Characterization of a unique attachment organelle: Single-cell force spectroscopy of Giardia duodenalis trophozoites.

By guest authors Gubesh Gunaratnam and Philipp Jung.

Gubesh Gunaratnam^a,  Ricarda Leisering^b,  Ben Wieland^a, Johanna Dudek^c, Nicolai Miosge^c, Sören L. Becker^a, Markus Bischoff^a, Scott C. Dawson^d, Matthias Hannig^c, Karin Jacobs^e,f, Christian Klotz^b, Toni Aebischer^b,  and  Philipp Jung^a  

a    Institute of Medical Microbiology and Hygiene, Saarland University, Homburg, Germany
b    Department of Infectious Diseases, Unit 16 Mycotic and Parasitic Agents and Mycobacteria, Robert Koch-Institute, Berlin, Germany
c    Clinic of Operative Dentistry and Periodontology, Saarland University, Homburg, Germany
d    Department of Microbiology and Molecular Genetics, University of California Davis, Davis, USA
e    Experimental Physics, Saarland University, Saarbrücken, Germany
f    Max Planck School, Matter to Life, Heidelberg, Germany

*Email: gubesh.gunaratnam@uni-saarland.de

Link to publication: Characterization of a unique attachment organelle: Single-cell force spectroscopy of Giardia duodenalis trophozoites

#Done with a FLEX: The Flexible Research AFM

The unicellular parasite Giardia duodenalis exists as a substrate colonizing active form (trophozoite) in the small intestine of humans. At the interface of substrate and cell body, it uses an attachment organelle, the ventral disc, for reversible attachment to the human host epithelium. This attachment organelle is unique to Giardia species. Relying on the function of the ventral disc, the trophozoite is capable of binding to different kinds of artificial substrates, including plain, positively charged, or hydrophobic glass. Different theoretical approaches have been suggested to describe this microbe’s mode of attachment, but until now there is no consensus. The most discussed one among them is a biophysical suction mechanism, but its existence has not been approved either. Therefore, technologies on a single-cell level are demanded that enable the description of such rarely seen attachment behaviors.

In our recent manuscript, we used the single-cell force spectroscopy (SCFS) technique, which was based on a FlexAFM (Nanosurf AG, Liestal, Switzerland) and a microfluidic control system (MFCS; Cytosurge AG, Glattbrugg, Switzerland) to study the detachment characteristics of individual Giardia trophozoites adhering to a flat glass substrate. To our knowledge, utilizing SCFS to investigate suction-based microbial attachment mechanisms is a novelty.

We combined micro-channeled FluidFM micropipettes with a 2 µm aperture and the negative pressure option of the MFCS, with the FlexAFM set-up and its inverted microscope integration for a correlative SCFS technique to approach Giardia attachment on a single-cell level. We could successfully determine important AFM parameters: The adhesion force, de-adhesion work, localization of the adhesion force (L-F_adh), and the cell detachment length.

During the retraction phase of the FluidFM micropipette and the pulling of the trophozoite from the flat glass surface, we observed a distinct major bond rupture of the trophozoite. This major peak corresponded to the maximum adhesion force and L-F_adh. For this type of attachment, we found an average maximum adhesion force of 7.7 nN. On the other side, the force signature during the detachment of adherent Candida albicans and spread- out adherent human keratinocytes were completely different. Representative force curves of C. albicans showed a characteristic pattern with multiple peaks of different forces, indicative of multiple molecular bond breakages at the interface of cell and substratum. There was no correlation between L-F_adh and the cell detachment length. Force curves of keratinocytes had maximum adhesion forces which were always followed by smaller detachment events and forces. Both types of retraction curves were clearly not the case for G. duodenalis trophozoites. In contrast, the close correlation between L-F_adh and the cell detachment length demonstrated an undescribed attachment mechanism for G. duodenalis.

In conclusion, our SCFS-based study is a new approach to describe the microbial detachment characteristics and attachment forces of the parasite G. duodenalis. Our results are compatible with a suction-based attachment mechanism but are incompatible with a significant contribution of cell-surface-ligand receptor interactions that define adhesion processes of other eukaryotic/prokaryotic cells. This work paves the way for studying attachment characteristics based on the FlexAFM-based FluidFM set-up also of other parasitic cells that might follow their own rules of attachment modes.

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