Ehrlichia chaffeensis Uses Its Surface Protein EtpE to Bind GPI-Anchored Protein DNase X and Trigger Entry into Mammalian Cells

Abstract

Ehrlichia chaffeensis, an obligatory intracellular rickettsial pathogen, enters and replicates in monocytes/macrophages and several non-phagocytic cells. E. chaffeensis entry into mammalian cells is essential not only for causing the emerging zoonosis, human monocytic ehrlichiosis, but also for its survival. It remains unclear if E. chaffeensis has evolved a specific surface protein that functions as an ‘invasin’ to mediate its entry. We report a novel entry triggering protein of Ehrlichia, EtpE that functions as an invasin. EtpE is an outer membrane protein and an antibody against EtpE (the C-terminal fragment, EtpE-C) greatly inhibited E. chaffeensis binding, entry and infection of both phagocytes and non-phagocytes. EtpE-C-immunization of mice significantly inhibited E. chaffeensis infection. EtpE-C-coated latex beads, used to investigate whether EtpE-C can mediate cell invasion, entered both phagocytes and non-phagocytes and the entry was blocked by compounds that block E. chaffeensis entry. None of these compounds blocked uptake of non-coated beads by phagocytes. Yeast two-hybrid screening revealed that DNase X, a glycosylphosphatidyl inositol-anchored mammalian cell-surface protein binds EtpE-C. This was confirmed by far-Western blotting, affinity pull-down, co-immunoprecipitation, immunofluorescence labeling, and live-cell image analysis. EtpE-C-coated beads entered bone marrow-derived macrophages (BMDMs) from wild-type mice, whereas they neither bound nor entered BMDMs from DNase X-/- mice. Antibody against DNase X or DNase X knock-down by small interfering RNA impaired E. chaffeensis binding, entry, and infection. E. chaffeensis entry and infection rates of BMDMs from DNase X-/- mice and bacterial load in the peripheral blood in experimentally infected DNase X-/- mice, were significantly lower than those from wild-type mice. Thus this obligatory intracellular pathogen evolved a unique protein EtpE that binds DNase X to enter and infect eukaryotic cells. This study is the first to demonstrate the invasin and its mammalian receptor, and their in vivo relevance in any ehrlichial species.

Ehrlichia chaffeensis Exploits Canonical and Noncanonical Host Wnt Signaling Pathways To Stimulate Phagocytosis and Promote Intracellular Survival

“…These domains associated with internalization were not examined in other TRPs but are predicted to be similar. Notably, another study reported that E. chaffeensis uses its surface protein EtpE to bind GPI-anchored protein DNase X to trigger entry via CD147 and hnRNP-K recruitment and actin mobilization (74,75). The domains in TRPs responsible for internalization are structurally distinct from EtpE and are not predicted to interact with DNase X.…”

Rickettsial endosymbiont in the “early‐diverging” streptophyte green alga Mesostigma viride

“…, Mohan Kumar et al. ). The Sca proteins are highly conserved among members of the Rickettsiales, suggesting an important functional role in the adhesion and/or invasion in these bacteria (Blanc et al.…”

“…, Mohan Kumar et al. ). BLAST analyses of M. viride and its rickettisal symbiont transcriptomes against these surface proteins revealed the presence of homologs to ku70 and rompB ( sca5 ), suggesting that the bacterial entry into the alga is mediated by the binding of the rickettsial rOmpB to the algal Ku70.…”

Discovery of in vivo Virulence Genes of Obligatory Intracellular Bacteria by Random Mutagenesis

“…Of remaining ORFs, ∼60 ORFs of the HF strain are likely required for macrophage infection, as orthologs of E. chaffeensis are essential for infection of macrophages including type IV secretion system apparatus (23 ORFs) and effector proteins (3), two-component regulatory system (6), and outer membrane proteins (porin, lipoproteins, invasin; ∼30 ORFs) etc. (Kumagai et al, 2006(Kumagai et al, , 2008Huang et al, 2008;Mohan Kumar et al, 2013;Rikihisa, 2015Rikihisa, , 2017Lin et al, 2016;Sharma et al, 2017;Teymournejad et al, 2017;Yan et al, 2018). Of the remaining ∼330 genes, most encode hypothetical proteins or proteins with unknown functions.…”

“…No known pathogen-associated molecular patterns (PAMPs) including endotoxin, peptidoglycan, flagella, common pili, or exotoxin has been detected in Ehrlichia spp., yet they induce acute and/or chronic inflammatory cytokines production in MyD88-dependent, but Toll-like receptors (TLR)-independent manner (Koh et al, 2010;Miura et al, 2011;Rikihisa, 2015). Several virulence factors of E. chaffeensis, which are required for bacterial entry, survival and proliferation of macrophages, have been demonstrated, including ehrlichial invasin that binds host cell receptor and triggers its entry into host cells, three type IV secretion system effectors, three pairs of two-component regulatory system, and outer membrane porins for nutrient acquisition (Kumagai et al, 2006(Kumagai et al, , 2008Mohan Kumar et al, 2013;Rikihisa, 2015Rikihisa, , 2017Lin et al, 2016;Sharma et al, 2017;Teymournejad et al, 2017;Yan et al, 2018). However, whether Ehrlichia spp.…”