Identification of ABC transporters in vancomycin-resistantEnterococcus faeciumas potential targets for antibody therapy

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<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 cross-reacted 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.

Unraveling the Protein Targets of Vancomycin in Living S. aureus and E. faecalis Cells

“…faecalis encodes a large number of sugar-uptake systems such as ABC transporters that support bacteria to ferment nonabsorbed sugars in the gastrointestinal tract . The ABC transporter identified here represents a thus far uncharacterized protein that is most likely not involved in antibiotic resistance as it is present in both VRE and VSE strains . This is supported by gene expression data that show that there is no increased transcription upon incubation of bacteria with vancomycin (see Table S2 ([SI]).…”

“…40 The ABC transporter identified here represents a thus far uncharacterized protein that is most likely not involved in antibiotic resistance as it is present in both VRE and VSE strains. 41 This is supported by gene expression data that show that there is no increased transcription upon incubation of bacteria with vancomycin (see Table S2 ([SI]).…”

Where is the difference between an epidemic and a high endemic level with respect to nosocomial infection control measures? An analysis based on the example of vancomycin-resistant Enterococcus faecium in hematology and oncology departments

“…Our search yielded 36 outbreaks appropriate for this study [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49]. The mean duration of 31 outbreaks with information about its duration was 11 months (range 1 to 36 months).…”

Advances and Prospects in Vaccine Development against Enterococci

“…Lipoproteins are substrate-binding proteins that deliver the substrate to the corresponding ABC transporters [104]. The immunogenicity of the ABC transporters has also been studied against E. faecium and Streptococcus pneumoniae [105,106]. In S. pneumoniae, PsaA, a homolog of PsaAfm, was a promising vaccine candidate with broad coverage, administrated either solely or as a carrier protein with a synthetic oligosaccharide from S. pneumoniae serotype 14 [105].…”