Ehrlichia TRP120 HECT E3 ubiquitin ligase modulation of host cell pathways

点击翻译按钮获取中文摘要

Project Summary/Abstract Ehrlichia chaffeensis (E. ch.) is a gram-negative, obligately intracellular bacterium and etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging, life-threatening, tick-borne zoonosis. E. ch. preferentially infects mononuclear phagocytes and survives intracellularly by subverting innate immune defenses mediated in part by tandem repeat protein (TRP) effectors. TRPs are secreted intracellularly during infection via a type-1 secretion system where they interact in a context-dependent manner with a diverse array of host cell proteins involved in essential cellular processes. The molecular features that determine the continuum of protein-protein interactions that occur during host-pathogen interplay, including those attributed to ehrlichial TRPs, are known to involve posttranslational modifications (PTMs). The scientific premise of this proposal is based on our previous research demonstrating that the E. ch. infection is dependent on exploitation of host PTM pathways for infection, the discovery that E. ch.TRP120 moonlights as a HECT-type E3 ubiquitin (Ub) ligase that tags host proteins for Ub-mediated degradation, identification of novel TRP120-host protein interactions, enhancement of infection through iRNA knockdown of selected TRP120 targets, and demonstration that Wnt and Notch pathways are critical for ehrlichial infection. The long-term goal of this research is to understand the role of host- and pathogen- derived PTMs in host cell reprogramming and infection by E. ch. The objectives of this proposal are to determine the functional role of TRP120 HECT E3 Ub ligase activity during infection, demonstrate TRP120 ubiquitination and degradation of specific host cell targets, and to further expand and define the repertoire of TRP120 Ub ligase substrates. We hypothesize that E. ch. TRP120 HECT E3 ligase modulates host cell signaling and transcriptional repressors via a Ub-mediated degradation mechanism to promote host cell survival and ehrlichial infection. Aim 1 is to determine the role of TRP120 HECT E3 Ub ligase in degrading host cell transcription and signaling regulators; Aim 2 will identify novel TRP120 HECT E3 Ub ligase substrates degraded during infection. This research is significant because it will addresses how intracellular pathogens, particularly those with small genomes and a limited number of effector proteins, interface with eukaryotic cells via PTMs and use pathogen- encoded Ub ligases to manipulate complex and evolutionarily conserved host cell pathways to promote infection. We have established an excellent model to investigate the mechanisms whereby intracellular pathogens influence the stability of cellular regulators though pathogen-encoded Ub ligases. Understanding the interactions between E. ch. HECT-type E3 Ub ligases and host processes/pathways will further define TRP moonlighting roles in pathobiology, and how pathogens mimic eukaryotic protein function to exploit host cell pathways for their benefit.