Atomic force microscopy-based characterization of Giardia duodenalis attachment (POP-WS)

Abstract text

Introduction

Trophozoites of the parasitic protozoan Giardia duodenalis (GD) have a strong attaching behaviour on artificial surfaces and on the epithelial surface of the human duodenum, a well-known prerequisite for GD infections. Despite the profound knowledge about the cellular structures involved in attachment and the pivotal roles of the ventral disc, the lamellipodium-like ventrolateral flange and the beating flagellas, the quantitative adhesive forces of single trophozoites have not been reported yet.

Objectives

Here, the maximum detachment forces (MDF) of single GD trophozoites on a smooth glass surface (RMS = 1.1 ± 0.2 nm) were determined. A direct comparison with the detachment behaviours of other eukaryotic species (i.e. Candida albicans yeast cells and human oral keratinocytes) was done to lay the foundation for a theoretical explanation of the attachment mode of GD.

Materials & Methods

Fluidic force microscopy (FluidFM)-based single-cell force spectroscopy was used to quantify the adhesion parameters of single GD WB C6 trophozoites to glass. Briefly, surface-bound trophozoites were loaded with a FluidFM micropipette, simultaneously sucked by negative pressure and gradually pulled and removed from the surface by retraction of the micropipette. Recorded force-distance curves were evaluated for the MDF, the cell-detachment length and curve characteristics. For comparison, experiments with C. albicans ATCC 10231 and K2 keratinocytes were conducted.

Results

MDFs for GD trophozoites were with 8.2 ± 4.3 nN comparable to the values determined for C. albicans (7.5 ± 4.6 nN), but clearly lower than those seen with oral keratinocytes (55.8 ± 43.9 nN). Interestingly, our GD retraction curves displayed a gradual force increase on the pulled cell until the MDF was reached close to the cell-detachment length. For the other investigated species, however, a clear distance between the MDF and the cell-detachment length was observed with intermediate detachment forces probably caused by the rupture of single or few binding partners. Importantly, this pattern was never detected for GD trophozoites.

Conclusion

Our data indicate a unique adhesion mechanism for GD trophozoites on artificial surfaces that likely are dependent on the ventral disc contributing a major part to the overall adhesion force. Research with GD ventral disc mutants and with porous or nanorough surfaces is ongoing to confirm this hypothesis.