Michelle L. Gee

Cell-Cell Communication between Malaria-Infected Red Blood Cells via Exosome-like Vesicles

Cell-cell communication is an important mechanism for information exchange promoting cell survival for the control of features such as population density and differentiation. We determined that Plasmodium falciparum-infected red blood cells directly communicate between parasites within a population using exosome-like vesicles that are capable of delivering genes. Importantly, communication via exosome-like vesicles promotes differentiation to sexual forms at a rate that suggests that signaling is involved. Furthermore, we have identified a P. falciparum protein, PfPTP2, that plays a key role in efficient communication. This study reveals a previously unidentified pathway of P. falciparum biology critical for survival in the host and transmission to mosquitoes. This identifies a pathway for the development of agents to block parasite transmission from the human host to the mosquito.

Self-organization of bacterial biofilms is facilitated by extracellular DNA

Twitching motility-mediated biofilm expansion is a complex, multicellular behavior that enables the active colonization of surfaces by many species of bacteria. In this study we have explored the emergence of intricate network patterns of interconnected trails that form in actively expanding biofilms of Pseudomonas aeruginosa. We have used high-resolution, phase-contrast time-lapse microscopy and developed sophisticated computer vision algorithms to track and analyze individual cell movements during expansion of P. aeruginosa biofilms. We have also used atomic force microscopy to examine the topography of the substrate underneath the expanding biofilm. Our analyses reveal that at the leading edge of the biofilm, highly coherent groups of bacteria migrate across the surface of the semisolid media and in doing so create furrows along which following cells preferentially migrate. This leads to the emergence of a network of trails that guide mass transit toward the leading edges of the biofilm. We have also determined that extracellular DNA (eDNA) facilitates efficient traffic flow throughout the furrow network by maintaining coherent cell alignments, thereby avoiding traffic jams and ensuring an efficient supply of cells to the migrating front. Our analyses reveal that eDNA also coordinates the movements of cells in the leading edge vanguard rafts and is required for the assembly of cells into the “bulldozer” aggregates that forge the interconnecting furrows. Our observations have revealed that large-scale self-organization of cells in actively expanding biofilms of P. aeruginosa occurs through construction of an intricate network of furrows that is facilitated by eDNA.

Hydrophobicity effects in the condensation of water films on quartz

Abstract The surface forces of thin water films condensed onto crystalline quartz plates have been investigated by ellipsometric measurements of film thickness as a function of disjoining pressure. Quartz substrates ranging from fully hydroxylated (contact angle − 0°) to completely dehydroxylated (contact angle − 45°) were used and the results obtained related to the theoretically predicted van der Waals and electrostatic forces present in the system. Water films on fully hydroxylated quartz are much thicker than expected, whereas films on fully dehydroxylated quartz are close to the Lifschitz prediction of dispersion forces. As the extent of dehydroxylation decreases, the adsorption isotherm approaches that obtained on fully hydroxylated quartz.

Coupled electrostatic, hydrodynamic, and mechanical properties of bacterial interfaces in aqueous media.

The interactions of bacteria with their environment are governed by a complex interplay between biological and physicochemical phenomena. The main challenge is the joint determination of the intertwined interfacial characteristics of bacteria such as mechanical and hydrodynamic softness, interfacial heterogeneity, and electrostatic properties. In this study, we have combined electrokinetics and force spectroscopy to unravel this intricate coupling for two types of Shewanella bacterial strains that vary according to the nature of their outer, permeable, charged gel-like layers. The theoretical interpretation of the bacterial electrokinetic response allows for the estimation of the hydrodynamic permeability, degree of interfacial heterogeneity, and volume charge density for the soft layer that constitutes the outer permeable part of the bacteria. Additionally, the electrostatic interaction forces between an AFM probe and the bacteria were calculated on the basis of their interfacial properties obtained from advanced soft particle electrokinetic analysis. For both bacterial strains, excellent agreement between experimental and theoretical force curves is obtained, which highlights the necessity to account for the interfacial heterogeneity of the bioparticle to interpret AFM and electrokinetic data consistently. From the force profiles, we also derived the relevant mechanical parameters in relation to the turgor pressure within the cell and the nature of the bacterial outer surface layer. These results corroborate the heterogeneous representation of the bacterial interface and show that the decrease in the turgor pressure of the cell with increasing ionic strength is more pronounced for bacteria with a thin surface gel-like layer.

Interactions of surfactant monolayers across hydrocarbon liquids

The forces between surfactant-coated monolayer surfaces across hydrocarbon liquids have been measured. The aim was to study the short-range and adhesion forces between various hydrocarbon surfaces across various hydrocarbon liquids, and the effects of polar additives such as water and alcohols. Single-chained, double-chained and fluid-chained surfactants were used, and the oils were either pure straight-chained alkanes, branched alkanes, or a polydisperse mixture. It was found that the force-laws are oscillatory between close-packed solid-crystalline monolayers. Otherwise, they are monotonically attractive down to surface separations of ca. 1 nm, below which they are monotonically repulsive. There are interesting correlations between the forces, the structure of the surface monolayers, and the properties of the intervening liquids. At separations <2 nm the measured force laws are not simply describable by current theories of van der Waals forces. A possible explanation is additional entropic orientational interactions of the solvent molecules and monolayer chains which enhance the attraction.

Imaging the action of antimicrobial peptides on living bacterial cells

Antimicrobial peptides hold promise as broad-spectrum alternatives to conventional antibiotics. The mechanism of action of this class of peptide is a topical area of research focused predominantly on their interaction with artificial membranes. Here we compare the interaction mechanism of a model antimicrobial peptide with single artificial membranes and live bacterial cells. The interaction kinetics was imaged using time-lapse fluorescence lifetime imaging of a fluorescently-tagged melittin derivative. Interaction with the synthetic membranes resulted in membrane pore formation. In contrast, the interaction with bacteria led to transient membrane disruption and corresponding leakage of the cytoplasm, but surprisingly with a much reduced level of pore formation. The discovery that pore formation is a less significant part of lipid-peptide interaction in live bacteria highlights the mechanistic complexity of these interactions in living cells compared to simple artificial systems.

Ellipsometric studies of alkane adsorption on quartz

Abstract The surface forces in thin liquid films of the n-alkanes from pentane to octane inclusive on quartz were investigated by ellipsometric measurement of the equilibrium film thicknesses of these liquids adsorbed from undersaturated vapor onto a smooth quartz substrate. The experimental adsorption isotherms were found to be in excellent agreement with the macroscopic van der Waals interactions as predicted by the Lifshitz theory.

Interaction forces between α-alumina fibres in aqueous electrolyte measured with an atomic force microscope

Abstract The surface charging properties of polycrystalline α-alumina fibres in aqueous electrolyte solutions have been investigated by direct force and streaming potential measurements. The presence of both Al and Si on the surface of the fibres resulted in a chemically heterogeneous surface. The heterogeneous distribution of Si resulted in large attractive forces between the fibres at moderate to low pH values and a pzc/iep at a pH value of approximately 5.5. The origin of this force was electrostatic in nature as the force profiles were well described by the DLVO theory of colloid stability. The agreement between the direct force and streaming potential measurements was good both in terms of the magnitude of the potentials and the position of the pzc/iep. By acid washing the fibres the chemical heterogeneity of the surface was reduced and the attractive force profiles at lower pH values were not observed. Instead repulsive forces were observed which were well described by DLVO theory at all separation distances greater than 8 nm. At smaller separation distances an additional repulsive force was measured which was attributed to the presence of a Al(OH)3 like layer on the surface of the alumina. The acid washing treatment also resulted in a shift in the pH at which the pzc/iep occurred to a value of 6.5, presumably due to a lower surface silica concentration.

Time-resolved evanescent wave-induced fluorescence anisotropy for the determination of molecular conformational changes of proteins at an interface

We have shown that the molecular conformation of a protein at an interface can be probed spatially using time-resolved evanescent wave-induced fluorescence spectroscopic (TREWIFS) techniques. Specifically, by varying the penetration depth of the evanescent field, variable-angle TREWIFS, coupled with variable-angle evanescent wave-induced time-resolved fluorescence anisotropy measurements, allow us to monitor how fluorescence intensity and fluorescence depolarization vary normal to an interface as a function of time after excitation. We have applied this technique to the study of bovine serum albumin (BSA) complexed noncovalently with the fluorophore 1-anilinonaphthalene-8-sulfonic acid. The fluorescence decay varies as a function of the penetration depth of the evanescent wave in a manner that indicates a gradient of hydrophobicity through the adsorbed protein, normal to the interface. Restriction of the fluorescent probe’s motion also occurs as a function of distance normal to the interface. The results are consistent with a model of partial protein denaturation: at the surface, an adsorbed BSA molecule unfolds, thus optimizing protein–silica interactions and the number of points of attachment to the surface. Further away, normal to the surface, the protein molecule maintains its coiled structure.

Atomic Force Microscopy Reveals the Mechanobiology of Lytic Peptide Action on Bacteria

Increasing rates of antimicrobial-resistant medically important bacteria require the development of new, effective therapeutics, of which antimicrobial peptides (AMPs) are among the promising candidates. Many AMPs are membrane-active, but their mode of action in killing bacteria or in inhibiting their growth remains elusive. This study used atomic force microscopy (AFM) to probe the mechanobiology of a model AMP (a derivative of melittin) on living Klebsiella pneumoniae bacterial cells. We performed in situ biophysical measurements to understand how the melittin peptide modulates various biophysical behaviors of individual bacteria, including the turgor pressure, cell wall elasticity, and bacterial capsule thickness and organization. Exposure of K. pneumoniae to the peptide had a significant effect on the turgor pressure and Youngs modulus of the cell wall. The turgor pressure increased upon peptide addition followed by a later decrease, suggesting that cell lysis occurred and pressure was lost through destruction of the cell envelope. The Youngs modulus also increased, indicating that interaction with the peptide increased the rigidity of the cell wall. The bacterial capsule did not prevent cell lysis by the peptide, and surprisingly, the capsule appeared unaffected by exposure to the peptide, as capsule thickness and inferred organization were within the control limits, determined by mechanical measurements. These data show that AFM measurements may provide valuable insights into the physical events that precede bacterial lysis by AMPs.