Alpha Enolase 1 Ubiquitination and Degradation Mediated by Ehrlichia chaffeensis TRP120 Disrupts Glycolytic Flux and Promotes Infection.

Bing Zhu,Jere W Mcbride

doi:10.3390/pathogens10080962

Bing Zhu, Jere W Mcbride

Open Access

https://doi.org/10.3390/pathogens10080962

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Abstract

Ehrlichia chaffeensis modulates numerous host cell processes, including gene transcription to promote infection of the mononuclear phagocyte. Modulation of these host cell processes is directed through E. chaffeensis effectors, including TRP120. We previously reported that TRP120 moonlights as a HECT E3 Ub ligase that ubiquitinates host cell transcription and fate regulators (PCGF5 and FBW7) to promote infection. In this study, we identified a novel TRP120 substrate and examined the relationship between TRP120 and α-enolase (ENO1), a metalloenzyme that catalyzes glycolytic pathway substrate dehydration. Immunofluorescence microscopy and coimmunoprecipitation demonstrated interaction between ENO1 and TRP120, and ubiquitination of ENO-1 by TRP120 was detected in vivo and in vitro. Further, ENO-1 degradation was observed during infection and was inhibited by the proteasomal inhibitor bortezomib. A direct role of TRP120 Ub ligase activity in ENO-1 degradation was demonstrated and confirmed by ectopic expression of TRP120 HECT Ub ligase catalytic site mutant. siRNA knockdown of ENO-1 coincided with increased E. chaffeensis infection and ENO-1 knockdown disrupted glycolytic flux by decreasing the levels of pyruvate and lactate that may contribute to changes in host cell metabolism that promote infection. In addition, we elucidated a functional role of TRP120 auto-ubiquitination as an activating event that facilitates the recruitment of the UbcH5 E2 ubiquitin-conjugating enzyme. This investigation further expands the repertoire of TRP120 substrates and extends the potential role of TRP120 Ub ligase in infection to include metabolic reprogramming.

Highlights

Ehrlichia chaffeensis is a Gram-negative, obligately intracellular bacterium and etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging, life-threatening, tickborne zoonosis [1,2]
TRP120 has been observed in the host cell cytosol and nucleus during infection, where it interacts with an array of host proteins including many involved in post-translational modifications, such as enzymes required for ligation and conjugation of ubiquitin (Ub) and ubiquitin-like modifier (SUMO) [4,9]
(~4 fold) compared to uninfected cells. These results demonstrate that TRP120 and ENO-1 interact, and ENO-1 levels decrease during E. chaffeensis infection

Summary

Introduction

Ehrlichia chaffeensis is a Gram-negative, obligately intracellular bacterium and etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging, life-threatening, tickborne zoonosis [1,2]. TRPs are secreted intracellularly during infection by the type-1 secretion system, where they interact in a spatial and context-dependent manner with a diverse array of host cell proteins involved in essential cellular processes [4,5,6,7,8,9]. We reported that TRP120 moonlights as a HECT-type E3 ubiquitin (Ub) ligase that ubiquitinates host proteins for proteasomal degradation [15]. Inhibiting HECT Ub ligase activity significantly decreased TRP120 interactions and recruitment of host proteins to ehrlichial inclusions and negatively impacted E. chaffeensis infection, demonstrating an important role during infection [15]. We have shown that TRP120 directly targets FBW7 for ubiquitination, resulting in FBW7 degradation by the ubiquitin–proteasome pathway to maintain the stability of Notch and other oncoproteins involved in cell proliferation and apoptosis regulation [16]

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