A core UPS molecular complement implicates unique endocytic compartments at the parasite–host interface in Giardia lamblia

ABSTRACT

Unconventional protein secretion (UPS) plays important roles in cell physiology. In contrast to canonical secretory routes, UPS does not generally require secretory signal sequences and often bypasses secretory compartments such as the ER and the Golgi apparatus. Giardia lamblia is a protist parasite with reduced subcellular complexity which releases several proteins, some of them virulence factors, without canonical secretory signals. This implicates UPS at the parasite–host interface. No dedicated machinery nor mechanism(s) for UPS in Giardia are currently known, although speculations on the involvement of endocytic organelles called PV/PECs, have been put forth. To begin to address the question of whether PV/PECs are implicated in virulence-associated UPS and to define the composition of molecular machinery involved in protein release, we employed affinity purification and mass spectrometry, coupled to microscopy-based subcellular localization and signal correlation quantification to investigate the interactomes of 11 reported unconventionally secreted proteins, all predicted to be cytosolic. A subset of these are associated with PV/PECs. Extended and validated interactomes point to a core PV/PECs-associated UPS machinery, which includes uncharacterized and Giardia-specific coiled-coil proteins and NEK kinases. Finally, a subset of the alpha-giardin protein family was enriched in all PV/PECs-associated protein interactomes, highlighting a previously unappreciated role for these proteins at PV/PECs and in UPS. Taken together, our results provide the first characterization of a virulence-associated UPS protein complex in Giardia lamblia at PV/PECs, suggesting a novel link between these primarily endocytic and feeding organelles and UPS at the parasite–host interface.

KEYWORDS:

• Giardia lamblia
• virulence
• unconventional protein secretion
• interactome
• peripheral endocytic compartments

Acknowledgements

We thank the Microscopy Imaging Center of the University of Bern, particularly Dr Yury Belyaev for training and advice, and members of BIOP of the EPFL and ScopeM of ETH Zurich for their help setting up an image analysis pipeline during the ZIDAS workshop. We thank the Proteomics and Mass Spectrometry Core Facility (PMSCF) of the Department for Biomedical Research at the University of Bern for generating all proteomics data. Funding for this project is provided by Swiss National Science Foundation grant numbers PR00P3_179813, PR00P3_179813/2 and PR00P3_179813/3 awarded to CF.

Author contributions

EAB, CDW, and CF designed experiments. EAB and CDW performed all experiments. EAB produced the figures and tables, did the data analysis, acquired all microscopy data and wrote the first draft of this manuscript. EAB, CDW, and CF revised the manuscript. All authors authorize this submission.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data deposition

Construct sequence data are deposited online to this location. Proteomics data are deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2022) partner repository with the dataset identifiers PXD035195 (cross-linking co-IP conditions) and PXD035190 (native co-IP conditions), both freely accessible through http://www.ebi.ac.uk/pride

SUPPLEMENTARY MATERIAL

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2023.2174288

Data availability statement

All data presented in this manuscript are freely accessible.

Additional information

Funding

The work was supported by the Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung [PR00P3_179813].