Abstract
Ehrlichia chaffeensis has type 1 and 4 secretion systems (T1SS and T4SS), but the substrates have not been identified. Potential substrates include secreted tandem repeat protein (TRP) 47, TRP120, and TRP32, and the ankyrin repeat protein, Ank200, that are involved in molecular host–pathogen interactions including DNA binding and a network of protein–protein interactions with host targets associated with signaling, transcriptional regulation, vesicle trafficking, and apoptosis. In this study we report that E. chaffeensis TRP47, TRP32, TRP120, and Ank200 were not secreted in the Agrobacterium tumefaciens Cre recombinase reporter assay routinely used to identify T4SS substrates. In contrast, all TRPs and the Ank200 proteins were secreted by the Escherichia coli complemented with the hemolysin secretion system (T1SS), and secretion was reduced in a T1SS mutant (ΔTolC), demonstrating that these proteins are T1SS substrates. Moreover, T1SS secretion signals were identified in the C-terminal domains of the TRPs and Ank200, and a detailed bioinformatic analysis of E. chaffeensis TRPs and Ank200 revealed features consistent with those described in the repeats-in-toxins (RTX) family of exoproteins, including glycine- and aspartate-rich tandem repeats, homology with ATP-transporters, a non-cleavable C-terminal T1SS signal, acidic pIs, and functions consistent with other T1SS substrates. Using a heterologous E. coli T1SS, this investigation has identified the first Ehrlichia T1SS substrates supporting the conclusion that the T1SS and corresponding substrates are involved in molecular host–pathogen interactions that contribute to Ehrlichia pathobiology. Further investigation of the relationship between Ehrlichia TRPs, Ank200, and the RTX exoprotein family may lead to a greater understanding of the importance of T1SS substrates and specific functions of T1SS in the pathobiology of obligately intracellular bacteria.
Highlights
Members of the family Anaplasmataceae consist of a group of Gram-negative obligately intracellular alphaproteobacteria belonging to the order Rickettsiales, and are responsible for various arthropod-borne diseases of mammalian hosts including ehrlichioses and anaplasmoses
Human monocytotropic the ehrlichiosis (HME) is an emerging life-threatening tick-borne zoonosis caused by Ehrlichia chaffeensis, which exhibits tropism for mononuclear phagocytes, and survives by evading the innate host defenses, most likely by secreting multiple effectors into the host cell
CHAFFEENSIS -SECRETED tandem repeat protein (TRP) AND ankyrin repeat protein (Ank) PROTEINS IN type 4 secretion system (T4SS) Expression of E. chaffeensis-secreted TRP and Ank proteins in A. tumefaciens We have previously demonstrated that TRP120, TRP47, TRP32, and Ank200 are secreted in E. chaffeensis-infected cells (Popov et al, 2000; Doyle et al, 2006; Luo et al, 2008; Zhu et al, 2009)
Summary
Members of the family Anaplasmataceae consist of a group of Gram-negative obligately intracellular alphaproteobacteria belonging to the order Rickettsiales, and are responsible for various arthropod-borne diseases of mammalian hosts including ehrlichioses and anaplasmoses. Human monocytotropic the ehrlichiosis (HME) is an emerging life-threatening tick-borne zoonosis caused by Ehrlichia chaffeensis, which exhibits tropism for mononuclear phagocytes, and survives by evading the innate host defenses, most likely by secreting multiple effectors into the host cell T4SS gene clusters (generally clustered into two to five groups) have been identified in the members of family Anaplasmataceae (Ohashi et al, 2002; Collins et al, 2005; Hotopp et al, 2006; Mavromatis et al, 2006; Alvarez-Martinez and Christie, 2009), and expression of Ehrlichia T4SS genes is upregulated during infection (Cheng et al, 2008). Using the Cre recombinase reporter assay for translocation (CRAfT) system developed in A. tumefaciens (Vergunst et al, 2000), translocation of an ankyrin repeat protein of Anaplasma phagocytophilum, AnkA, was recently reported (Lin et al, 2007)
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