ABSTRACT
Ehrlichia chaffeensis (E. ch.) is a gram-negative, obligately intracellular bacterium and the 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. Within the last decade, our laboratory has identified
a multitude of molecular ehrlichial TRP-host interactions, many that are new to science, illuminating the breath
and complexity of pathogen-host interaction dynamics that occur during infection. We have shown that E. ch. is
dependent on activation of conserved eukaryotic signaling pathways including Notch and Wnt for infection.
However, understanding the molecular basis of Notch activation events, whereby E. ch. repurposes Notch
signaling for infection remains a major gap in our knowledge. Thus, the purpose of this investigation is to define
the molecular and cellular mechanisms E. ch. has evolved to repurpose Notch signaling for infection. We propose
that E. ch. TRP120 has distinct eukaryotic protein interaction modules known as short linear motifs (SLiMs) that
mimic Notch ligand function and directly engage cognate receptors to exploit Notch signaling for infection. This
investigation will address our limited understanding of the functionally diverse roles of protein interaction modules
in the continuum of host-pathogen interactions and cellular reprogramming. The long-term goal of this research
is to define the molecular basis of Ehrlichia host cell mimicry, and the mechanisms involved in infection and
immune evasion. The objective of this proposal is to determine the molecular interactions involved and functional
mechanisms whereby E. ch. TRP120 Notch ligand mimicry establishes and promotes infection by inhibiting host
cell apoptosis. We hypothesize that E. ch. TRP120 has a Notch SLiM mimetic that activates Notch signaling to
upregulate anti-apoptotic regulators (MCL1 and NICD), thereby inhibiting mitochondrial apoptotic signaling and
caspase activation. Aim 1 will define the E. ch. TRP120 Notch SLiM mimetic and investigate receptor binding
and signaling; Aim 2 will examine E. ch. TRP120 Notch-upregulated MCL1 inhibition of mitochondrial apoptosis;
and Aim 3 will investigate the role of E. ch. Notch stabilization of XIAP and inhibition of caspase activation. This
investigation will extend our knowledge of the molecular interactions by which E. ch. TRP120 surface protein
exploits Notch signaling to inhibit apoptosis and promote host cell survival and infection. The significance of this
research is defining the mechanistic strategies whereby intracellular pathogens with small genomes and a limited
number of effector proteins, have evolved host mimicry modules to repurpose host cell signaling to manipulate
downstream host defense mechanisms for infection. A molecular understanding of E. ch. pathobiology will also
facilitate development of novel therapeutic approaches for Ehrlichia spp. and intracellular pathogens that utilize
SLiM mimicry or exploit conserved cellular pathways for infection and immune evasion.