Ehrlichia chaffeensis and E. canis hypothetical protein immunoanalysis reveals small secreted immunodominant proteins and conformation-dependent antibody epitopes

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.

Type 1 secretion system and effectors in Rickettsiales

“…While O. tsutsugamushi Ank1 and Ank6 localize to the nucleus in an importin β1-dependent manner, O. tsutsugamushi Ank13 co-opts eukaryotic RaDAR (RanGDP-ankyrin repeats) nuclear import ( Evans et al., 2018 ; Adcox et al., 2021 ). In addition, newly identified immunoreactive proteins from E. chaffeensis and E. canis ORFeomes (complete set of open reading frames) are also predicted to be secreted by the T1SS and are discussed in greater detail below ( Table 3 ) ( Luo et al., 2020 ; Luo et al., 2021 ), highlighting the largely uncharacterized roles of T1SS components and substrates in pathobiology and immunity to rickettsial pathogens.…”

“…Strong TRP- and Ank-specific antibody responses are observed in humans and dogs during infection, and antibodies directed at linear epitopes of E. chaffeensis TRP proteins are protective against ehrlichial infection ( Chen et al., 1997 ; Li et al., 2001 ; McBride et al., 2003 ; Kuriakose et al., 2012 ). Notably, recent advances have identified novel repertoires of undiscovered major immunoreactive proteins from E. chaffeensis and E. canis ORFeomes (complete set of open reading frames of the genome), many of which are predicted to be T1SS effectors with confirmation-dependent antibody epitopes ( Luo et al., 2020 ; Luo et al., 2021 ). These findings reveal the underappreciated and largely uncharacterized roles of the T1SS effector proteins in immunity to Ehrlichia and related obligates.…”

EhrlichiaNotch signaling induction promotes XIAP stability and inhibits apoptosis

“…E. chaffeensis immune evasion strategies are mediated, in part, by tandem repeat proteins (TRPs). TRPs are type 1 secretion system (T1SS) effectors that also elicit strong host antibody responses during infection (19–22). Notably, TRP120 decorates the surface of dense-cored ehrlichiae and has multiple moonlighting functions including as a transcription factor, HECT E3 ubiquitin ligase, and cellular signaling ligand mimetic to repurpose host cell signaling (2, 9, 13–16, 18, 23).…”

Ehrlichia TRP effectors: moonlighting, mimicry and infection

“…secretes many effector proteins by the type 1 secretion system (T1SS) as recently predicted by bioinformatic analysis (Luo et al . 2020 ). TRPs contain type 1 secretion signal sequences located in the C-terminal domain and were the first ehrlichial proteins to be identified as T1SS substrates using an E. coli complemented with the hemolysin secretion system (Fig.…”