Identification of an immunodominant ABC transporter in methicillin-resistant Staphylococcus aureus infections

Abstract

Immunoblotting sera from 26 patients with septicemia due to an epidemic strain of methicillin-resistant Staphylococcus aureus (EMRSA-15), 6 of whom died, revealed an immunodominant EMRSA-15 antigen at 61 kDa. There was a statistically significant correlate (P < 0.001) between survival and immunoglobulin G to the 61-kDa band. The antigen was identified by sequencing positive clones obtained by screening a genomic expression library of EMRSA-15 with pooled sera from patients taken after the septicemic episode. Eluted antibody reacted with the 61-kDa antigen on immunoblots. The amino terminus was obtained by searching the S. aureus NCTC 8325 and MRSA strain COL databases, and the whole protein was expressed in Escherichia coli TOP 10F'. The derived amino acid sequence showed homology with ABC transporters, with paired Walker A and Walker B motifs and 73% homology to YkpA from Bacillus subtilis. Epitope mapping of the derived amino acid sequence with sera from patients who had recovered from EMRSA-15 septicemia delineated seven epitopes. Three of these epitopes, represented by peptides 1 (KIKVYVGNYDFWYQS), 2 (TVIVVSHDRHFLYNNV), and 3 (TETFLRGFLGRMLFS), were synthesized and used to isolate human recombinant antibodies from a phage antibody display library. Recombinant antibodies against peptides 1 and 2 gave logarithmic reductions in organ colony counts, compared with control groups, in a mouse model of the infection. This study suggests the potential role of an ABC transporter as a target for immunotherapy.

Figures

FIG. 1

Staphylococcal ABC transporter DNA and amino acid sequences. The YkpA protein amino acid sequence shown underneath for comparison. Walker A (A1 and A2) and B (B1, and B2) motifs in bold.

FIG. 1

Staphylococcal ABC transporter DNA and amino acid sequences. The YkpA protein amino acid sequence shown underneath for comparison. Walker A (A1 and A2) and B (B1, and B2) motifs in bold.

FIG. 2

Immunoblots of EMRSA-15 with paired sera from five patients with EMRSA-15 infection: case 1, pre- and postinfection sera showing IgM (lanes 1 and 2, respectively) and IgG (lanes 3 and 4); case 2, pre- and postinfection sera showing IgM (lanes 5 and 6, respectively) and IgG (lanes 7 and 8); cases 3, 4, and 5, preinfection (lanes 9, 11, and 13, respectively) and postinfection (lanes 10, 12, and 14, respectively) IgG. The 61-kDa band is marked.

FIG. 3

Immunoblots of EMRSA-15 showing one of the sera used to screen the phage expression library (lane 1), the subfraction of this serum which bound to and was then eluted from the positive clone (lane 2), the rabbit serum before (lane 3) and after (lane 4) immunization with the recombinant ABC protein. Bands at 97 and 61 kDa are marked.

FIG. 4

Elution of the ABC transporter from the Ni-NTA column by increasing levels of imidazole (125 to 200 mM fractions marked). Numbers on the left represents molecular mass markers (in kilodaltons). W, wash fraction, obtained after the flowthrough and before elution with imidazole, using 10 mM imidazole as instructed by the manufacturer.