFGE was performed using a previously described method with ramped pulse times of 5 s and 45 s.

Mouse peritonitis model
OG1RF and OG1RFDace were tested following our previously published method [60]. In brief, mice were injected intraperitoneally with appropriate dilutions of bacteria (BHI or BHIS), premixed with sterile rat fecal extract (SRFE) and were observed for 5 days for survival. Two-fold inocula (range of ,1610 8 -1610 9 CFU/ml) of both test bacteria were used to compare animal survival/mortality. LD 50 was determined using six mice per group and by the method of Reed and Muench [59].

Statistics
To compare the mean 6 SD values of the adherence results, an unpaired t test was used. Percentages (%) of OG1RF versus OG1RFDace present in mixed infection vegetations were analyzed by the paired t test. Similar to the method previously described for E. faecalis and E. faecium endocarditis using a mixed infection [23,29], the mean virulence index of the mutant relative to WT