Antigenic variation of Ehrlichia chaffeensis resulting from differential expression of the 28-kilodalton protein gene family

Abstract

The transcriptional activity and allele variation of the 28-kDa outer membrane protein gene (p28) of Ehrlichia chaffeensis were analyzed to determine the mechanism of the antigenic variation of the 28-kDa outer membrane proteins. Reverse transcriptase PCR amplification of mRNA indicated that 16 of the 22 members of the p28 multigene family were transcribed. Amino acid sequence analysis indicated that the p28-19 protein was produced in vitro in the Arkansas strain. The p28-19 gene and its promoter region were sequenced and compared in 12 clinical isolates of E. chaffeensis to determine allele variation. The variation of the p28-19 gene among the isolates is limited to three types represented by strains Arkansas, 91HE17, and Sapulpa, respectively. These results indicate that the majority of the p28 genes are active genes and that antigenic variation of the E. chaffeensis 28-kDa proteins may result from differential expression of the p28 gene family members rather than gene conversion.

FIG. 1.

Transcriptional analysis of the p28 genes and their intergenic sequences (sp) by RT-PCR. PCR amplification of the DNA template with Taq polymerase is shown as a positive control. On the left of each panel is a DNA standard(s) in base pairs. M, 1-kb DNA ladder markers.

FIG. 2.

Growth rate of E. coli. E. coli harboring plasmid pKK232-8 or pKK1314 was cultivated in LB medium containing ampicillin or LB medium containing both ampicillin and chloramphenicol (Ch). The standard deviation of the value at each time point was obtained from triplicates of one experiment. OD, optical density.

FIG. 3.

Growth curves of E. coli. E. coli harboring plasmid pKK1819, pKK1819S, pKK70, or pKK232-8 was cultivated in LB medium containing ampicillin or LB medium containing both ampicillin and chloramphenicol (Ch). The standard deviation of the value at each time point was obtained from triplicates of one experiment. OD, optical density.

FIG. 4.

(A) Clustal alignment of the promoter region of the p28-19 gene of the E. chaffeensis isolates studied. Low-passage isolates V1, V4, and V7 represent the three genetically defined groups Arkansas, 91HE17, and Sapulpa, respectively. (B) Clustal alignment of the coding region of the p28-19 gene of E. chaffeensis isolates. V1, V4, and V7 represent the three groups Arkansas, 91HE17, and Sapulpa, respectively.

FIG. 5.

Phylogenetic analysis of the alleles of p28-19 of the p28 multigene family of E. chaffeensis isolates. p28-15 to p28-18, which are the closest genes to p28-19, were used as the outgroup. Bootstrap values are shown at the nodes of the branches.