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

Human monocytic ehrlichiosis (HME) is caused by an obligatory intracellular bacterium, E. chaffeensis, and is one of the most prevalent, life-threatening emerging infectious zoonoses in the United States. The mechanisms through which E. chaffeensis invades and establishes an intracellular niche are not well understood but are dependent on secreted ehrlichial effector proteins. The significance of this study is in addressing how intracellular pathogens, particularly those with small genomes such as Ehrlichia, exploit a limited number of secreted effector proteins such as tandem repeat proteins (TRPs) to manipulate complex eukaryotes and to regulate host cell processes through molecular pathogen-host interplay. The results of our studies highlight the broader role of ehrlichial TRPs in promoting infection and help define the mechanisms through which obligately intracellular bacteria modulate host cell function for survival. ABSTRACT Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes. The mechanisms involved in E. chaffeensis infection of the host cell and evasion of host defenses are not fully defined, but a subset of type 1 secreted tandem repeat protein (TRP) effectors play important roles. Recently, we determined molecular interactions of TRP120, TRP47, and TRP32 with the eukaryotic host cell. In this investigation, we used yeast two-hybrid analysis to reveal that another E. chaffeensis tandem repeat protein, TRP75, interacts with a diverse group of human proteins associated with organismal and tissue homeostasis, multiple metabolic processes and regulation, response to reactive oxygen species, signal transduction, and protein modifications. Thirteen identified host target proteins associated with actin cytoskeleton reorganization or apoptosis were examined in detail and confirmed to interact with TRP75 at different levels as determined by coimmunoprecipitation assays. These protein interactions were visualized by immunofluorescence confocal microscopy during infection and colocalized with Ehrlichia morulae with different intensities. Moreover, small interfering RNAs (siRNAs) (n = 86) were used to knock down identified TRP75-interacting host proteins separately, and their influence on ehrlichial infection was investigated by real-time quantitative PCR (qPCR). Knockdown of 74/86 (86%) TRP75 target proteins had a significant negative effect on ehrlichial infection. The results of this study further support the idea of a role of Ehrlichia TRPs as effectors that interact with a complex array of host proteins to promote ehrlichial infection. IMPORTANCE Human monocytic ehrlichiosis (HME) is caused by an obligatory intracellular bacterium, E. chaffeensis, and is one of the most prevalent, life-threatening emerging infectious zoonoses in the United States. The mechanisms through which E. chaffeensis invades and establishes an intracellular niche are not well understood but are dependent on secreted ehrlichial effector proteins. The significance of this study is in addressing how intracellular pathogens, particularly those with small genomes such as Ehrlichia, exploit a limited number of secreted effector proteins such as tandem repeat proteins (TRPs) to manipulate complex eukaryotes and to regulate host cell processes through molecular pathogen-host interplay. The results of our studies highlight the broader role of ehrlichial TRPs in promoting infection and help define the mechanisms through which obligately intracellular bacteria modulate host cell function for survival.