Cell wall polymers of Enterococcus faecalis and uses thereof

Abstract

The present invention relates to enterococcal cell wall polymers and their uses in the prevention and therapy of bacterial infection.

Claims

1. A method for inducing an immune response to Enterococcus faecalis in a subject, the method comprising administering, to the subject an enterococcal cell wall component or an antibody that specifically recognizes the enterococcal cell wall component, wherein the enterococcal cell wall component is -D-GalpNAc-ribitol having the following structure: ##STR00005## wherein R.sub.1 is selected from -D-Glcp and -L-Rhap, and R.sub.2 is selected from H and -D-Glcp.

Description

(1) The present invention will now be further described in the following preferred non-limiting examples with reference to the accompanying figures. For the purposes of the present invention, all references as cited herein are incorporated in their entireties.

(2) FIG. 1 shows opsonophagocytosis and complement deposition of E. faecalis mutants in WTA biosynthesis. A: Opsonophagocytic killing of E. faecalis V583 and WTA biosynthesis mutants in the presence of 1.7% absorbed baby rabbit complement alone, complement in combination with human white blood cells (WBC), or complement, WBC and specific rabbit Ab. Percentage of killing was determined as a relative to colony-forming units from control tubes containing bacteria and WBCs. B: Phagocytosis of FITC-labeled, formalin killed bacteria after 15 min of incubation with human serum. C: C3b deposition on enterococcal bacterial cells measured by FACS after incubation with human serum.

(3) FIG. 2 shows the SDS-PAGE of enzymatically digested cell wall extracts. A Staining with periodic acid and Schiff's reagent (PAS). B Staining with Stains All. Lane 1: molecular mass marker; lane 2: E. faecalis V583 wild type; lane 3: E. faecalis V5831172.

(4) FIG. 3 shows the NMR spectroscopy of two preferred oligosaccharides (OS) I and II of the present invention. A .sup.1H NMR spectra of the anomeric region of OS I and OS II. ROESY spectra of V583 wt OS I (B) and OS II (C) are shown below. Positive signals are depicted in green indicating .sup.1H,.sup.1H connectivities through covalent bonds, negative signals are depicted in blue indicating .sup.1H,.sup.1H ones through the space. All measurements were conducted at 27 C. in D.sub.2O at 700 MHz with the internal standard acetone (.sub.H 2.225).

(5) FIG. 4 shows the structures of OS I (A) and OS II (B).

EXAMPLES

Methods and Materials

(6) Bacterial Strains, Growth Conditions, and Medium

(7) E. faecalis was cultivated at 37 C. without agitation in tryptic soy broth (TSB) (Carl Roth, Karlsruhe) which was supplemented with the respective antibiotics as indicated. Short term cultures were grown on tryptic soy agar (TSA) and stored at 4 C. Cultures for isolation of enterococcal cell wall polysaccharides were grown to stationary phase for 18 h and harvested by centrifugation. E. coli cultures were cultivated aerobically in Luria Bertani (LB) medium (Carl Roth, Karlsruhe) which was supplemented with antibiotics as indicated.

(8) Construction of an EF1172 Gene Insertion Mutant in E. faecalis V583

(9) A targeted insertion mutant of gene EF_1172 in E. faecalis V583 was done as described by Rigottier-Gois et al. (Rigottier-Gois L, Alberti A, Houel A, Taly J-F, Palcy P, Manson J, et al. Large-Scale Screening of a Targeted Enterococcus faecalis Mutant Library Identifies Envelope Fitness Factors. PLoS ONE. 2011 Dec. 15; 6(12):e29023), and the localization of the targeted insertion was verified by PCR and Southern blot as described previously.

(10) Sera and Abs in Complement Binding Studies

(11) Normal pooled human serum was obtained from healthy volunteers, who gave informed consent. Baby rabbit serum was obtained from Cederlane Laboratories. Clq-depleted serum was purchased from Quidel. Heat-inactivation of serum was performed by incubation for 20 min at 56 C. Serum was depleted of specific Ab to E. faecalis V583 by absorption with bacterial cells at 4 C. for 60 min. After absorption, bacteria were pelleted by centrifugation and the supernatant was passed through a 0.2 m filter.

(12) Opsonophagocytic Killing Assay

(13) Bacterial opsonophagocytosis by human white blood cells (WBCs) was measured as described previously (Theilacker C, et al. Serodiversity of Opsonic Antibodies against Enterococcus faecalis-Glycans of the Cell Wall Revisited. PLoS ONE. 2011; 6(3):e17839). Briefly, bacterial strains were grown to mid-logarithmic phase (OD.sub.600=0.4) in TSB and diluted with RPMI supplemented with 15% fetal calf serum. WBCs were purified from the blood of healthy volunteers by sedimentation with heparin-dextran buffer and remaining erythrocytes were removed by hypotonic lysis in 1% NH.sub.4Cl solution. Baby rabbit serum diluted 1:15 served as complement source. To remove natural Abs against target strains the complement was absorbed with E. faecalis V583 as described above. Rabbit serum raised against heat-killed E. faecalis V583 at a dilution of 1:2500 served as Ab source. In control tubes, Ab, complement, or PMNs were omitted from the assay. For the measurement of opsonophagocytosis, equal volumes of 2.510.sup.6 PMNs, 2.510.sup.6 CFUs bacteria, complement and heat-inactivated immune rabbit serum were combined. After 90 min incubation, the reaction was stopped at 4 C., PMNs were lyzed by hypotonic lysis, and viable cell counts were determined by plating of serial dilutions.

(14) Phagocytosis Assay

(15) The phagocytosis assay was performed as described with modifications (Rooijakkers S H, et al. Immune evasion by a staphylococcal complement inhibitor that acts on C3 convertases. Nat Immunol. 2005 September; 6(9):920-7). Human pooled serum was absorbed with the E. faecalis as described above and diluted in RPMI with addition of 0.05% human serum albumin (HSA) to the respective concentration. Next, 7.510.sup.4 freshly isolated human neutrophils and 7.510.sup.5 FITC-labeled, heat-killed E. faecalis V583 wild type or E. faecalis V5831172 were added and incubated for 15 min at 37 C. while shaking at 750 rpm. The reaction was stopped by adding 1.5% ice-cold paraformaldehyde in RPMI 0.1% HSA. Phagocytosis was analyzed by flow cytometry (FACSCalibur; Becton Dickinson) measuring fluorescence of 10,000 gated neutrophils. Heat-inactivated serum was used as a control for complement-independent phagocytosis.

(16) C3b Deposition on E. faecalis

(17) E. faecalis strains were grown to an OD.sub.600 of 0.5 in TSB and washed in HEPES.sup.++ buffer (20 mM HEPES, 140 mM NaCl, 5 mM CaCl.sub.2, 2.5 mM MgCl.sub.2) with 0.1% BSA. 12.510.sup.5 bacteria were incubated with serum for 30 min while shaking at 900 rpm. Bacteria were washed with PBS supplemented with 0.1% BSA. C3b deposition was detected using mouse anti-human C3d Abs (Quidel) and FITC-conjugated goat anti-mouse IgG (Protos). Fluorescence of 10,000 bacteria was measured by flow cytometry. Classical and lectin pathway was abolished by incubation bacteria with complement in the presence HEPES.sup.++ buffer plus 5 mM MgCl.sub.2 and 10 mM EGTA, pH 7.5. For specific inactivation of the classical pathway, Clq depleted serum was used (Quidel). The C3 convertase complex (C4bC2a) was measured using a monoclonal antibody to human C4/C4d (Quidel) and goat polyclonal anti-mouse FITC conjugate as secondary antibody (Dako). MASP-2 deposition was detected using a goat polyclonal anti-MASP-2 Ab (Santa Cruz Biotechnology).

(18) Isolation of Cell-Wall Polysaccharide

(19) For isolation of enterococcal cell wall polysaccharides, bacteria were cultivated as described above. Bacteria were washed and resuspended in digestion buffer (PBS plus 20 mM CaCl.sub.2, 20 mM MgCl.sub.2, NaN.sub.3 0.05%) and cleaved enzymatically with mutanolysin (0.01 mg/mL) and lysozyme (0.5 mg/mL) at 37 C. for 18 h. Afterwards, insoluble material was removed by centrifugation and the supernatant was treated with nucleases (DNase I and RNase, final concentration 0.1 mg/mL) for 18 h at 37 C. Proteins were degraded by digestion with proteinase K (0.1 mg/mL) for 8 h at 56 C. The supernatant was extensively dialyzed (10 ku MMCO) against H.sub.2O and lyophilized. For size exclusion chromatography (SEC), the sample was dissolved in 50 mM ammoniumcarbonate buffer (pH 8.8; NaN.sub.3 0.02%) and applied on a Sephacryl S 200 column (1.6100 cm, GE Healthcare). Hexose and phosphorus content were measured as described previously (Theilacker C, et al. Opsonic antibodies to Enterococcus faecalis strain 12030 are directed against lipoteichoic acid. Infect Immun. 2006 October; 74(10):5703-12), and positive fractions eluting at a K.sub.av of 0.29 and 0.31, respectively, were combined, dialyzed and lyophilized. The resulting material was dissolved in 20 mM NaHCO.sub.3 (pH 8.0, NaN.sub.3 0.02%) and subjected to anion-exchange chromatography (Q Sepharose FF, GE Healthcare).

(20) To cleave phosphodiester bonds, 10 mg sample was dissolved in 50 L 48% HF and incubated at 4 C. for 2 d. The material was neutralized and separated by SEC on Sephadex G50 (1.6100 cm column, Biorad). Fractions of the lowest molecular mass were further purified by SEC on Biogel P2 (1120 cm Column, Biorad), followed by high-performance anion-exchange chromatography (HPAEC, Dionex) applying a CarboPak PA 100 column (9250 mm) and an ED50 electrochemical detector (Dionex). Data analysis was performed using the Chromeleon Version 6.6 software.

(21) SDS-PAGE

(22) For SDS-PAGE of crude enterococcal cell wall carbohydrates 40 mL culture was spun down and the pellet was digested with mutanolysin, lysozyme, nucleases and proteinase K as described above. After extensive dialysis against H.sub.2O, 100 g of the lyophilized material was separated by a precasted 10% Nupage Novex BisTris gel (Invitrogen) in Nupage SDS-MES running buffer (Invitrogen). Carbohydrates were stained with Stains All or periodic acid Schiff's (PAS) reagent as described previously (Theilacker C, Kaczynski Z, Kropec A, Sava I, Ye L, Bychowska A, et al. Serodiversity of Opsonic Antibodies against Enterococcus faecalis-Glycans of the Cell Wall Revisited. PLoS ONE. 2011; 6(3):e17839; Hancock L E, Gilmore M S. The capsular polysaccharide of Enterococcus faecalis and its relationship to other polysaccharides in the cell wall. Proc Natl Acad Sci USA. 2002 Feb. 5; 99(3):1574-9).

(23) General and Analytical Chemical Methods

(24) Qualitative and quantitative analyses of neutral sugars were performed by gas chromatography (GC) of the hydrolyzed and peracetylated alditol acetates as described previously (Haseley S R, Holst O, Brade H. Structural and serological characterisation of the O-antigenic polysaccharide of the lipopolysaccharide from Acinetobacter haemolyticus strain ATCC 17906. Eur J Biochem. 1997 Mar. 15; 244(3):761-6). GC separations were conducted with an Agilent GC System (6890N) equipped with a poly-(5%-diphenyl-95%-dimethyl)-siloxan SPB-5-capillary column (30 m, 0.32 mm i.d.). Signals were detected by flame ionization and analyzed with the Agilent ChemStation Version B 01.01 software. The absolute configuration of sugars was determined by GC of peracetylated (S)-2-butanolglycosides.

(25) Methylation analysis was carried out by analyzing the partially methylated alditol acetates of the HF-treated material by GC-MS (Ciucanu I, Kerek F. A Simple and Rapid Method for the Permethylation of Carbohydrates. Carbohydrate Research. 1984; 131(2):209-17). Briefly, 150 g of the HF-treated lyophilized material was three times methylated (methyl iodide) in water-free DMSO (stored over molecular sieve [4 ]) with addition of powdered NaOH. The mixture was kept for 1 h at 20 C. stirring. Then, the methylated polysaccharides were extracted three times with 2 mL chloroform, dried and hydrolyzed with 4 M CF.sub.3COOH for 4 h at 100 C. Subsequently the material was evaporated with deionized H.sub.2O to remove residual CF.sub.3COOH and reduced with sodium borodeuteride (18 h in the dark). Peracetylation was performed as described above, followed by GC-MS analysis.

(26) Nuclear Magnetic Resonance Spectroscopy

(27) Nuclear magnetic resonance (NMR) spectroscopy was conducted on an Avance III Bruker 700 MHz Ultrashield Plus spectrometer, applying standard software (Bruker). The working frequencies for .sup.1H-NMR experiments were 700.75 MHz, for .sup.13C-NMR experiments 176.20 MHz and for .sup.31P-NMR experiments 283.67 MHz. For H-D exchange, samples were repeatedly solved in D.sub.2O (99.9%) and evaporated under nitrogen, and for final measurements the sample was solved in D.sub.2O (99.99%). All spectra were recorded at 27 C. Chemical shifts are assigned in ppm and calibrated according to values of acetone (.sub.H: 2.225; .sub.C: 31.45) used as internal standard.

(28) Correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear-Overhauser-enhancement spectroscopy (NOESY) and rotating-frame nuclear-Overhauser-enhancement spectroscopy (ROESY) spectra were recorded with datasets (t2t1) of 2048512 bitpoints. TOCSY and NOESY experiments were conducted phase sensitive with mixing times of 400 ms and 180 ms respectively. Heteronuclear 2D .sup.1H.sup.13C correlations were performed by heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond correlation (HMBC), and heteronuclear single quantum correlation (HSQC) experiments with datasets of 4096512 bitpoints.

(29) Results

(30) E. faecalis Mutants Impaired in WTA Biosynthesis are More Susceptible to Opsonophagocytosis in the Absence of Specific Ab

(31) Insertional mutants of genes with high homology to teichoic acid glycerol (tag) genes tagO (EF2198), tagA (EF1173) and tagB (EF1172) have been constructed previously (Table 1) (Rigottier-Gois L, Alberti A, Houel A, Taly J-F, Palcy P, Manson J, et al. Large-Scale Screening of a Targeted Enterococcus faecalis Mutant Library Identifies Envelope Fitness Factors. PLoS ONE. 2011 Dec. 15; 6(12):e29023).

(32) TABLE-US-00001 TABLE 1 Analysis of homologies of B. subtilis W23 and S. aureus COL teichoic acid biosynthesis genes to E. faecalis V583 by the BLAST algorithm. B. subtilis S. aureus % Id % Si % Id % Si Function EF2198 TagO 24 40 41 65 UDP-N-acetylglucosamine: undecaprenyl-P N- acetylglucosaminyl-1-P transferase EF1173 TarA 38 60 33 59 N-acetylglucosaminyl- diphospho-undecaprenol N-acetyl-mannosaminyl- transferase EF1172 TagB 28 48 26 47 glycerophosphotransferase

(33) The inventors studied these mutants in a heterologous opsonophagocytic killing assay using 1.7% baby rabbit serum depleted of specific Ab by absorption to the target strain as complement source (FIG. 1). E. faecalis V583 wild type was resistant to complement-dependent opsonophagocytosis by human neutrophils. Only after addition of specific Ab, bacteria were readily killed. In contrast, insertional mutants of E. faecalis in tagO (EF2198), tagA (EF_1173) and tagB (EF_1172) were highly susceptible to complement-mediated killing and the addition of specific Ab did not further promote opsonophagocytosis (FIG. 1, A). Next, the inventors incubated FITC-labeled, heat-killed E. faecalis cells with absorbed human serum to measure the uptake by human neutrophils by FACS analysis. At serum concentrations up to 10%, the rate of phagocytosed E. faecalis V5831172 was much higher compared to wild type bacteria (FIG. 1B).

(34) Inactivation of tagB (EF1172) in E. faecalis V583 Leads to Increased C3b Deposition by the Lectin Pathway

(35) To test whether higher phagocytic uptake of E. faecalis V5831172 was due to increased complement deposition, the inventors measured the amount of C3b bound to bacterial cells by flow cytometry. Human serum depleted of specific Ab by absorption with the target strain was used as complement source. Compared to wild type bacteria, higher concentrations of C3b bound to E. faecalis V5832198 (tagO homologue) and to E. faecalis V5831172 (FIG. 1C) were detected. C3b is the final product of the classical, lectin and alternative pathway of complement activation. Classical and lectin pathway both lead to the formation of the C3 convertase complex C4bC2b, which cleaves C3 into C3a and C3b. Ca.sup.2+ is an essential cofactor for the activation of classical and lectin pathway.

(36) Sensitivity to Complement Induced Opsonophagocytic Killing is Associated with Altered Cell Envelope Carbohydrates

(37) To investigate this hypothesis, the inventors characterized the structure of the cell wall-associated polysaccharides of E. faecalis wild-type and V5831172. To this end, cell wall fragments after depolymerization of peptidoglycan by mutanolysin and lysozyme were separated by SDS-PAGE and stained by PAS and Stains All (FIG. 2). SDS-PAGE of the cell wall fragments of the wild type strain revealed a broad band around 60 ku. In E. faecalis V5831172, in contrast, this band migrated distinctly slower and was not stained by cationic dye Stains All, suggesting a loss of negative charge motifs (FIG. 2).

(38) Cell Wall Extracts of E. faecalis V5831172 Contain Less Phosphorus and Glucosamine but More Rhamnose

(39) Cell wall extracts of the E. faecalis V583 wild type and its insertional mutant V5831172 were further purified by chromatography. First, the material was separated by SEC. Despite the different migration pattern on SDS PAGE, cell wall extracts of the wild type and mutant eluted at similar volumes in SEC, indicating a similar molecular mass. Material from E. faecalis V5831172, however, contained less phosphorus. For further analysis, fractions of the major carbohydrate-containing peak were combined (K.sub.Av 0.30) and subjected to anion-exchange chromatography. Material from E. faecalis wild type eluted as a major, phosphorus-containing peak at 175 mM NaCl from Q Sepharose. In contrast, the major peak in E. faecalis V5831172 did not bind to the anion-exchange column, again suggesting a loss of negative charge. Compositional analysis of the purified extracts revealed that polysaccharide of both strains contained Rha, Glc, GalN, GlcN, ribitol, and phosphate. Comparison of the molar ratios of sugars, ribitol and phosphate revealed that the V5831172 polysaccharide contained approximately fourfold less galactosamine and ribitol, and app. 2.5-fold less phosphate compared to the wild type. On .sup.1H NMR spectroscopy, anomeric proton signals of the cell wall polysaccharide of E. faecalis wild type and V5831172 differed, but heterogeneity of the anomeric region precluded a detailed analysis without further degradation of the molecule.

(40) Cell-Wall Polysaccharide of E. faecalis V5831172 Lacks Covalently Bound WTA

(41) To further investigate the structure of both cell-wall polysaccharides, phosphodiester bonds were hydrolyzed by aqueous HF and the hydrolysate was fractioned by SEC (Supplemental FIGS. 1 and 3 A). In comparison to the cell wall fragments of the wild type strain, the elution profile of the mutant polysaccharide lacked a low-molecular mass peak eluting near the total column volume. This low-molecular mass material from the wild type strain was further purified by size exclusion chromatography and compositional analysis was performed. It confirmed the presence of D-GalN, L-Rha, D-Glc, and ribitol as typical components of a ribitol-containing TA (Table 2). Further separation of this material by HPAEC revealed the presence of two different oligosaccharides designated OS I and OS II, which were then isolated by preparative HPAEC.

(42) TABLE-US-00002 TABLE 2 Compositional analysis of accessory cell wall polymers of E. faecalis after dephosphorylation with HF and SEC on Sephadex G50. In E. faecalis V583 1172 pool 3 was absent. Rha Glc Ribitol GlcN GalN Ala Glu Lys V583 wt 2116 643 N.D. 280 154 112 28 28 pool 1 V583 wt 363 170 27 198 178 972 274 287 pool 2 V583 wt 589 690 1180 N.D. 1668 14 6 7 pool 3 V5831172 3337 762 N.D. 268 123 129 30 28 pool 1 V5831172 524 110 N.D. 10 6 64 16 16 pool 2 N.D.: not detected. Rharhamnose, Glcglucose, GlcNglucosamine, GalNgalactosamine, Alaalanine, Gluglutamine, Lyslysine. Concentrations are expressed as nmol/mg.

(43) The .sup.1H NMR spectrum of OS I (see FIG. 4) showed two different signals in the anomeric region, i.e., at 4.88 (A1) that was assigned as the anomeric proton of -L-Rhap and at 4.58 (B1) which was annotated as anomeric proton of -D-GalpN. The signal at 1.27 (A6) was assigned to the methyl protons of Rha. The .sup.1H NMR spectrum of OS II (see FIG. 4) comprised three different signals in the anomeric region. One signal at 4.59 (E1) represented the anomeric proton of a -D-GalpN residue, another at 4.98 was assigned to the anomeric proton of -D-Glcp (D1), and that at 4.45 (F1) to the anomeric proton of -D-Glcp. The .sup.1H NMR spectra displayed signals at 2.06 (OS I) and 2.03 (OS II), indicating N-acetylation of GalpN in both samples. Thus, the corresponding structures were the trisaccharide -L-Rhap-(1.fwdarw.3)--D-GalpNAc-(1.fwdarw.1)-ribitol for OS I and the branched tetrasaccharide -D-Glcp-(1.fwdarw.4)-[-D-Glcp-(1.fwdarw.3)-]-D-GalpNAc-(1.fwdarw.1)-ribitol for OS II.