Internalization of the Active Subunit of the Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Is Dependent upon Cellugyrin (Synaptogyrin 2), a Host Cell Non-Neuronal Paralog of the Synaptic Vesicle Protein, Synaptogyrin 1.

Kathleen Boesze-Battaglia,Bruce J Shenker,Lisa P Walker,Hsin-Yao Tang,Anuradha Dhingra,Konstantin Kandror

doi:10.3389/fcimb.2017.00469

Abstract

The Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is a heterotrimeric AB2 toxin capable of inducing lymphocytes, and other cell types, to undergo cell cycle arrest and apoptosis. Exposure to Cdt results in binding to the cell surface followed by internalization and translocation of the active subunit, CdtB, to intracellular compartments. These events are dependent upon toxin binding to cholesterol in the context of lipid rich membrane microdomains often referred to as lipid rafts. We now demonstrate that, in addition to binding to the plasma membrane of lymphocytes, another early and critical event initiated by Cdt is the translocation of the host cell protein, cellugyrin (synaptogyrin-2) to the same cholesterol-rich microdomains. Furthermore, we demonstrate that cellugyrin is an intracellular binding partner for CdtB as demonstrated by immunoprecipitation. Using CRISPR/cas9 gene editing we established a Jurkat cell line deficient in cellugyrin expression (JurkatCg−); these cells were capable of binding Cdt, but unable to internalize CdtB. Furthermore, JurkatCg− cells were not susceptible to Cdt-induced toxicity; these cells failed to exhibit blockade of the PI-3K signaling pathway, cell cycle arrest or cell death. We propose that cellugyrin plays a critical role in the internalization and translocation of CdtB to critical intracellular target sites. These studies provide critical new insight into the mechanism by which Cdt, and in particular, CdtB is able to induce toxicity.

Highlights

Intracellular-acting microbial toxins face the common need to gain entry into the cytosol of host target cells
In this paper we demonstrate that in concert with cytolethal distending toxin (Cdt) binding to cholesterol and association with membrane microdomains, cellugyrin levels increase and the protein translocates from the cytoplasm to cholesterol rich membrane microdomains
In previous studies we have demonstrated that the ability of Cdt to intoxicate cells was dependent upon toxin association with cholesterol-rich membrane microdomains

Summary

Introduction

Intracellular-acting microbial toxins face the common need to gain entry into the cytosol of host target cells. The exact role for CdtA in binding to cells is not clear, but several studies have suggested that this subunit may recognize a range of targets including fucose moieties and glycosphoingolipids, among others (Nesic et al, 2004; Mise et al, 2005) It should be noted the Cdt binding occurs in the context of cholesterol/sphingomyelin-rich membrane microdomains, commonly referred to as lipid rafts. This association is the result of the CdtC subunit’s ability to recognize and bind to cholesterol via cholesterol recognition sequences known as CRAC sites (Guerra et al, 2005; BoeszeBattaglia et al, 2009, 2015; Eshraghi et al, 2010; Zhou et al, 2012; Lai et al, 2013). These observations are significant as membrane cholesterol rich microdomains have been shown to serve a number of relevant functions including concentrating toxins on the cell surface and providing access to molecular pathways associated with endocytosis and signaling (Cherukuri et al, 2001; Dykstra et al, 2003)

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Conclusion