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Ehrlichia chaffeensis exploits host SUMOylation pathways to mediate effector-host interactions and promote intracellular survival.

ABSTRACT: Ehrlichia chaffeensis is an obligately intracellular Gram-negative bacterium that selectively infects mononuclear phagocytes. We recently reported that E. chaffeensis utilizes a type 1 secretion (T1S) system to export tandem repeat protein (TRP) effectors and demonstrated that these effectors interact with a functionally diverse array of host proteins. By way of these interactions, TRP effectors modulate host cell functions; however, the molecular basis of these interactions and their roles in ehrlichial pathobiology are not well defined. In this study, we describe the first bacterial protein posttranslational modification (PTM) by the small ubiquitin-like modifier (SUMO). The E. chaffeensis T1S effector TRP120 is conjugated to SUMO at a carboxy-terminal canonical consensus SUMO conjugation motif in vitro and in human cells. In human cells, TRP120 was selectively conjugated with SUMO2/3 isoforms. Disruption of TRP120 SUMOylation perturbed interactions with known host proteins, through predicted SUMO interaction motif-dependent and -independent mechanisms. E. chaffeensis infection did not result in dramatic changes in the global host SUMOylated protein profile, but a robust colocalization of predominately SUMO1 with ehrlichial inclusions was observed. Inhibiting the SUMO pathway with a small-molecule inhibitor had a significant impact on E. chaffeensis replication and recruitment of the TRP120-interacting protein polycomb group ring finger protein 5 (PCGF5) to the inclusion, indicating that the SUMO pathway is critical for intracellular survival. This study reveals the novel exploitation of the SUMO pathway by Ehrlichia, which facilitates effector-eukaryote interactions necessary to usurp the host and create a permissive intracellular niche.

SUBMITTER: Dunphy PS

PROVIDER: S-EPMC4187855 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Ehrlichia chaffeensis exploits host SUMOylation pathways to mediate effector-host interactions and promote intracellular survival.

Dunphy Paige Selvy PS Luo Tian T McBride Jere W JW

Infection and immunity 20140721 10

Ehrlichia chaffeensis is an obligately intracellular Gram-negative bacterium that selectively infects mononuclear phagocytes. We recently reported that E. chaffeensis utilizes a type 1 secretion (T1S) system to export tandem repeat protein (TRP) effectors and demonstrated that these effectors interact with a functionally diverse array of host proteins. By way of these interactions, TRP effectors modulate host cell functions; however, the molecular basis of these interactions and their roles in e ...[more]

PMID: 25047847

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Project description:UNLABELLED:Ehrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP) effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD) occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey Significant differences in canonical Notch signaling gene expression levels (>40%) were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs) against ?-secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4) expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival. IMPORTANCE:E. chaffeensis is an obligately intracellular bacterium and the etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. Mechanisms by which E. chaffeensis establishes intracellular infection and avoids innate host defenses are not understood, but functionally relevant host-pathogen interactions with type 1 secreted TRP effectors are essential for the ehrlichial cellular reprogramming strategy. This study provides further insight into the molecular strategies used by obligately intracellular pathogens such as E. chaffeensis, which have small genomes and a limited number of effector proteins and exploit evolutionarily conserved host cell programs such as Notch signaling to promote infection and intracellular survival.

Project description: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.

Project description:Ehrlichia chaffeensis, a cholesterol-rich and cholesterol-dependent obligate intracellular bacterium, partially lacks genes for glycerophospholipid biosynthesis. We found here that E. chaffeensis is dependent on host glycerolipid biosynthesis, as an inhibitor of host long-chain acyl CoA synthetases, key enzymes for glycerolipid biosynthesis, significantly reduced bacterial proliferation. E. chaffeensis cannot synthesize phosphatidylcholine or cholesterol but encodes enzymes for phosphatidylethanolamine (PE) biosynthesis; however, exogenous NBD-phosphatidylcholine, Bodipy-PE, and TopFluor-cholesterol were rapidly trafficked to ehrlichiae in infected cells. DiI (3,3'-dioctadecylindocarbocyanine)-prelabeled host-cell membranes were unidirectionally trafficked to Ehrlichia inclusion and bacterial membranes, but DiI-prelabeled Ehrlichia membranes were not trafficked to host-cell membranes. The trafficking of host-cell membranes to Ehrlichia inclusions was dependent on both host endocytic and autophagic pathways, and bacterial protein synthesis, as the respective inhibitors blocked both infection and trafficking of DiI-labeled host membranes to Ehrlichia In addition, DiI-labeled host-cell membranes were trafficked to autophagosomes induced by the E. chaffeensis type IV secretion system effector Etf-1, which traffic to and fuse with Ehrlichia inclusions. Cryosections of infected cells revealed numerous membranous vesicles inside inclusions, as well as multivesicular bodies docked on the inclusion surface, both of which were immunogold-labeled by a GFP-tagged 2×FYVE protein that binds to phosphatidylinositol 3-phosphate. Focused ion-beam scanning electron microscopy of infected cells validated numerous membranous structures inside bacteria-containing inclusions. Our results support the notion that Ehrlichia inclusions are amphisomes formed through fusion of early endosomes, multivesicular bodies, and early autophagosomes induced by Etf-1, and they provide host-cell glycerophospholipids and cholesterol that are necessary for bacterial proliferation.

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Ehrlichia chaffeensis exploits host SUMOylation pathways to mediate effector-host interactions and promote intracellular survival.

Publications

Ehrlichia chaffeensis exploits host SUMOylation pathways to mediate effector-host interactions and promote intracellular survival.

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