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

The unicellular parasite Giardia duodenalis is the causative agent of giardiasis, a gastrointestinal disease with global spread. In its trophozoite form, G. duodenalis can adhere to the human intestinal epithelium and a variety of other, artificial surfaces. Its attachment is facilitated by a unique microtubule-based attachment organelle, the so-called ventral disc. The mechanical function of the ventral disc, however, is still debated. Earlier studies postulated that a dynamic negative pressure under the ventral disc, generated by persistently beating flagella, mediates the attachment. Later studies suggested a suction model based on structural changes of the ventral discs, substrate clutching or grasping, or unspecific contact forces. In this study, we aim to contribute to the understanding of G. duodenalis attachment by investigating detachment characteristics and determining adhesion forces of single trophozoites on a smooth glass surface (RMS = 1.1 ± 0.2 nm) by fluidic force microscopy (FluidFM)-based single-cell force spectroscopy (SCFS). Briefly, viable adherent trophozoites were approached with a FluidFM micropipette, immobilized to the micropipette aperture by negative pressure, and detached from the surface by micropipette retraction while retract force curves were recorded. These force curves displayed novel and so far undescribed characteristics for a microorganism, namely, gradual force increase on the pulled trophozoite, with localization of adhesion force shortly before cell detachment length. Respective adhesion forces reached 7.7 ± 4.2 nN at 1 µm s-1 pulling speed. Importantly, this unique force pattern was different from that of other eukaryotic cells such as Candida albicans or oral keratinocytes, considered for comparison in this study. The latter both displayed a force pattern with force peaks of different values or force plateaus (for keratinocytes) indicative of breakage of molecular bonds of cell-anchored classes of adhesion molecules or membrane components. Furthermore, the attachment mode of G. duodenalis trophozoites was mechanically resilient to tensile forces, when the pulling speeds were raised up to 10 µm s-1 and adhesion forces increased to 28.7 ± 10.5 nN. Taken together, comparative SCSF revealed novel and unique retract force curve characteristics for attached G. duodenalis, suggesting a ligand-independent suction mechanism, that differ from those of other well described eukaryotes.

Using Knock-Out Mutants to Investigate the Adhesion of Staphylococcus aureus to Abiotic Surfaces.

The adhesion of Staphylococcus aureus to abiotic surfaces is crucial for establishing device-related infections. With a high number of single-cell force spectroscopy measurements with genetically modified S. aureus cells, this study provides insights into the adhesion process of the pathogen to abiotic surfaces of different wettability. Our results show that S. aureus utilizes different cell wall molecules and interaction mechanisms when binding to hydrophobic and hydrophilic surfaces. We found that covalently bound cell wall proteins strongly interact with hydrophobic substrates, while their contribution to the overall adhesion force is smaller on hydrophilic substrates. Teichoic acids promote adhesion to hydrophobic surfaces as well as to hydrophilic surfaces. This, however, is to a lesser extent. An interplay of electrostatic effects of charges and protein composition on bacterial surfaces is predominant on hydrophilic surfaces, while it is overshadowed on hydrophobic surfaces by the influence of the high number of binding proteins. Our results can help to design new models of bacterial adhesion and may be used to interpret the adhesion of other microorganisms with similar surface properties.

Human blood plasma factors affect the adhesion kinetics of Staphylococcus aureus to central venous catheters.

Staphylococcus aureus is a common cause of catheter-related blood stream infections (CRBSI). The bacterium has the ability to form multilayered biofilms on implanted material, which usually requires the removal of the implanted medical device. A first major step of this biofilm formation is the initial adhesion of the bacterium to the artificial surface. Here, we used single-cell force spectroscopy (SCFS) to study the initial adhesion of S. aureus to central venous catheters (CVCs). SCFS performed with S. aureus on the surfaces of naïve CVCs produced comparable maximum adhesion forces on three types of CVCs in the low nN range (~ 2-7 nN). These values were drastically reduced, when CVC surfaces were preincubated with human blood plasma or human serum albumin, and similar reductions were observed when S. aureus cells were probed with freshly explanted CVCs withdrawn from patients without CRBSI. These findings indicate that the initial adhesion capacity of S. aureus to CVC tubing is markedly reduced, once the CVC is inserted into the vein, and that the risk of contamination of the CVC tubing by S. aureus during the insertion process might be reduced by a preconditioning of the CVC surface with blood plasma or serum albumin.