Trafficking and intracellular processing of exogenous and endogenous proteins: VacA toxin from Helicobacter pylori as a tool

INTRODUCTION Eukaryotic cells are complex systems, which require a continuous flow of communication and material exchange between the intracellular and extracellular environments. The importance of a precise and fine-tuned cellular organization is demonstrated by the development of intracellular aggregates, such Particle-rich Cytoplasmic Structure (PaCS), as a result of altered trafficking or the failure of the degradation quality system to manage misfolded proteins. PaCSs were firstly identified in Helicobacter pylori (Hp) infected human superficial gastric epithelium and are present in a variety of cultured cell lines and ex vivo tissue. Inside PaCSs, it was found out the presence of Vacuolation toxin A (VacA), which has been recognized to be the key of the bacterium's ability to adapt in a hostile environment. VacA is a multifunctional toxin, with a similar structure and cleavage to the AB family of bacterial toxins. Intracellularly VacA can promote the formation of large vacuoles, arising from late endosome, but can alter also mitochondria functioning and intracellular calcium signaling, suggesting that the toxin could achieved the Endoplasmic Reticulum (ER). AIM The aims of this work are: to deeply characterize aggregates such as PaCSs, Lafora Bodies (LBs), Justanuclear Quality Control (JUNQ) and Insoluble Protein Deposit (IPOD) in order to investigate their nature and development; to elucidate the effect of trafficking inhibitor on VacA-induced vacuolization; to investigate VacA trafficking at different time. RESULTS Our results demonstrated that these aggregates are different inclusions, even if they share some similarities either structural or cytochemical. We observed that PaCSs and LBs are glycogen and laforin positive. A comprehensive analysis of JUNQ and IPOD showed that these aggregates have different ultrastructure from PaCSs and that they are mutually exclusive. Considering that inside PaCSs, VacA is accumulated together with polyubiquitin proteins and proteasome components, we evaluate if the toxin is ubiquitinated inside cells. Using histidine-tagged ubiquitin and metal affinity purification, we found out two bands, which might represent ubiquitinated VacA. We next analyzed the effect of eight different trafficking inhibitors, among which, EGA virtually abolish VacA-induced vacuolization. EGA effects was achieved also when vacuolization was already promoted, but it did not prevent toxin binding or internalization process. Binding-on-ice results support the idea that VacA can exploit the retrograde movement to reach ER. A preincubation time with EGA showed that the inhibitor can relocated the toxin to other organelles. CONCLUSION We demonstrated that PaCSs are unique aggregates and that VacA could be ubiquitinated inside cytoplasm. Our VacA ubiquitination and trafficking results supports the idea that the toxin could escape ER, reach the cytoplasm, ubiquitinated and later accumulated inside PaCSs. This approach could be a cell’s tool to prevent VacA cytotoxic effect and promote degradation, making vacuoles only an intermediate step of toxin trafficking. Moreover, we found out that EGA, a newly inhibitor with a powerful effect on several toxins, virtually abolished the toxin activity in all experiments. Its effects, associated to a low toxicity in mouse model, make it look like a potent inhibitor and interesting tool to develop therapeutic strategy against bacterial toxins