A Novel Role for D-Alanylation of Lipoteichoic Acid of Enterococcus faecalis in Urinary Tract Infection

Background Enterococci are the third most common cause of healthcare-associated infections, which include urinary tract infections, bacteremia and endocarditis. Cell-surface structures such as lipoteichoic acid (LTA) have been poorly examined in E. faecalis, especially with respect to urinary tract infections (UTIs). The dlt operon is responsible for the D-alanylation of LTA and includes the gene dltA, which encodes the D-alanyl carrier protein ligase (Dcl). The involvement of LTA in UTI infection by E. faecalis has not been studied so far. Here, we examined the role of teichoic acid alanylation in the adhesion of enterococci to uroepithelial cells. Results In a mouse model of urinary tract infection, we showed that E. faecalis 12030ΔdltA mutant colonizes uroepithelial surfaces more efficiently than wild type bacteria. We also demonstrated that this mutant adhered four fold better to human bladder carcinoma cell line T24 compared to the wild type strain. Bacterial adherence could be significantly inhibited by purified lipoteichoic acid (LTA) and inhibition was specific. Conclusion In contrast to bacteraemia model and adherence to colon surfaces, E. faecalis 12030ΔdltA mutant colonized uroepithelial surfaces more efficiently than wild-type bacteria. In the case of the uroepithelial surface the adherence to specific host cells could be prevented by purified LTA. Our results therefore suggest a novel function of alanylation of LTA in E. faecalis.


Introduction
Enterococci are the third most common cause of healthcareassociated infections, which include urinary tract infections, bacteremia and endocarditis. Risk groups for invasive enterococcal infections include neonates, Intensive Care Unit (ICU) patients and immunocompromised hosts [1]. The increasing identification of enterococcal strains resistant to multiple antibiotics in recent decades represents a serious threat to therapy and emphasizes the need for a better understanding of the pathogenicity of these microbes.
Bacterial adherence is an important step in the disease process by facilitating colonization and translocation across the mucosal barrier, which eventually results in systemic dissemination within the host [2]. Like other Gram-positive bacteria (e.g., Staphylococcus aureus), the surface of E. faecalis is rich in exposed adhesins that mediate binding to human receptors or to various components of the extracellular matrix (ECM); they are thus considered a member of MSCRAMM-microbial surface component-recognizing adhesive matrix molecules [3]. Basically, MSCRAMMs are cell wall-anchored surface proteins that have characteristic immunoglobulin-like folds [4].
Attachment of microorganisms to mucosal surfaces of the urinary tract is important for the pathogenesis of UTI, because the mechanical removal of colonizing bacteria by the urine flow is an important innate defense mechanism. In the process of bacterial cell adherence, infectious agents interfere with specific molecules on epithelial cells [5][6]. Furthermore, it has been shown that the tendency of certain bacteria to infect specific tissues is often related to their ability to adhere to a specific target cell.
While UTI-specific virulence factors of E. coli have been studied extensively, relatively little is known about E. faecalis cell-surface structures with respect to UTIs [7-10 & 11]. In a model of ascending UTI, the presence of an enterococcal surface protein encoded by an acquired gene was shown to increase the persistence of bacteria in the urinary bladders of mice [7] whereas [9] demonstrated that E. faecalis has greater tropism for the kidneys. Using a similar model, the MSCRAMM adhesion of collagen of E. faecalis (Ace) was recently identified as a putative virulence factor involved in colonization of renal tissue [12][13]. In another study, Torelli et al. reported that a PavA-like fibronectinbinding protein in E. faecalis called enterococcal fibronectinbinding protein A (EfbA) contributes to the pathogenesis of enterococcal UTIs. The EfbA mutant shows attenuated colonization in a mouse model of mounting UTI [11]. Ebp, a biofilm-associated pilus, produces immunogenic and pleomorphic pili [14] and also plays a role in UTI pathogenesis [15].
Generally, the functions of most E. faecalis cell surface structures have been poorly investigated, especially with respect to UTIs [7][8][9][10]16]. The enterococcal cell wall contains peptidoglycan (PG) and a number of accessory cell-wall polysaccharides and glycoconjugates such as LTA wall teichoic acid (WTA), a rhamnose-containing cell-wall polysaccharide epa (enterococcal polysaccharide antigen) [17][18][19][20] and a diheteroglycan [21]. Lipoteichoic acid (LTA) is an amphiphilic polymer consisting of polyglycerolphosphate and a glycolipid anchor inserted into the cell membrane [22][23]. The dlt operon includes four genes and is responsible for the D-alanylation of LTA [24]. One of these genes, dltA, encodes the D-alanyl carrier protein ligase (Dcl), which activates the D-alanine and ligates it to the D-alanyl carrier protein (Dcp). The involvement of LTA in UTI infection by E. faecalis has not been studied. Here we examined the role of teichoic acid alanylation in the adhesion of enterococci to uroepithelial cells.

Bacterial strains and chemicals
E. faecalis 12030 strain is a clinical isolate obtained in Cleveland, OH. It is a strong biofilm producer and is opsonized by antibodies against its LTA [25]. E. faecalis 12030DdltA mutant and its complemented strain E. faecalis 12030DdltAcompl were kindly provided by C. Theilacker and F. Fabretti. All bacterial strains were grown at 37uC without agitation in Caso Bouillon (Carl Roth), and reagents were obtained from Sigma.

LTA extraction
LTA from E. faecalis 12030 wild type were isolated as described [26] and with modifications by Theilacker et al., 2006 [25]. Bacterial cells were grown in tryptic soy broth, harvested after 3 h and resuspended in 0.1 M citrate buffer (pH 4.7). The harvested cells were disrupted using glass beads (Beadbeater; Glenn Mills, Clifton, NJ) and stirred with an equal volume of n-butanol for 30 min at room temperature. After centrifugation, the aqueous layer was dialyzed against 0.1 M ammonium acetate (pH 4.7) and lyophilized. The material was redissolved in 15% n-propanol in 0.1 M ammonium acetate (pH 4.7) and applied to an octyl Sepharose column for hydrophobic interaction chromatography. Bound material was eluted with a gradient of 15-80% n-propanol. Fractions were assayed for total phosphorus and by immunoblot assay [26].

Invasion/Adherence assay
For the adherence assay, cells were cultivated in 24-well plates to a density of approximately 1610 5 cells/well for 16 h. Bacteria were inoculated at (OD 600 nm , 0.1) and grown to mid-log phase (OD 600 nm , 0.4) at 37uC in Caso Bouillon. T24 cells were incubated with bacteria for 2 h at a multiplicity of infection of 100:1. After infection of the monolayer, human bladder carcinoma cells were washed five times with bovine serum albumin (PBS, Biochrom AG) and lysed with DMEM F-12 Ham + 5% FBS + 0.25% Triton-X100 buffer for 15 min. Surface adherent and intracellular bacteria were enumerated by quantitative bacterial counts. Six replicates (wells) of each stimulation were prepared and the full experiment was repeated 3 times.

Inhibition assays
For inhibition of enterococcal binding to T24 cells, purified LTA (10-500 mg/mL) from wild type E. faecalis 12030 was added to the cells 30 min before infection. The assay was then performed as described above.

Mouse Urinary Tract Infection model
Female BALB/C mice (6-8 weeks old, Charles River, Sulzfeld, Germany) were used for the experiments. To prepare the inoculum, bacteria were grown in 5 mL for , 16 h at 37uC with gentle shaking. For inoculum preparation the overnight culture was diluted 1:100 in 50 mL fresh medium and grown to an OD 600nm , 0.3 at 37uC with shaking. The cells were pelleted for 10 min (6.0006g at 10uC) and resuspended in half of the volume of 0.9% saline. Further dilutions were prepared in Caso Bouillon and then plated to determine the actual inoculum. Before starting the experiment, the bladder of the mouse was emptied by catheter (20 mm). Subsequently, isoflurane-anesthetized mice were infected via urethral catheterization with 100 mL of the bacterial suspension (3-5610 8 CFU/mL). After 2 h the same amount of bacteria was injected again to achieve consistent infection. The urethral catheter was removed immediately after this step, and all animals had free access to food and water during the course of study. Mice were euthanized by CO 2 inhalation at 24 h and 48 h after transurethral challenge. The urinary bladder and kidney pairs were excised, weighed, and homogenized in 1 mL of 0.9% saline + 0.025% Triton X-100, and dilutions were plated onto Caso Agar without antibiotics. The CFU/100 mg of bacteria for each tissue (bladder or kidney) was calculated for each animal.

Statistical analysis
If not stated otherwise, the experiments were repeated three times. All cell culture tests were made by multigroup comparisons using ANOVA (PRISM4, GraphPad software). P value less than 0.05 was considered significant. Comparisons among groups were made using the Mann-Whitney U test. P value less than 0.05 was considered significant.

Ethics Statement
All animal experiments were performed in compliance with the German animal protection law (TierSchG). The mice were housed and handled in accordance with good animal practice as defined by FELASA and the national animal welfare body GV-SOLAS.

Results
E. faecalis 12030DdltA mutant shows strong adherence to uroepithelial cells To investigate the importance of LTA D-alanylation to in vitro adherence to uroepithelia, we used T24 human bladder cell lines, E. faecalis 12030DdltA mutant and the wild type E. faecalis 12030. As control, the E. faecalis 12030DdltA complemented strain was used. As shown in Figure 1a, compared to the wild type strain (0.6610 6 CFU/mL), the E. faecalis 12030DdltA mutant showed up to four fold increased adherence (2.9610 6 CFU/mL, p,0.001) to uroepithelial cells. The complemented mutant strain E. faecalis 12030DdltAcompl showed a partially restored effect of the mutation (1.2610 6 CFU/mL, p,0.001).

Bacterial adherence could be inhibited by purified LTA
To study the specific interaction between bacteria and a stillunknown receptor, T24 cells were first incubated with 100 mg/mL purified LTA from wild type E. faecalis 12030 strain to saturate binding sites and subsequently they were inoculated with bacteria. As shown in Figure 1b, the E. faecalis 12030DdltA mutant with LTA treatment showed significantly less adherence (1.25610 6 CFU/mL, p,0.01) to the bladder epithelial cell line as compared to without LTA treatment (2.9610 6 CFU/mL). In the wild type, no significant differences in adherence properties were observed using purified LTA and a dose dependent reduction of adherence using LTA could also demonstrated in Figure 1c.

E. faecalis 12030DdltA colonizes uroepithelial surfaces more efficiently than wild type bacteria
To study the effect of lipoteichoic acid on urinary tract infection, a modified mouse urinary infection model was used [9]. The wild type strain E. faecalis 12030 and the mutant E. faecalis 12030DdltA were tested. The bacterial counts in the bladder and in both kidneys were determined after 24 h and 48 h of incubation as described elsewhere [9]. As shown in Figure 2 after 24 h, the mutant colonized the bladder (4610 5 CFU/ 100 mg) significantly better compared to the wild type strain (9610 3 CFU/100 mg), i.e. up to 1.5 log more bacteria (p,0.001). This difference in colonization behaviour between mutant and wild type strains was 2.4 logs higher in kidneys (p,0.001). The mutant showed 3610 6 CFU/100 mg tissue compared to wild type 9610 3 CFU/100 mg. We observed colonization differences between mutant and wild type strains to bladder and kidney epithelia after the prolonged incubation time of 48 h. A statistically significant difference (p,0.05) was observed only in kidneys. Here the mutant colonized up to 9610 5 CFU/100 mg tissue whereas the wild type achieved only 1610 3 CFU/100 mg. In fact, the E. faecalis 12030DdltA mutant colonized the kidneys at both time points considerably better than the wild-type strain.

Discussion
Attachment of microorganisms to mucosal surfaces of the urinary tract is important for the pathogenesis of UTI because the mechanical removal of colonizing bacteria by the urine flow is an important innate defense mechanism. In the process of bacterial cell adherence, infectious agents interfere with specific molecules on epithelial cells [17,20,[5][6]27,28]. While UTI-specific virulence factors of E. coli have been studied extensively, relatively little is known about E. faecalis cell-surface structures with respect to UTIs [7-10 & 11].
In a model of ascending UTI, the presence of an enterococcal surface protein was shown to increase the persistence of bacteria in the urinary bladders of mice without histological changes [7] whereas Kau et al. demonstrated that E. faecalis has greater tropism for the kidneys [9]. Using a similar model, the MSCRAMMs of E. faecalis such as collagen adhesion protein (Ace) [12][13], PavA-like fibronectin-binding protein (EfbA) [11], and a biofilm-associated pilus (Ebp) [14,15] contributed to the pathogenesis of enterococcal UTIs.
The D-Ala-LTA formation racks up several functions, such as cationic homeostasis maintenance, integration of metal cations faecalis 12030DdltA complemented strain were tested for their ability to adhere to and/or invade T24 uroepithelial cells. Bacterial titers in logarithmic scale (log scale). T24 cells were cultivated in 24-well plates to a density of 1610 5 cells/well and incubated for 2 h with bacteria grown to mid-log phase (OD 600nm , 0.4) at a bacteriato-cell ratio of 100:1. Total cell-associated bacteria include surface-adherent and intracellular bacteria. Bars represent average 6 S.E. The E. faecalis 12030DdltA mutant shows strongly increased adherence to uroepithelial cells compared to the wild type (p,0.001). B) Treatment of cells with purified LTA. E. faecalis 12030 wild type and the E. faecalis 12030DdltA mutant were tested. Concentration of purified LTA was 100 mg/mL. The adherence to uroepithelial cells was significantly reduced by E. faecalis 12030DdltA mutant (p,0.01) after LTA treatment and no differences were obtained by wild type strain. In each experiment 6 replicates (wells) of each stimulation were prepared. The full experiment was repeated 3 times. Multigroup comparisons were made by ANOVA (PRISM4, GraphPad software). P-values of ,0.05 (*), ,0.005 (**) and ,0.0005 (***) were considered statistically significant. C) Dose-dependent reduction in adhesion using purified LTA. E. faecalis 12030DdltA mutant were tested with increasing concentrations of LTA (10 -500 mg/mL). doi:10.1371/journal.pone.0107827.g001 and inflection of autolytic actions and several other electromechanical properties of the bacterial envelope when the surface of gram-positive bacteria is exposed to disparate microenvironments. The cell wall of gram-positive bacteria contains different types of anionic molecules. Two different teichoic acids are present on the surface of gram-positive bacteria: lipoteichoic acid (LTA), an amphiphilic polymer non-covalently inserted into the cellular membrane, and wall teichoic acid (WTA), covalently linked to the peptidoglycan of the cell wall. These two molecules are synthesized by separate biosynthetic pathways that have been well characterized in Bacillus subtilis and other gram-positive bacteria [29]. Nevertheless, D-alanyl-ester substituents of WTA originally derive from LTA and are later transferred to WTA by transacylation. It is a single dlt operon encoding the genes responsible for the Dalanine incorporation [30][31][32].
The dltA operon has been studied in several gram-positive bacteria revealing an identical organization [33]. Bacteria with mutations in the dlt operon showed a variety of phenotypic changes that could be attributed to the resulting charge modification of their cell surface. The lack of D-alanine esters resulted in a stronger negative net charge, because D-alanine esters introduce positively charged groups into the otherwise negatively charged teichoic acids [34]. In B. subtilis and S. aureus, the absence of D-alanine has been shown to cause alterations in the activity of autolytic enzymes [35,36]. Also possible is an altered host immune response, leading to enhanced proliferation/persistence.
In this work, the E. faecalis 12030DdltA mutant has been investigated with regard to adherence to T24 bladder carcinoma cells. Surprisingly, we observed that it displayed more than fourfold increased adherence to human bladder carcinoma cells compared to the wild-type strain. We could partially restore this adherence to 1.310 6 CFU/mL using a complemented mutant strain (Figure 1a). Moreover, using purified LTA it was possible to significantly inhibit adherence to human bladder cells, demonstrating the specificity of binding (Figure 1b). A dose dependent reduction of adherence using LTA could also be demonstrated (data not shown). Interestingly, in contrast to our previous data, which showed that the same mutant exhibits less binding to Caco2 colonic epithelial cells than wild-type bacteria, here we obtained higher adherence to uroepithelial cells [3,25,37]. In addition, these data are in contrast to previous studies using S. aureus and Listeria monocytogenes, in which elimination of D-alanylation of LTA impaired adherence to mammalian cells [38][39][40].
To assess the relevance of these results and the differences in vivo, a modified mouse urinary infection model was used; our results confirmed that the E. faecalis 12030DdltA mutant colonized the kidneys significantly better than the wild-type strain after 24 and 48 h ( Figure 2). In addition, the mutant colonized the bladder significantly better after 24 and 48 h albeit the differences observed at 48 h were not statistically significant. This is assumed by the fact that E. faecalis usually does not persist in the bladder, resulting in decrease number of colony counts, because of clearance of bacteria from the bladder by mechanical forces of urine flow. However, the same E. faecalis 12030DdltA mutant demonstrated less colonization in a mouse sepsis model [37]. This disparate finding suggests the existence of specific receptors on bladder and/or uroepithelial cell surfaces. Our results therefore show that D-alanylation of LTA can decrease adherence to specific host cells and therefore suggest a novel function of alanylation of LTA in E. faecalis. Differential expression of the dlt locus may allow enterococci to adapt to specific ecological niches.