Julia M. Ross

Novel experimental study of receptor-mediated bacterial adhesion under the influence of fluid shear.

Dynamic adhesion of cells to surfaces is a vital step in a variety of biochemical and physiological phenomena. Bacterial adhesion is responsible not only for problems associated with biofouling and biofilm formation in the biochemical industry but also in the initiation of certain infectious diseases. In this study, we report the effect of critical parameters, such as receptor and ligand densities and shear rate, on receptor-mediated dynamic bacterial adhesion. Adhesion of a pathogenic strain of Staphylococcus aureus to immobilized collagen was studied. The receptor density on the cell surface was varied by harvesting cells at different growth times and was quantified using flow cytometry. Dynamic adhesion experiments were conducted over a range of physiologically relevant shear rates (50 to 1500 s(-1)) using a parallel-plate flow chamber. Video microscopy coupled with digital image processing was employed to quantify adhesion. A semiquantitative comparison between experimental results and theoretical data obtained using a previously proposed mathematical model was also performed. The results suggest that dynamic adhesion is dependent on receptor density and shear rate, but independent of ligand density. This report demonstrates the feasibility of using bacteria to study fundamental aspects of receptor-mediated dynamic adhesion.

Erosion from Staphylococcus aureus Biofilms Grown under Physiologically Relevant Fluid Shear Forces Yields Bacterial Cells with Reduced Avidity to Collagen

ABSTRACT An estimated 65% of infective diseases are associated with the presence of bacterial biofilms. Biofilm-issued planktonic cells promote blood-borne, secondary sites of infection by the inoculation of the infected sites with bacteria from the intravascular space. To investigate the potential role of early detachment events in initiating secondary infections, we studied the phenotypic attributes of Staphylococcus aureus planktonic cells eroding from biofilms with respect to expression of the collagen adhesin, CNA. The collagen-binding abilities of S. aureus have been correlated to the development of osteomyelitis and septic arthritis. In this study, we focused on the impact of CNA expression on S. aureus adhesion to immobilized collagen in vitro under physiologically relevant shear forces. In contrast to the growth phase-dependent adhesion properties characteristic of S. aureus cells grown in suspension, eroding planktonic cells expressed invariant and lower effective adhesion rates regardless of the age of the biofilm from which they originated. These results correlated directly with the surface expression level of CNA. However, subsequent analysis revealed no qualitative differences between biofilms initiated with suspension cells and secondary biofilms initiated with biofilm-shed planktonic cells. Taken together, our findings suggest that, despite their low levels of CNA expression, S. aureus planktonic cells shed from biofilms retain the capacity for metastatic spread and the initiation of secondary infection. These findings demonstrate the need for a better understanding of the phenotypic properties of eroding planktonic cells, which could lead to new therapeutic strategies to target secondary infections.

Fluid shear regulates the kinetics and receptor specificity of Staphylococcus aureus binding to activated platelets

The interaction between surface components on the invading pathogen and host cells such as platelets plays a key role in the regulation of endovascular infections. However, the mechanisms mediating Staphylococcus aureus binding to platelets under shear remain largely unknown. This study was designed to investigate the kinetics and molecular requirements of platelet-S. aureus interactions in bulk suspensions subjected to a uniform shear field. Hydrodynamic shear-induced collisions augment platelet-S. aureus binding, which is further potentiated by platelet activation with stromal derived factor-1β. Peak adhesion efficiency occurs at low shear (100 s−1) and decreases with increasing shear. The molecular interaction of platelet αIIbβ3 with bacterial clumping factor A through fibrinogen bridging is necessary for stable bacterial binding to activated platelets under shear. Although this pathway is sufficient at low shear (≤400 s−1), the involvement of platelet gpIb and staphylococcal protein A through von Willebrand factor bridging is essential for optimal recruitment of S. aureus cells by platelets in the high shear regime. IgG plays an inhibitory role in the adhesion process, presumably by interfering with the binding of von Willebrand factor to staphylococcal protein A. This study demonstrates that platelet activation and a fluid-mechanical environment representative of the vasculature affect platelet-S. aureus cell-adhesive interactions pertinent to the process of S. aureus-induced bloodstream infections.

Staphylococcus aureus Adhesion via Spa, ClfA, and SdrCDE to Immobilized Platelets Demonstrates Shear-Dependent Behavior

Objective—The objective of this study is to delineate the molecular mechanisms responsible for Staphylococcus aureus–platelet adhesion as a function of physiologically relevant wall shear stresses. Methods and Results—A parallel plate flow chamber was used to quantify adhesion of wild-type, Spa−, ClfA− and SdrCDE− strains to immobilized platelet layers. In the absence of plasma, adhesion increases with increasing wall shear rate from 100 to 5000 seconds−1. The presence of plasma significantly enhances adhesion at all shear levels. Addition of exogenous fibrinogen yields adhesion levels similar to plasma in the lower shear regimes, but has a diminishing effect on potentiating adhesion at higher shear rates. Alternatively, as shear rate increases von Willebrand factor (VWF) plays an increasingly significant role in mediating binding. Conclusions—Addition of plasma proteins potentiates S aureus–platelet interactions at all shear rates examined. Whereas fibrinogen plays a significant role in all shear regimes, VWF mediation becomes increasingly important as wall shear rate increases. Fibrinogen binding is dependent on bacterial adhesins ClfA and SdrCDE whereas Spa is the dominant receptor for VWF.

Shear Stress Prevents Fibronectin Binding Protein-Mediated Staphylococcus aureus Adhesion to Resting Endothelial Cells

ABSTRACT Fibronectin binding proteins (FnBP) on the surface ofStaphylococcus aureus have previously been shown to mediate adherence of the organism to resting endothelial cells in static adhesion assays. However, in this study using well-defined flow assays, we demonstrate that physiologic levels of shear stress prevent FnBP-mediated adhesion of S. aureus 8325-4 to resting endothelial cells. This result suggests that mechanical forces present in vivo may influence the ability of staphylococci to bind endothelial cell surfaces.

Shear Stress Affects the Kinetics of Staphylococcus aureus Adhesion to Collagen

Staphylococcus aureus is a major human pathogen that has been shown to bind collagen under static conditions. However, many staphylococcal infections are hematogenously acquired and adhesion events may be influenced by shear stress. In this study, we used a dynamic experimental system consisting of a parallel‐plate perfusion chamber and phase‐contrast video microscope to study the effects of shear stress on the adhesion kinetics of intact S. aureus to collagen surfaces in vitro. The adhesion of S. aureus Phillips to collagen types I, II, and IV was investigated over a physiologically relevant range of wall shear stresses at 37 °C. S. aureus PH100, a collagen adhesin‐deficient mutant strain, was used as a control strain for the experiments. We found that S. aureus Phillips could adhere to collagens I, II, and IV at wall shear stresses less than 15 dyn/cm2 and that the kinetics of the adhesion process were wall shear stress‐dependent. Similar studies with PH100 demonstrated that these cells are unable to adhere firmly to collagen surfaces. Transient interactions between PH100 and the collagen surfaces were observed at low levels of shear stress suggesting that S. aureus may also interact with collagen by an alternative mechanism that does not lead to firm adhesion.

Quantification of Staphylococcal-Collagen Binding Interactions in Whole Blood by Use of a Confocal Microscopy Shear-Adhesion Assay

In bloodborne staphylococcal infections, bacteria and platelets often combine, forming thrombi on the subendothelium, where collagen is exposed. In this process, the collagen serves as a potential binding surface for Staphylococcus aureus. However, the extent and importance of S. aureus-collagen binding interactions in the development of infected thrombi is uncertain. We quantified S. aureus adhesion to collagen in a whole-blood suspension under defined physiologically relevant fluid shear conditions. S. aureus-collagen binding interactions, mediated by both the S. aureus collagen adhesin (CNA) and protein A-von Willebrand factor (vWf), were evaluated using mutant strains and antibody-blocking techniques. The position of adherent bacteria (at the collagen surface or above the surface in the platelet aggregate) was measured using confocal laser microscopy. Results demonstrated significant CNA-collagen interactions and protein A-vWf-collagen binding interactions under physiological shear conditions. We conclude that collagen binding interactions are important in the development of infected thrombi.

Hydrodynamic Shear and Collagen Receptor Density Determine the Adhesion Capacity of S. aureus to Collagen

AbstractDynamic bacterial adhesion has recently gained significant attention due to its role in the initiation of infectious diseases. Staphylococcus aureus binding to collagen has been shown to be an important event in the pathogenesis of infection. Staphylococcal strains have exhibited wide variability in their level of collagen binding, which may be a result of the collagen receptor expression level. In this study, the dynamic adhesion to collagen for several S. aureus strains was quantified at physiological wall shear rates in a parallel-plate flow chamber. In addition, the collagen receptor density was quantified for each strain. An existing theoretical framework was used to analyze the dependence of adhesion on receptor density. Intrinsic kinetic adhesion parameters were determined and demonstrated to be strong functions of receptor density for all strains. These results suggest that staphylococcal adhesion to collagen is heavily dependent on the receptor density. Using this analytical approach it is possible to predict the dynamic adhesion of S. aureus to collagen in vitro by only measuring the collagen receptor density.

Differential Kinetics and Molecular Recognition Mechanisms Involved in Early Versus Late Growth Phase Staphylococcus aureus Cell Binding to Platelet Layers under Physiological Shear Conditions

BACKGROUND Staphylococcus aureus adhesion to platelets via microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) is a critical first step in vascular infection. The molecular mechanisms governing adhesion are influenced by the repertoire of MSCRAMMs expressed on the bacterial surface and the fluid mechanical shear rates present in the vasculature. We compared the predominant adhesion mechanisms between early and late growth phase S. aureus under physiological shear conditions. METHODS A parallel-plate flow chamber was used to quantify the adhesion of early and late growth phase S. aureus to immobilized platelet layers as a function of wall shear rate. Specifically, we evaluated the influence of clumping factor (Clf) A, ClfB, serine-aspartate repeats, fibronectin-binding proteins (Fnbps), and protein A in supporting S. aureus adhesion to platelets. The ability of the plasma proteins fibrinogen and fibronectin to act as bridging molecules was also investigated. RESULTS Our results demonstrate a markedly elevated binding efficiency for late growth phase staphylococci to immobilized platelets, compared with that of the early growth phase cells in the high shear regime. During the late growth phase, fibrinogen in concert with von Willebrand factor (vWF) potentiates S. aureus-platelet binding via shear-dependent mechanisms. By contrast, fibrinogen, but not vWF, supports the adhesion of early growth phase S. aureus at the high wall shear rates. During the early growth phase, ClfA is identified as the dominant staphylococcal adhesion receptor, with Fnbps playing a supporting role. CONCLUSION The results presented here demonstrate a differential mechanism and binding efficiency for the adhesion of early versus late growth phase S. aureus to immobilized platelets.

Fluorescent silica particles for monitoring oxygen levels in three‐dimensional heterogeneous cellular structures

Bacterial biofilms are a major obstacle challenging the development of more effective therapies to treat implant infections. Oxygen availability to bacterial cells has been implicated in biofilm formation and planktonic cell detachment; however, there are insufficient tools available to measure oxygen concentrations within complex three‐dimensional structures with ∼1 µm resolution. Such measurements may complement measures of biofilm structure and cell activity to provide a more comprehensive understanding of biofilm biology. Thus, we developed oxygen‐sensing microparticles specifically designed to characterize oxygen transport through the volume of bacterial biofilms. The Stöber method was used to synthesize monodisperse silica microparticles of approximately the same size as a bacterium (∼1 µm). Two fluorophores, oxygen‐sensitive Ru(Ph2phen3)Cl2, and the reference fluorophore Nile blue chloride were immobilized on the surface of the particles. We demonstrate application of the microparticles toward measuring the oxygen concentration profiles within a live Staphylococcus aureus biofilm. Biotechnol. Bioeng. 2012; 109: 2663–2670.