Ehrlichia chaffeensis Tandem Repeat Effector Targets Differentially Influence Infection

Abstract

Ehrlichia chaffeensis infects mononuclear phagocytes and survives intracellularly by exploiting host cell processes to evade host defenses. The mechanisms involved are not fully defined, but appear to rely largely on a subset of tandem repeat proteins (TRP) effectors. E. chaffeensis TRPs are type 1 secreted effectors that interact with a functionally diverse group of host cell targets associated with various biological processes. In this study, we investigated the influence of TRP host target proteins on ehrlichial infection by RNA interference. In total, 138 TRP-interacting host proteins identified by yeast two-hybrid were targeted by siRNA and the infection level determined by real-time qPCR. Knockdown of 124 (89%) TRP target proteins had significant influence on infection either by inhibiting (85%) or promoting (15%) ehrlichial infection. Notably, knockdown of 18 host proteins which interacted with TRP120 promoted the infection, suggesting that these targets may be degraded to promote infection. Host proteins that interact with TRPs are involved in cellular processes, including cell signaling, vesicle trafficking and intracellular transport, transcriptional regulation, metabolism, protein posttranslational modification, and apoptosis. Selected host targets were examined by immunofluorescent microscopy during infection and were found to localize with the morulae, or in the host cell cytoplasm adjacent to morulae. This study confirms that the majority of host proteins known to interact with TRP effectors influence infection and further extends the current knowledge that E. chaffeensis TRPs participate in a complex array of host protein interactions in order to reprogram the host cell and promote intracellular survival.

Ehrlichia chaffeensis TRP120 Effector Targets and Recruits Host Polycomb Group Proteins for Degradation To Promote Intracellular Infection

“…Quantification of E. chaffeensis by qPCR. E. chaffeensis infection was evaluated by real-time qPCR as previously described (25). Briefly, E. chaffeensis-infected THP-1 cells (48 hpi) were pelleted, washed with PBS, and lysed in SideStep lysis and stabilization buffer (Agilent, Santa Clara, CA) at room temperature, and lysates were used in qPCRs.…”

“…Based on sequence similarity and position within the cluster, mammalian Hox genes have been assigned to 13 paralogous groups, and each cluster has 9 to 11 members (24). E. chaffeensis TRP120 interacts with the PCGF component of PRC1, and a previous study demonstrated that knockdown of PCGF5 enhances ehrlichial infection (25). Thus, we investigated the functional relevance of this interaction to better understand the role of PcGs and PRC-associated functions during E. chaffeensis infection.…”

Ehrlichia chaffeensis TRP75 Interacts with Host Cell Targets Involved in Homeostasis, Cytoskeleton Organization, and Apoptosis Regulation To Promote Infection

“…Our Y2H data also identified some closely related or function-associated interacting partners of E. chaffeensis TRPs, including a member of solute carrier family, solute carrier family 4 member 7 (SLC4A7). We previously identified solute carrier family 2 member 3 (SLC2A3) interacting with TRP120, solute carrier family 25 member 42 (SLC25A42) and family 46 member 3 (SLC46A3) interacting with TRP47, and solute carrier family 43 member 3 (SLC43A3) interacting with TRP32 ( 12 , 13 , 23 ). The solute carrier (SLC) consists of a large series of membrane transport proteins, including 52 families and over 400 members identified so far.…”

“…TRP120 interacts with intercellular adhesion molecule 3 (ICAM3) ( 12 ), which promotes drug resistance via inhibition of apoptosis, and with protein phosphatase 3 regulatory subunit B alpha (PPP3R1), which positively regulates TNF-related apoptosis ( 41 , 42 ). Moreover, TRP32, TRP47, and TRP120 interact with a wide range of immunoglobulins and ribosomal proteins, such as IGL, IGKC, IGHA1, IGHV, IGLL1, IGLL5, RPL24, and RPL11 ( 12 , 13 , 23 ), which are associated with stabilization of antiapoptotic protein expression and p53-mediated apoptosis, respectively ( 43 , 44 ). Most of TRP75 target proteins involved in apoptosis exhibited strong interactions with TRP75 by coimmunoprecipitation and immunofluorescence analysis.…”

Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response

“…TRPs are immunoreactive proteins that contain protective linear antibody epitopes in the tandem repeat (TR) domains. In addition, previous studies have demonstrated that TRP effectors interact with a diverse array of host proteins involved in biological processes such as cell signaling, intracellular transport, metabolism, protein post-translational modification, apoptosis, and regulation of transcription [ 2 ]. Notably, E .…”