Identification of ABC transporters in vancomycin-resistant Enterococcus faecium as potential targets for antibody therapy1

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Identi¢cation of ABC transporters in vancomycin-resistant Enterococcus faecium as potential targets for antibody therapy1 James Burnie a

a;b;


, Tracey Carter b , Gordon Rigg b , Samantha Hodgetts b , Michael Donohoe b , Ruth Matthews a;b

Infectious Diseases Research Group, University of Manchester, Oxford Road, Manchester M13 9WL, UK NeuTec Pharma plc, Central Manchester Healthcare Trust, Oxford Road, Manchester M13 9WL, UK

b

Received 30 January 2002 ; received in revised form 13 March 2002; accepted 15 March 2002 First published online 25 April 2002

Abstract The occurrence of an outbreak of septicaemias due to vancomycin-resistant Enterococcus faecium (VRE), in Manchester, UK, provided an opportunity to examine the antibody responses in patients infected by the same strain. Immunoblotting sera from 24 cases, six of whom died, showed an immunodominant cluster of antigens at 34, 54 and 97 kDa, with a statistically significant correlate between survival and immunoglobulin G to the 34 and 97 kDa bands (P 6 0.05). Screening a genomic expression library of VRE with seropositive serum and peritoneal dialysate from a survivor gave a recombinant clone with two contiguous open reading frames, the derived amino acid sequences of which both showed sequence homologue with ABC transporters, with a Walker A and Walker B motif and the signature sequence LSGGQ. The first open reading frame (putative VRE ABC1) showed 57% homologue with YbxA from Bacillus subtilis. A partial sequence (putative VRE ABC2) was also obtained, in the same recombinant clone, of a second ABC transporter with 72% homologue with ybaE from B. subtilis. Affinity selection with the seropositive serum and peritoneal dialysate used to screen the library showed that the eluted antibody bound to the 97, 54, 34 and 30 kDa bands. Direct amino acid sequencing identified this as a possible ABC transporter. Rabbit antiserum against peptides representing Walker A and an area adjacent to the Walker B site crossreacted with bands at 34, 54, 97, 110 kDa and at 30, 34 and 54 kDa respectively. This therefore appeared to be an immunodominant complex of ABC transporters of which the smallest was the 30 kDa antigen. Epitope mapping of this antigen with seropositive patients’ sera delineated three linear epitopes (KVGIV, FGPKNF and RVAI). The Walker A site represented by peptide 1 (GHNGSGKSTLAKTIN), epitope RVAI represented by peptides 2 (MRRVAIAGVLAMPRE) and 3 (ELSGGQMRRVAIAGV), epitope KVGIV represented by peptide 4 (LKPIRKKVGIVFQFP), and recombinant VRE ABC1 and VRE ABC2 expressed in Escherichia coli pBAD were then used to isolate human genetically recombinant antibodies from a phage antibody display library. An assessment of the protective potential of these antibodies was carried out in a mouse model of the infection. This study suggests that an ABC transporter homologue could be a target for antibody therapy against VRE infections. A 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : ABC transporter; Vancomycin-resistant Enterococcus faecium; Therapeutic antibody

1. Introduction Enterococcus faecium accounts for 10^15% of enterococcal isolates, and is the third most common pathogen in hospital patients and responsible for 12% of nosocomial

* Corresponding author. Tel. : +44 (161) 276-8827; Fax : +44 (161) 276-8826. E-mail address : [email protected] (J. Burnie). 1

The EMBL accession number for the sequence reported in this paper is AJ428868.

infections, with an associated mortality of 13.7% [1]. It is characterised by antibiotic resistance with reported rates of 50% resistant to vancomycin, 52.1% to high-level gentamicin, 58.3% to streptomycin and 97% to penicillin [2]. Vancomycin resistance has been associated with a poor prognosis in patients with bacteraemia, survival falling to 24% compared to 59% in cases of bacteraemia due to vancomycin-sensitive strains [3], although this ¢nding was contested when adjustments were made for the severity of the illness [4]. Hospitals in Northern England have been swept by a strain of vancomycin-resistant E. faecium [5] (VRE), which

0928-8244 / 02 / $22.00 A 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 9 2 8 - 8 2 4 4 ( 0 2 ) 0 0 3 0 4 - 8

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commonly caused septicaemia in infected neutropenic and renal patients. Di⁄culties in treatment led one a¡ected teaching hospital, in Manchester, UK, to use chloramphenicol as front line therapy in neutropenic patients with a VRE septicaemia, with mixed success. These experiences con¢rmed the lack of clinical response previously reported by others [6] and catalysed the search for new therapeutic agents. Before the antibiotic era, antibodies, in the form of immune serum therapy, were widely used to treat a range of bacterial infections [7]. In the case of Enterococcus faecalis and E. faecium, complement-dependent neutrophil-mediated killing was enhanced by anti-enterococcal antibodies raised in rabbits or from the sera of patients with endocarditis [8^10]. Phagocytosis-resistant strains of E. faecium have been identi¢ed [9,11]. This resistance was mediated by bacterial carbohydrate and reversed in the presence of complement by a polyclonal rabbit antiserum or derived antibody fragments against one of these strains [11]. The potential of capsular polysaccharides as targets for opsonophagocytic antibodies and as a vaccine candidate have also been reported [12,13]. Recent developments in antibody engineering have included the use of immunoglobulin mRNA from the antibody-secreting cells of patients who have recovered from a speci¢c infection to produce phage antibody display libraries [14^16]. The cDNA from the immunoglobulin genes encoding heavy- and light-chain variable domains are linked together to produce a library of human genetically recombinant antibodies. The retention of the antigen-binding capacity of these antibody fragments allows them to be matched with speci¢c antigens or their epitopes by a⁄nity puri¢cation (panning). The aim then becomes the identi¢cation of antigens associated with potentially therapeutic antibodies. This study describes the identi¢cation of an immunodominant ABC transporter homologue complex in VRE, associated with potentially therapeutic antibodies.

2. Materials and methods 2.1. Immunoblotting An antigen preparation was made from a clinical isolate of VRE grown in brain heart infusion broth (Oxoid, Basingstoke, UK) at 37‡C and fragmented as previously described [17]. The VRE was identi¢ed by Gram stain, rapid ID 32 Strep (bioMe¤rieux, Basingstoke, UK) and con¢rmed as the endogenous hospital outbreak strain [5] by pulsed-¢eld gel electrophoresis following SmaI digestion. It was resistant to ampicillin, vancomycin, teicoplanin and gentamicin. The following sera were examined by immunoblotting. Group 1: Control sera. Sera from hospital inpatients with no evidence of infection or colonisation by VRE (n = 60).

Group 2: Faecal carriers. All had either leukaemia (n = 29) or chronic renal failure (n = 5). Despite faecal carriage, there was no evidence of related sepsis as judged by pyrexia or positive urine, wound or blood culture. Group 3: Septicaemias (survivors). Sera from clinically septicaemic patients with blood culture con¢rmed VRE, successfully treated by chloramphenicol (n = 18). Patients were either neutropenic with acute myeloid leukaemia (n = 12) or non-neutropenic with chronic renal failure and concomitant abdominal sepsis (n = 6). In all cases, sera were examined within 72 h after starting therapy. In nine cases, additional sera were available, taken before the ¢rst positive blood culture. Group 4: Septicaemias (fatal). Sera from patients, who had acute myeloid leukaemia, and were neutropenic, who died from a VRE septicaemia despite chloramphenicol therapy and had at least one positive blood culture within 72 h of death (n = 6). Sera were collected within 72 h of starting therapy. There was no obvious clinical discriminator between the leukaemic patients who survived and those who died. They were septicaemic, neutropenic, treated by chloramphenicol therapy and infected by the hospital endogenous strain. Patients’ sera were examined at a dilution of 1:10 against immunoblots of VRE, as described previously [17]. IgM and IgG were assayed separately, except for the control Group 1 where they were combined. Blots were analysed as described previously [14,15]: antibody was recorded as present if the intensity of the bound antibody was s 50 mm by re£ectance densitometry (Chromoscan 3; Joyce Loebl) ; where multiple sequential sera were tested, titres were reported as constant if the variation in height of the trace remained within 5 mm whereas a rising antibody response was recorded if there was an increase of at least 30 mm. 2.2. Identi¢cation of antigens from a VRE genomic library Chromosomal DNA from the clinical isolate of VRE was partially digested by SauIIIa and fragments in the size range 2^6 kbp were inserted into the expression vector lambda ZAP Express (Stratagene Ltd., Cambridge, UK) essentially as described by Young and Davies [18]. The library was screened with a serum and peritoneal dialysate £uid (1:100 IgG) from a patient on chronic ambulatory peritoneal dialysis who survived the infection, having had positive blood and peritoneal cultures. The peritoneal dialysate contained antibodies dominant for the bands at 54 and 97 kDa (Fig. 1, lane 1) and the serum contained IgG for the bands at 34 and 97 kDa (Fig. 2, lane 7). Positive clones were detected by alkaline phosphatase-conjugated goat anti-human immunoglobulin (IgG; 1:5000; Sigma, Poole, UK). Antibodies in the screening serum and peritoneal dialysate (1:10) were a⁄nity puri¢ed against positive recombinant plaques. Bound antibody was then eluted with glycine bu¡er pH 2.8 and screened against an immu-

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ing the biotinylated fusion proteins were probed with a negative control serum, a late serum from a septicaemiasurviving patient (as illustrated in Fig. 2, lane 4), the peritoneal dialysate from an infected patient (Fig. 1, lane 1) and the serum originally used to screen the VRE library (Fig. 2, lane 7). Both VRE ABC 1 and VRE ABC 2 were also cloned into the vector pBAD by means of a pBADTA-TOPO cloning kit (Invitrogen Corp., Oxon, UK), protein induction initiated by 0.02% arabinose and the V5tagged recombinant protein con¢rmed by probing with a monoclonal antibody to the V5 tag (1:5000). 2.4. Epitope mapping Fig. 1. Immunoblot of VRE against: the peritoneal dialysate of an infected patient (lane 1); after a⁄nity selection (lane 2); rabbit pre- (lane 3) and post-immunisation (lane 4) with peptide 1; and rabbit pre- (lane 5) and post-immunisation (lanes 6 and 7) with peptide 2. Molecular masses are marked in kDa.

noblot of VRE. Direct amino acid sequencing was performed on the 34 and 97 kDa bands by the Biochemistry Department, University of Cambridge, Cambridge, UK. For DNA sequencing the positive clones (Sequenase version 2.0 kit; United States Biochemical, Cambridge, UK), the ¢rst set of annealing reactions was done with universal primers, T3 and T7; subsequent primers were derived from the sequences obtained from both the coding and non-coding strands. Similar proteins to the VRE ABC transporters were identi¢ed using a BLAST search on the National Center for Biotechnology Information, Baylor College of Medicine, BLAST WWW Server, BCM search launcher, by the BLAST P+BEAUTY program. 2.3. Construction of fusion proteins Gene fusion techniques were used to con¢rm the immunogenicity of VRE ABC1 and VRE ABC2. The PCR primers for VRE ABC1 were 5P GGA GTA ATC ATG GAG CC and 5P CGG ATG TCC ATA ACC AAT CCA CC and for VRE ABC2 5P GGT TAT GGA CAT CCG and 5P GAT CAA GTC CTG CCG TTG G. DNA from positive clones was ampli¢ed and the resulting products were ligated into the expression vector Pin Point Xa1 T-Vector to produce a fusion of the antigen with the 13 kDa biotinylation tag sequence of the vector (Promega, Madison, WI, USA). The ligation mixture was used to transform Escherichia coli JM109. Potential clones were examined by restriction analysis to con¢rm the presence of an insert of the correct size. Clones containing inserts of the correct size were grown to early exponential phase in Luria^Bertani broth and induced with 1 mM IPTG and growth continued for 2 h. E. coli cells were collected by centrifugation and examined for the expression of biotinylated fusion proteins by immunoblot analysis with streptavidin-conjugated alkaline phosphatase. Colonies express-

This procedure, described by Geyson et al. [19], was applied to the amino acid sequence derived for VRE ABC 2. It was synthesised as a series of overlapping nonapeptides on polyethylene pins using an epitope scanning kit (Cambridge Research Biochemicals, Cambridge, UK), the ¢rst nonapeptide consisting of residues 1^9, the second residues 2^10 etc. An IgG ELISA reaction (OD405 ) was used to determine the reactivity of each peptide with patients’ sera (1:200) after 30 min incubation. Sera were examined from both survivors (n = 3) and fatal cases (within 72 h of death; n = 3) of septicaemia due to VRE, as well as serum from a hospitalised control patient who had no evidence of VRE colonisation or infection (n = 2) (Table 1). 2.5. Rabbit antisera Polyclonal rabbit antisera were produced against two synthetic peptides in the animal facility at Manchester University: GHNGSGKSTLAKTIN (peptide 1) representing the Walker A site and MRRVAIAGVLAMPRE (peptide 2) an area before the Walker B site incorporating

Fig. 2. Immunoblots of VRE with paired sera from three patients who recovered form septicaemia, showing the IgG present early in the infection (lanes 1, 3 and 5 representing the three respective patients) and later in the infection (lanes 2, 4 and 6 respectively corresponding to the same patients). Lane 7 is the antibody used for screening the VRE library. Molecular masses are marked in kDa.

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Table 1 Epitope map showing sites at which the mean OD value in survivors was at least 2 standard deviations above the mean OD value for fatal cases or uninfected hospital inpatient controls Well No.

Epitope sequence

Hospital inpatient controls (n = 2) Septicaemia patients who died (n = 3) Septicaemia patients who survived (n = 3)

50 51 52 53 54 74 75 76 77 116 117 118 119 120 121

KVGIV KVGIV KVGIV KVGIV KVGIV FGPKNF FGPKNF FGPKNF FGPKNF RVAI RVAI RVAI RVAI RVAI RVAI

0.259 X 0.206 0.279 X 0.228 0.250 X 0.182 0.285 X 0.189 0.342 X 0.235 0.445 X 0.108 0.321 X 0.288 0.274 X 0.231 0.334 X 0.309 0.296 X 0.244 0.384 X 0.347 0.444 X 0.227 0.365 X 0.113 0.389 X 0.192 0.365 X 0.317

0.259 X 0.038 0.278 X 0.028 0.264 X 0.019 0.356 X 0.037 0.440 X 0.034 0.392 X 0.028 0.356 X 0.07 0.279 X 0.048 0.383 X 0.042 0.319 X 0.075 0.492 X 0.106 0.541 X 0.062 0.419 X 0.015 0.429 X 0.048 0.472 X 0.073

0.674 X 0.393 0.775 X 0.345 0.551 X 0.264 0.732 X 0.434 0.902 X 0.550 0.717 X 0.425 0.809 X 0.543 0.676 X 0.411 0.807 X 0.499 0.694 X 0.444 0.837 X 0.528 1.053 X 0.582 0.839 X 0.407 0.847 X 0.426 0.771 X 0.421

The overlapping amino acid sequences were derived by a comparison of ¢rst and last peptide sequences and were used to de¢ne the epitopes.

the epitope RVAI. Each peptide was dissolved in deionised water at a concentration of 2 mg ml31 and diluted 1:1 with Freund’s complete adjuvant (Sigma) before injection into New Zealand white rabbits. Animals were boosted every 2 weeks with the peptide at a concentration of 1 mg ml31 in Freund’s incomplete adjuvant (Sigma). Rabbits were bled after four immunisations and the sera puri¢ed by centrifugation. Pre-immunisation sera and sera from the sequential bleeds were immunoblotted at 1 in 40 against the VRE extract. 2.6. Preparation of human genetically recombinant antibodies A phage antibody display library, derived from a patient who had recovered from E. faecium sepsis, was panned as previously described [14^16], enriching for antigen-speci¢c recombinant antibodies against peptides 1 and 2, ELSGGQ MRRVAIAGV (peptide 3) and LKPIR KKVGIV FQFP (peptide 4) by panning against immunotubes coated with peptide (10 mg ml31 ). To obtain phages expressing recombinant antibodies against recombinant VRE ABC 1 and ABC 2, the latter were run on SDS^ PAGE gels, immunoblotted and overlaid with the phage library diluted 1 in 2 in 2% skimmed milk. Bound antibodies were eluted by triethylamine (14 Wl ml31 water) neutralised by 500 Wl of 1 M Tris HCl pH7.4 and rescued by exponential phase E. coli TG1. Phages were typed by BstNI ¢ngerprinting. 2.7. Animal assessment The VRE was grown overnight at 37‡C in brain heart infusion broth, washed in saline, and the concentration (determined by haemocytometer and plating of dilutions on blood agar) adjusted so that 1U109 CFU were injected as a 0.1-ml bolus into the lateral tail vein of female CD1

mice (22^24 g; Charles River). Two hours after challenge, randomised groups of ten animals were given intravenously 200 Wl of phage types 1^6 (peptide 2), 7 (peptide 3), 8^11 (peptide 4), 12^14 (VRE ABC 1) and 15 and 16 (VRE ABC 2) or a negative control phage (expressing antibody which did not bind E. faecium). The phage dosage was limited to 9 5U108 phage to avoid toxicity due to the phage itself. Bacterial cell counts were determined from the kidney, liver and spleen, expressed as the mean log10 CFU per gram plus standard deviation, and the treatment and negative control group counts were then compared using the Mann^Whitney U-test. For all tests, a P value of 6 0.05 was considered signi¢cant.

3. Results 3.1. Immunblotting patient sera Immunoblotting sera against VRE showed bands of apparent molecular mass ranging from 24 to 150 kDa (Table 2). In patients who recovered from a VRE septicaemia (Group 3), bands at 34, 54, 57 and 97 kDa were recognised by v 50%. In patients with VRE faecal carriage (Group 2), the 97 kDa was immunodominant, 21 (61%) having IgG and 12 (35%) having IgM to this band, followed by the band at 34 kDa, 15 (44%) having IgG and 8 (24%) having IgM. In contrast only two of the six fatal cases of VRE septicaemia (Group 4) had IgG to the 97 kDa band (33%) whilst none had an antibody to the 34 kDa band. The increased frequency of IgG to the 34 and 97 kDa bands in survivors of VRE septicaemia (Group 3) was statistically signi¢cant compared to the control group (Group 1) (P 6 0.0001 in each case) and fatal cases (Group 4) (P = 0.0333 and P = 0.0295 respectively). The increased frequency of IgG against the 54 kDa band in survivors

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Table 2 Immunoblot analysis of patients’ sera against VRE No. of patients with indicated antibody Antigens (apparent molecular mass; kDa)

150 130 110 97 89 87 85 83 81 76 73 71 67 65 59 57 54 50 48 40 34 30 29 26 24

Controls (Group 1)

Faecal carriers (Group 2)

(n = 60)

(n = 34)

IgM and IgG

IgM

IgG

IgM

IgG

IgM

IgG

0 0 0 3 0 0 2 0 4 3 0 0 2 0 0 7 4 0 0 4 1 2 0 0 1

0 0 1 12 1 0 1 0 4 0 0 0 1 0 0 4 4 0 0 1 8 0 0 0 1

0 0 5 21 4 0 1 0 3 1 0 1 4 0 0 10 12 0 0 4 15 1 0 1 3

1 1 1 5 1 1 1 2 4 0 1 2 4 0 1 5 10 0 3 0 2 0 1 0 0

3 1 4 16 5 1 1 1 3 1 1 2 3 2 1 9 14 2 3 6 11 4 1 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 2 0 2 0 0 0 2 2 0

1 0 1 2 1 0 0 0 0 0 0 0 0 0 0 0 2 0 2 1 0 0 2 0 0

(Group 3) was also statistically signi¢cant compared to controls (Group 1) (P 6 0.0001) and faecal carriers (Group 2) (P = 0.0087). The increased frequency of IgM to the 54 kDa band in survivors of septicaemia (Group 3) was statistically signi¢cant compared to controls (Group 1) (P 6 0.0001) and faecal carriers (Group 2) (P = 0.0022). The increased frequency of IgM to the 97 kDa band in survivors of septicaemia (Group 3) was statistically signi¢cant compared to controls (Group 1) (P = 0.0187) (Fisher’s exact two-tailed test; P 9 0.05). Serial sera were available from nine patients who survived the VRE septicaemia and the majority of these showed rising antibody levels (IgG and/or IgM) to the bands at 34 kDa (seven patients), 54 kDa (seven patients) and 97 kDa (all nine patients), as illustrated in Fig. 2. 3.2. Identi¢cation of VRE antigens Screening the VRE genomic library with the peritoneal dialysate from a patient infected with VRE gave a positive clone. A⁄nity selection, either with the original serum (Fig. 2, lane 7) or the corresponding peritoneal dialysate (Fig. 1, lane 1), demonstrated that when the bound antibody was eluted it cross-reacted strongly with the 97 kDa band and weakly with the bands at 54, 34 and 30 kDa on

VRE septicaemias : survivors (Group 3)

VRE septicaemias : fatal cases (Group 4)

(n = 18)

(n = 6)

immunoblot (Fig. 1, lane 2). The derived amino acid sequence produced two contiguous open reading frames with both proteins containing ATP-binding domains and sequences homologous to the ABC transporter proteins [20,21]. One of these proteins (putative VRE ABC1) was a complete sequence (apparent molecular mass 30.6 kDa) whilst the other coded only for the amino terminal end of putative VRE ABC2 (apparent molecular mass of fragment 19 kDa) (Fig. 3). The consensus ribosomal-binding site of Bacillus subtilis is AAA GG AGG, which is normally distributed between positions 35 and 311. In the case of the present clone these areas were represented by GAA GG AGT (VRE ABC1) and AAA GG ATG (VRE ABC2). The putative VRE ABC1 ends with a lysine, which in B. subtilis is perceived as a common terminal amino acid [22]. The homologues for the genes encoding the putative VRE ABC1 and VRE ABC2 are given in Table 3. The closest database match for the gene encoding VRE ABC1 was ybxA (encoding a protein of apparent molecular mass 31.3 kDa) and for VRE ABC2 it was ybaE (encoding a protein of apparent molecular mass 30.4 kDa). In B. subtilis, the ybxA and ybaE genes belong to the same operon, which also includes a gene coding for a membrane spanning domain protein ybaF. These belong to the extruder

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Fig. 3. Enterococcal ABC transporter DNA and amino acid sequences. The YbxA and YbaE protein amino acid sequences are shown underneath for comparison. Epitopes are in italics.

subfamily 12 group of proteins [23]. The VRE genes encoding the putative ABC1 and ABC2 were also homologous to ykoD, a gene from B. subtilis encoding an ABC protein with an apparent molecular mass of 54.6 kDa. The remainder of the clone contained the carboxy end homologue of ribosomal protein L17 that is the next protein in the genomes of B. subtilis, Bacillus stearothermophilus, Bacillus halodurans and Mycoplasma capricolum [24^27]. A search of the un¢nished database of E. faecalis V583 found a contig 10502 (TIGR database) which contained a homologue similar to VRE ABC1 (79.2% identity over

279 amino acids, with apparent molecular mass of 30.71 kDa) and a homologue similar to VRE ABC2 (51.9% identity over 289 amino acids, with apparent molecular mass of 32.35 kDa). 3.3. Gene fusion constructs Expression of VRE ABC1 gave a band at 44 kDa, corresponding to a biotinylated recombinant protein of 31 kDa, the biotinylation tag being 13 kDa. Expression of VRE ABC2 gave a band at 32 kDa, corresponding to

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Table 3 Table of gene homologues Homologue VRE ribosomal sequence B. stearothermophilus L17 B. subtilis L17 B. halodurans rplQ/L17 M. capricolum L17 VRE ABC 1 YbxA (YbaD) B. subtilis StrpA Streptococcus pyogenes YbxA homologue B. halodurans Y179 Mycoplasma genitalium Protein O mth 133 M. thermoautotrophicum CbiO MJ1088 Methanococcus jannaschii CbiO OT3 Pyrococcus horikoshii YkoD B. subtilis VRE ABC 2 Yba E B. subtilis Cbi O Propionibacterium freudenreichii Z3339 M. capricolum Art P E. coli YkoD B. subtilis CbiO Salmonella typhimurium

Identity observed 66% 66% 63% 51%

in in in in

63 63 63 60

residues residues residues residues

PO7843 P20277 O50635 PIR S48596

57% 56% 48% 42% 42% 41% 40% 30%

in in in in in in in in

271 272 273 264 263 259 281 249

72% 45% 61% 42% 40% 40%

in 162 residues P70970 in 161 residues U13043, cobalt transport in 100 residues P14788, sulfate transport in 158 residues CAA 60101, arginine transport over 168 residues AJ002571 in 168 residues L12006, cobalt transport

residues residues residues residues residues residues residues residues

the tag plus a 19 kDa protein. Both constructs were positive with: the late serum from a septicaemia-surviving patient; the peritoneal dialysate from an infected patient; and the serum originally used to screen the VRE library. Each was negative with serum from the negative control group. The non-biotinylated, V5-tagged, arabinose-induced recombinant VRE ABC 1 produced a single band at 31 kDa and VRE ABC 2 a band at 22 kDa when probed with the V5 monoclonal antibody. 3.4. Epitope mapping Epitope mapping identi¢ed three sites at which sera from patients who survived VRE septicaemia produced a mean optical density (OD), over three or more consecutive wells, at least 2 standard deviations above that of sera from septicaemia patients who died or uninfected hospital inpatient controls (Table 1). A comparison with the derived sequences from ybaE demonstrated that each epitope was highly conserved except FGPKNF, which is represented by FGPKMF in B. subtilis. Comparison with VRE ABC 1 demonstrated that RVAI was the only conserved epitope and this was used in peptides 2 and 3 for panning. 3.5. Antigen speci¢city of rabbit antisera Rabbit immunisation with peptide 1 gave rise to antiserum, which cross-reacted with bands at 34, 54, 97 and 110 kDa (Fig. 1, lanes 3 and 4), while immunisation with peptide 2 gave antiserum-recognising bands at 30, 34 and 54 kDa (Fig. 1, lanes, 5^7). Direct amino acid sequencing of the 34 kDa band gave MEP (the amino end of VRE

P40735 AF082738 AB017508 P47425, haemolysin secretion AE000928, cobalt transport U67551, cobalt transport PIR G64435, cobalt transport AJ002571

Fasta score

Reference

200 202 196 147

[25] [26] [27] [51]

818 802 698 590 521 506 501 436

[23,26,52,53] [54] [26] [55] [38,56] [42] [43] [23,26,52,53]

749 317 311 273 255 253

[23,26,52,53] [43] [51] [56] [23,26,52,53] [44]

ABC 1), while direct amino acid sequencing of the 97 kDa band gave REFSLQNT, showing an 87.5% identity with an area from Mdr50, a Drosophila P-glycoprotein/multidrug resistance gene homologue which is a member of the ABC super family [28]. 3.6. Human recombinant antibodies BstNI ¢ngerprinting of the PCR-ampli¢ed recombinant antibody inserts showed that before panning, the library was highly heterogeneous. After four rounds of panning: no focusing occurred against peptide 1; peptide 2 produced multiple copies of six types (DNA ¢ngerprint types 1^6); peptide 3 focused a single type (type 7); peptide 4, four types (types 8^11); VRE ABC1, three types (types 12^14); VRE ABC2, two types (types 15 and 16). Each of these was tested for activity in vivo. 3.7. Assessment in mice Phage antibody activity was assessed in terms of a statistically signi¢cant reduction (P 6 0.05 Mann^Whitney U-test) in organ colony counts (kidney, liver and spleen) when compared to the negative control phage. Phage type 6 (against peptide 2) showed a statistically signi¢cant reduction in colony counts in all three organs in four independently conducted sets of experiments (P 6 0.05). Phage types 9 and 10 (both against peptide 4) showed a signi¢cant reduction in all three organs; phage type 14 (against recombinant protein VRE ABC 1) in two organs (spleen and kidney); phage types 12 and 13 (against recombinant protein VRE ABC 1) and phage types 15 and 16 (against recombinant protein VRE ABC 2) in one organ (spleen).

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Table 4 Results of in vivo assessment of the human recombinant antibodies Expt No.a

Antibody type (DNA ¢ngerprint)

1

negative type 1 type 2 type 3 negative type 1 type 2 type 3 negative type 4 type 5 type 6 type 7 type 15 negative type 6 type 12 type 13 type 15 type 16 negative type 6 type 13 type 14 type 15 negative type 6 type 8 type 9 type 10 type 11

2

3

4

5

6

Antibody speci¢city

control

5 5 5 5 5 5 5 5 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

peptide 2 peptide 2 peptide 2 control peptide 2 peptide 2 peptide 3 control peptide peptide peptide peptide ABC2

2 2 2 3

control peptide 2 ABC 1 ABC 1 ABC 2 ABC 2 control peptide 2 ABC 1 ABC 1 ABC 2 control peptide peptide peptide peptide peptide

No. of mice Organ colony counts (mean log10 CFU g31 X S.D.)

2 4 4 4 4

Kidney

Liver

Spleen

6.43 X 1.33 6.54 X 1.31 6.55 X 1.35 6.65 X 1.30 6.70 X 1.12 6.51 X 1.49 6.57 X 1.56 6.69 X 1.15 5.72 X 0.78 5.20 X 0.98b 5.16 X 1.08b 4.94 X 0.99b 5.66 X 1.67 4.70 X 0.80c 4.96 X 1.07 4.16 X 0.37b 4.46 X 0.81b 4.94 X 1.09 4.50 X 0.41 5.26 X 1.37 5.84 X 1.83 4.44 X 0.82c 5.15 X 1.26b 5.53 X 1.72b 5.04 X 1.04b 5.82 X 1.88 5.07 X 1.07b 5.43 X 1.62 4.99 X 1.02b 4.78 X 0.87c 5.57 X 1.73

6.74 X 1.83 6.42 X 1.25 6.44 X 1.54 6.70 X 1.14 6.68 X 1.59 6.57 X 1.32 6.23 X 1.02b 6.66 X 1.45 6.30 X 1.42 5.71 X 0.69b 5.96 X 0.94 5.04 X 1.26c 6.04 X 1.03 5.30 X 1.23c 5.57 X 0.59 4.99 X 0.86b 5.00 X 1.17b 4.94 X 0.92b 5.01 X 0.94b 5.49 X 1.34 5.38 X 1.39 4.08 X 1.16c 4.88 X 0.82b 5.20 X 1.28 4.73 X 0.64b 6.11 X 1.08 5.08 X 0.96c 5.55 X 1.59b 5.41 X 0.21b 5.27 X 1.19b 5.45b X 1.31

6.62 X 1.58 5.84 X 0.72b 6.31 X 1.12 5.91 X 0.87b 6.22 X 1.11 5.62 X 0.46b 5.64 X 0.78b 5.77 X 0.30b 4.66 X 0.82 4.72 X 0.89 5.01 X 1.11 3.78 X 0.85b 4.78 X 0.88 4.08 X 1.08b 5.04 X 1.08 4.04 X 0.95c 4.53 X 1.67b 4.36 X 0.98b 4.14 X 0.96b 4.67 X 0.79 4.41 X 1.32 3.24 X 0.29c 4.41 X 0.66 3.36 X 0.30c 3.73 X 0.69b 5.30 X 1.56 4.22 X 1.12c 4.51 X 1.54 5.15 X 1.36 4.10c X 1.06 4.43b X 1.56

a

The phage dose was 108 X 0.5 PFU. Mice were culled at 24 h in experiment 1 but at 48 h for all following experiments. Semi-logarithmic reduction compared to negative control. c Logarithmic reduction compared to negative control. b

All other phage antibodies, types 1^5 (panned against peptide 2), type 7 (peptide 3) and types 8 and 11 (peptide 4), showed no signi¢cant activity. Further evaluation of the data by examination of the reduction in mean organ colony counts, expressed as log10 CFU per gram con¢rmed a similar trend (Table 4). Phage type 6 (against peptide 2) demonstrated in the four experiments a mean logarithmic reduction in counts of 0.88^1.17 (spleen), 0.75^1.4 (kidney) and 0.58^1.3 (liver).

4. Discussion This study characterised the antibody response to VRE septicaemia, and identi¢ed a cluster of immunodominant putative ABC transporter proteins, which could be targets for potential antibody therapy. The antibody pro¢le in survivors involved in an outbreak of VRE septicaemias was compared with that of fatal cases, faecal carriers of VRE and uninfected hospital inpatients. There was a statistically signi¢cant increase in the frequency of IgG to the

34 and 97 kDa bands in survivors compared to fatal cases of VRE septicaemia and uninfected controls, and in the frequency of IgG and IgM against the 54 kDa band in survivors, compared to uninfected controls and faecal carriers of VRE. Serial sera from nine patients who survived the VRE septicaemia showed rising antibody levels (IgG and/or IgM) to the bands at 34 kDa (seven patients), 54 kDa (seven patients) and 97 kDa (all nine patients). Immunodominant bands at 54 and 98 kDa have previously been reported in enterococcal infections [17,29]. Screening a VRE genomic library with serum and peritoneal dialysate from a patient who survived VRE septicaemia and peritonitis and had antibody to the 34, 54 and 97 kDa bands, revealed a positive clone which, on DNA sequencing, comprised two contiguous open reading frames, both encoding putative ABC transporter proteins (VRE ABC1 and VRE ABC2). Members of the ABC (ATP-binding cassette) super family share a highly conserved protein, called the ABC module, that not only displays the Walker A and Walker B motifs common to all ATP-requiring proteins, but in addition, a short, highly

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conserved sequence (consensus LSGGQ) called the signature sequence or linker peptide, which is displayed uniquely by and thus identi¢es members of the ABC super family [30^32]. Both VRE ABC1 and VRE ABC2 had Walker A and Walker B sites and the signature sequence LSGGQ. Immunoblot analysis of the antibody eluted from this positive clone showed cross-reactivity with the 97, 54, 34 and 30 kDa bands. Rabbit immunisation with synthetic peptides representing the Walker A site (peptide 1) and a conserved epitope, RVAI, occurring just before the Walker B site (peptide 2), gave rise to antisera which cross-reacted with bands at 34, 54, 97 and 110 kDa (peptide 1), and bands at 30, 34 and 54 kDa (peptide 2). Direct amino acid sequencing of the 34 and 97 kDa bands also indicated homologue with ABC transporter proteins (with VRE ABC1 and a Drosophila ABC transporter protein respectively). This suggests that the 34, 54 and 97 kDa bands associated with antibody responses in patients, who recovered from VRE septicaemia, are part of an ABC transporter family in VRE. ABC transporters have also been identi¢ed as immunodominant antigens in infections due to E. faecalis [33,34] and methicillin-resistant Staphylococcus aureus [15]. In the latter, recovery from MRSA septicaemia was associated with seroconversion of surviving patients to an ABC transporter of 61 kDa (EMRSA-15 ABC protein) [15]. Multiple ABC transporters have been implicated as virulence factors in Gram-positive bacterial infections, pathogenesis being studied by in vivo expression and signaturetagged mutagenesis [35^37]. Homologue studies (Table 3) show the closest database match for the gene encoding VRE ABC1 was ybxA and for VRE ABC2, ybaE, both encoding monomeric ATPases in B. subtilis of apparent molecular mass 31.3 and 30.4 kDa respectively. The ybxA and ybaE genes belong to the same operon in B. subtilis, which also includes a gene coding for a membrane spanning domain protein ybaF. These are members of the subfamily 12 extruders, which have not been reported in E. coli [23]. It includes the product of ykoD (a dimeric ATPase with an apparent molecular mass of 54.6 kDa) in which the N- and C-terminal nucleotide-binding domains are related to ybxA and ybaE respectively. The VRE genes encoding the putative ABC1 and ABC2 also shared some homologue with ykoD. The ybxA gene is located upstream of the ybaE gene in the same operon, which has led to speculation that ykoD occurred by fusion of these genes after duplication of the operon. Nevertheless, the presence of an equivalent structure in the archaeon Methanobacterium thermoautotrophicum suggests that the fusion occurred early in evolution [38]. Recently MsrC, a member of the ABC transporter family, has been described from E. faecium [39^40]. This dimeric ATPase, associated with macrolide resistance, has an apparent molecular mass of 56 kDa and is homologous to MsrA from S. aureus which itself is homologous to the EMRSA-15 ABC protein [15]. Therapeutic activity has

187

been demonstrated by human recombinant antibodies against this latter molecule in a mouse model of infection [15]. The synthetic peptide (peptide 1) representing the Walker A site derived from VRE ABC2 was identical in nine out of 10 amino acids to the corresponding area in the previously described E. faecium MsrC [39^40], so that an antiserum raised against this peptide would be likely to cross-react with MsrC. The antiserum produced a band on the VRE immunoblot at 54 kDa, suggesting that this might be a homologue of E. faecium MsrC. An alternative explanation is that this 54 kDa band is a homologue of ykoD, which has an apparent molecular mass of 54.6 kDa. Another ATPase of 54 kDa has been cloned from E. faecium with a di¡erent sequence; this was shown to be present in cell wall preparations [41]. The function of the putative VRE ABC1 and VRE ABC2 proteins is at this stage unknown. There is some homology (Table 3) with proteins involved in cobalt transport [42^45], which in B. subtilis has been linked to tetracycline transport [46]. E. faecium possesses endogenous e¥ux pumps that excrete tetracycline [47]. Cobalt has been shown to activate an aminopeptidase in Streptococcus sanguis and Saccharomyces cerevisiae [48]. Both VRE ABC1 and VRE ABC2 had Walker A, Walker B and the linear peptide LSGGQ, which have all been shown to play an essential role in the transport process [31]. The VRE ABC1 sequence also demonstrated a short motif at the end of the L5 strand which is thought to be implicated in signal transduction to the ABC domain by sensing conformational changes in the membrane spanning domain upon substrate binding [30]. This switch region is marked in Fig. 3 and is centred on an invariant histidine and associated threonine. Epitope mapping identi¢ed three sites at which sera from patients who survived VRE septicaemia were signi¢cantly more reactive than sera from septicaemia patients who died or uninfected hospital inpatient controls. Two of these (KVGIV and RVAI) were successfully incorporated into synthetic peptides, peptide 4 (KVGIV) and peptides 2 and 3 (RVAI). Therapeutic activity of phages panned against these epitopes and the recombinant VRE ABC1 and VRE ABC2 was assessed in a mouse model of systemic VRE infection. Activity was greatest with phages panned against peptides 2 and 4 and the recombinant VRE ABC1 and VRE ABC2. It was least with phages panned against peptide 3. Phage type 6 showed the greatest activity with a statistically signi¢cant reduction in colony counts in all three organs in four independently conducted sets of experiments (P 6 0.05). This antibody was produced against synthetic peptide 2 which incorporated the immunodominant epitope RVAI, an epitope that was conserved between VRE ABC1 and VRE ABC2. This sequence is part of the linker peptide between LSGGQ and the Walker B site [30] (Fig. 3). The ABC transporter family comprises cell membraneassociated export and import systems, which participate in

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the import and secretion of many di¡erent molecules (sugars, amino acids, oligopeptides, carbohydrates, toxins, ions, drugs etc.) [20,21,23]. In E. faecium systems for enterocin A excretion [49] and antibiotic resistance [40,47] have been described. In E. faecalis an ABC transporter system has been implicated in polysaccharide biosynthesis [50]. A humoral response which blocks ABC transporter function by targeting shared epitopes common to multiple transporters would simultaneously interfere with numerous processes key to the survival of the bacteria and its ability to function as a pathogen. It would explain the widespread immunodominance of these molecules in Gram-positive infections [14,15,33,34] and act as a potent method by which a single antibody could produce a therapeutic e¡ect. The potential therapeutic activity of such antibodies is now under further investigation.

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