Ehrlichia chaffeensis and E. canis hypothetical protein immunoanalysis reveals small secreted immunodominant proteins and conformation-dependent antibody epitopes
Tian Luo, Jignesh Patel, Xiaofeng Zhang, David H. Walker, Jere W. McBride
Abstract
Abstract Immunomolecular characterization of Ehrlichia chaffeensis ( E. ch .) and E. canis ( E. ca .) has defined protein orthologs, including tandem repeat proteins (TRPs) that have immunodominant linear antibody epitopes. In this study, we combined bioinformatic analysis and cell-free protein expression to identify undiscovered immunoreactive E. ch . and E. ca . hypothetical proteins. Antigenicity of the E. ch . and E. ca . ORFeomes ( n = 1105 and n = 925, respectively) was analyzed by the sequence-based prediction model ANTIGENpro, and we identified ~250 ORFs in each respective ORFeome as highly antigenic. The hypothetical proteins ( E. ch . n = 93 and E. ca . n = 98) present in the top 250 antigenic ORFs were further investigated in this study. By ELISA, 46 E. ch . and 30 E. ca . IVTT-expressed hypothetical proteins reacted with antibodies in sera from naturally E. ch .-infected patients or E. ca .-infected dogs. Moreover, 15 E. ch . and 16 E. ca . proteins consistently reacted with a panel of sera from patients or dogs, including many that revealed the immunoreactivity of “gold standard” TRPs. Antibody epitopes in most (>70%) of these proteins exhibited partial or complete conformation-dependence. The majority (23/31; 74%) of the major immunoreactive proteins identified were small (≤250 aa), and 20/31 (65%) were predicted to be secreted effectors. Unlike the strong linear antibody epitopes previously identified in TRP and OMP orthologs, there were contrasting differences in the E. ch . and E. ca . antigenic repertoires, epitopes and ortholog immunoreactivity. This study reveals numerous previously undefined immunodominant and subdominant antigens, and illustrates the breadth, complexity, and diversity of immunoreactive proteins/epitopes in Ehrlichia .