Expression of members of the 28-kilodalton major outer membrane protein family of Ehrlichia chaffeensis during persistent infection

Abstract

The 28-kDa immunodominant outer membrane proteins (P28 OMPs) of Ehrlichia chaffeensis are encoded by a multigene family. As an indirect measure of the in vivo expression of the members of the p28 multigene family of E. chaffeensis, sera from two beagle dogs experimentally infected with E. chaffeensis were evaluated for the presence of specific antibodies to P28 OMPs by enzyme-linked immunosorbent assay. Antigenic peptides unique to each of the P28s were identified within the first hypervariable region of each P28 OMP. Serological responses to peptides derived from all P28 OMPs were detected from day 30 postinoculation to day 468 and from day 46 until day 159 in the two beagles. Although antibody titers to the peptides fluctuated, the peak response to all of the peptides appeared simultaneously in each dog. The antibody responses to another outer membrane protein of E. chaffeensis (GP120) showed similar temporal and quantitative changes. These data suggest that the P28 OMPs are expressed concurrently during persistent Ehrlichia infection.

FIG. 1.

Alignment of the amino acid sequences of HVR1 and adjacent sequences of E. chaffeensis P28 OMPs. The corresponding sequences of the synthetic peptides for each P28 OMP are underlined. Dots represent residues that were identical in the majority of sequences. Dashes indicate gaps that were introduced to facilitate alignment.

FIG. 2.

Protein immunoblotting of recombinant P28 proteins reacting with dog ACC serum obtained on day 88 postinoculation of E. chaffeensis diluted at 1:200 (top). The dog sera did not react with the Positope protein that was used as a negative control. Positive controls for each recombinant P28 protein reacting with antithioredoxin antibodies are shown on the bottom. The antithioredoxin did not react with P28-15 and P28-19 because these proteins were not thioredoxin fusion proteins.

FIG. 3.

Structures of truncated P28-19 proteins and their reactivity with dog sera. (A) Schematic diagram of the truncated P28-19 proteins illustrating three hypervariable regions (HVR1, HVR2, and HVR3) and their flanking sequences. Protein immunoblotting of sera from dog ACC (B) and dog ADJ (C) reacted with overlapping truncated recombinant proteins of P28-19: Δ1, Δ3, Δ2/3, and Δ1/2/3. The days postinoculation of E. chaffeensis when the sera were obtained are shown below the blots. The same amount of each protein was used in the Western blots for both dog sera.

FIG. 4.

Reaction of dog immune serum to P28-1′ proteins with synthetic peptides. The 22 synthetic peptides are shown on the x axis, and the OD values of the immune serum reacting with each peptide are shown on the y axis.

FIG. 5.

Reactivity of all 22 synthetic peptides derived from HVR1 of P28 OMPs with the sera of dog ACC. The 22 time points representing dog sera obtained on days 0 (prior to inoculation of E. chaffeensis), 8, 15, 23, 30, 38, 46, 53, 60, 67, 74, 81, 88, 95, 102, 110, 117, 131, 145, 159, 248, and 462 postinoculation of E. chaffeensis are shown on the x axis. The height of the bars represents the increase in OD of an infected dog serum reacted with a P28 peptide over the OD of the day 0 serum from the same dog reacting with the same peptide shown on the y axis. Each bar represents the reaction of a peptide with a serum collected at one time point. The reactions of the dog sera with recombinant proteins P28-5 and GP120 were included for comparison with the synthetic peptides and recombinant outer membrane proteins. The dashed lines indicated the 0.5-fold cutoff for a positive ELISA reaction.

FIG. 6.

The reactivity of 22 HVR1 synthetic peptides with the sera from dog ADJ. The 22 time points representing dog sera obtained on days 0 (prior to inoculation of E. chaffeensis), 8, 15, 23, 30, 38, 46, 53, 60, 67, 74, 81, 88, 95, 102, 110, 117, 131, 145, 159, 248, and 462 postinoculation of E. chaffeensis are shown on the x axis. Each bar represents the reaction of a peptide with one serum collected at one time point. The height of the bars represents the increase in OD of an infected dog serum reacted with a P28 peptide over the OD of the day 0 serum from the same dog reacting with the same peptide. The reactions of the dog sera with recombinant proteins P28-5 and GP120 were included for comparison with the antibody reaction to the synthetic peptides and recombinant outer membrane proteins. The dashed lines indicate the 0.5-fold cutoff for a positive ELISA reaction.