The accumulation and growth of Pseudomonas aeruginosa on surfaces is modulated by surface mechanics via cyclic-di-GMP signaling.

Attachment of bacteria onto a surface, consequent signaling, and accumulation and growth of the surface-bound bacterial population are key initial steps in the formation of pathogenic biofilms. While recent reports have hinted that surface mechanics may affect the accumulation of bacteria on that surface, the processes that underlie bacterial perception of surface mechanics and modulation of accumulation in response to surface mechanics remain largely unknown. We use thin and thick hydrogels coated on glass to create composite materials with different mechanics (higher elasticity for thin composites; lower elasticity for thick composites) but with the same surface adhesivity and chemistry. The mechanical cue stemming from surface mechanics is elucidated using experiments with the opportunistic human pathogen Pseudomonas aeruginosa combined with finite-element modeling. Adhesion to thin composites results in greater changes in mechanical stress and strain in the bacterial envelope than does adhesion to thick composites with identical surface chemistry. Using quantitative microscopy, we find that adhesion to thin composites also results in higher cyclic-di-GMP levels, which in turn result in lower motility and less detachment, and thus greater accumulation of bacteria on the surface than does adhesion to thick composites. Mechanics-dependent c-di-GMP production is mediated by the cell-surface-exposed protein PilY1. The biofilm lag phase, which is longer for bacterial populations on thin composites than on thick composites, is also mediated by PilY1. This study shows clear evidence that bacteria actively regulate differential accumulation on surfaces of different stiffnesses via perceiving varied mechanical stress and strain upon surface engagement.

Nanofiber-Based Substrate for a Triboelectric Nanogenerator: High-Performance Flexible Energy Fiber Mats.

Flexible and stretchable triboelectric nanogenerators (TENGs) are the next-generation systems for wearable and portable electronics. In this study, we have demonstrated an all nanofiber-based TENG for energy harvesting and biomechanical sensing applications. The TENG was prepared using the Forcespinning (FS) method to produce poly(vinylidene fluoride) (PVDF) and thermoplastic polyurethane (TPU) nanofiber (NF) membranes. The TPU nanofiber membranes were interfaced with a homogeneously sputtered gold nanofilm. The experimental characterization of the PVDF-TPU/Au NF-TENG revealed that surface interfaced with dispersed gold in a TPU fiber membrane produced a maximum open-circuit voltage of 254 V and a short-circuit current of 86 µA output at a 240 bpm load frequency, which was, respectively, 112 and 87% greater than bare PVDF-TPU NF-based TENG. All systems were composed of an active contact surface area of 3.2 × 2.5 cm2. Furthermore, the TENG was able to light up 75 LEDs (1.5 V of each) by the hand-tapping motion. The resistive load and capacitor test results exemplified a TENG offering a simple and high-performance self-chargeable device. Furthermore, we have tested the TENG's response for biomechanical movements at different frequencies, suggesting the TENG's potential to be also used as a cost-effective self-powered flexible body motion sensor.

Neuroprotective effects of the Chrysophyllum perpulchrum extract against an Alzheimer-like rat model of ß amyloid1-40 intrahippocampal injection.

OBJECTIVE: Alzheimer's disease (AD) is a threatening disease for African populations in the upcoming years because of the increase in their expectancy of life. Here, we investigated whether natural products from Chrysophyllum perpulchrum as catechin and two dimeric procyanidins (catechin + hexose) could prevent progression of oxidative stress and cognitive changes using an AD-like rat model induced by Aß1-40 injection into the hippocampal CA1 subfield. METHODOLOGY: Adult male Wistar rats were either microinjected with 1% ammonia as a vehicle (10 µL) or aggregated Aß1-40 at 10 µg bilateral hippocampus. On the 14th day of post-surgery, some Aß rats were treated with melatonin (10 mg/kg i.p.) or with the Chrysophyllum perpulchrum extract (300 mg/kg p.o.), and some sham-operated rats received the extract alone. Cognitive abilities were tested with Y-maze, object recognition test and Morris Water Maze. Oxidative stress markers as well as the level of activated microglial cells were assayed in the brain. RESULTS: Aß rats exhibited significant deficits of recognition memory and spatial learning. This was associated with an increase of microglia Iba 1 immunoreactivity as well as nitric oxide (NO), malondialdehyde and superoxide dismutase levels but not to the thiol content in the hippocampus, prefrontal cortex and septum of AD-like rats. The Chrysophyllum perpulchrum extract treatment mitigated Aß-induced cognitive impairments and reversed microglia overactivation and subsequent generation of oxidative stress markers. Interestingly, the neuroprotective actions of the Chrysophyllum perpulchrum extract seem to be comparable to the control drug melatonin used albeit with some more beneficial effects. CONCLUSION: These findings are preliminary and should be strengthened by more pharmacological studies of bioactive compounds of Chrysophyllum perpulchrum before being proposed as a promising drug against AD.

Evolutionary adaptations of biofilms infecting cystic fibrosis lungs promote mechanical toughness by adjusting polysaccharide production.

Biofilms are communities of microbes embedded in a matrix of extracellular polymeric substances, largely polysaccharides. Multiple types of extracellular polymeric substances can be produced by a single bacterial strain. The distinct polymer components of biofilms are known to provide chemical protection, but little is known about how distinct extracellular polysaccharides may also protect biofilms against mechanical stresses such as shear or phagocytic engulfment. Decades-long infections of Pseudomonas. aeruginosa biofilms in the lungs of cystic fibrosis patients are natural models for studies of biofilm fitness under pressure from antibiotics and the immune system. In cystic fibrosis infections, production of the extracellular polysaccharide alginate has long been known to increase with time and to chemically protect biofilms. More recently, it is being recognized that chronic cystic fibrosis infections also evolve to increase production of another extracellular polysaccharide, Psl; much less is known about Psl's protective benefits to biofilms. We use oscillatory bulk rheology, on biofilms grown from longitudinal clinical isolates and from genetically-manipulated lab strains, to show that increased Psl stiffens biofilms and increases biofilm toughness, which is the energy cost to cause the biofilm to yield mechanically. Further, atomic force microscopy measurements reveal greater intercellular cohesion for higher Psl expression. Of the three types of extracellular polysaccharides produced by P. aeruginosa, only Psl increases the stiffness. Stiffening by Psl requires CdrA, a protein that binds to mannose groups on Psl and is a likely cross-linker for the Psl components of the biofilm matrix. We compare the elastic moduli of biofilms to the estimated stresses exerted by neutrophils during phagocytosis, and infer that increased Psl could confer a mechanical protection against phagocytic clearance.

Mechanosensing of shear by Pseudomonas aeruginosa leads to increased levels of the cyclic-di-GMP signal initiating biofilm development.

Biofilms are communities of sessile microbes that are phenotypically distinct from their genetically identical, free-swimming counterparts. Biofilms initiate when bacteria attach to a solid surface. Attachment triggers intracellular signaling to change gene expression from the planktonic to the biofilm phenotype. For Pseudomonas aeruginosa, it has long been known that intracellular levels of the signal cyclic-di-GMP increase upon surface adhesion and that this is required to begin biofilm development. However, what cue is sensed to notify bacteria that they are attached to the surface has not been known. Here, we show that mechanical shear acts as a cue for surface adhesion and activates cyclic-di-GMP signaling. The magnitude of the shear force, and thereby the corresponding activation of cyclic-di-GMP signaling, can be adjusted both by varying the strength of the adhesion that binds bacteria to the surface and by varying the rate of fluid flow over surface-bound bacteria. We show that the envelope protein PilY1 and functional type IV pili are required mechanosensory elements. An analytic model that accounts for the feedback between mechanosensors, cyclic-di-GMP signaling, and production of adhesive polysaccharides describes our data well.

Neuroprotection of medial septal cholinergic neurons by memantine after intralateral septal injection of Aß1-40.

Alzheimer's disease (AD) is a progressive disorder of the brain that leads to memory loss, dementia, and death. Several lines of evidence suggest that the accumulation of amyloid-ß (Aß) peptides may trigger the dysfunction and degeneration observed in the AD brain. The basal forebrain, including the septal region, which regulates the excitability of the hippocampus and neocortex, is affected early in AD because its neurons are vulnerable to Aß peptides. In addition, connections between lateral and medial septal regions (medial septum and diagonal band of Broca) have been demonstrated in previous studies. To demonstrate the involvement of excitotoxicity in Aß-induced septal damage, we compared rats injected with Aß1-40 into the lateral septal region structure with rats treated with memantine (a noncompetitive NMDA receptor antagonist), before, during, and after Aß1-40 injection. Medial septal cholinergic neurons were immunochemically identified and their numbers were estimated using Image J cell count. Our results show that Aß1-40-treated animals have a significantly low number of medial septum and diagonal band of Broca cholinergic neurons compared with the Aß/memantine-treated group.

The extracellular polysaccharide Pel makes the attachment of P. aeruginosa to surfaces symmetric and short-ranged.

Biofilms are surface-mounted, multicellular communities of microbes. Biofilms are often associated with chronic infections that resist treatment, evade the immune system, and damage host tissue. An essential characteristic of the biofilm state is that constituent organisms are bound in a polymeric matrix. This matrix gives the system spatial structure and clusters bacteria near each other, facilitating intercellular interactions. The Pseudomonas aeruginosa strain PAO1 is widely studied as a model biofilm-forming organism. The polymeric matrix of PAO1 biofilms is dominated by two bacteria-produced extracellular polymers, Pel and Psl. We use a combination of optical and atomic force microscopy to examine the roles of these polymers in very early biofilm development. In agreement with other researchers, we find that Psl mediates strong attachment to a glass surface. We find that Pel alone can mediate some attachment, but not as permanent as that mediated by Psl. Unexpectedly, we find that Pel promotes symmetric attachment, in the form of rod-shaped bacteria lying down flat on the surface, and that the presence of Pel makes attachment forces more short-ranged than they are with Psl alone. We suggest that these effects may result from synergistic interactions of Pel with the Psl polymeric matrix.

Amyloid ß peptides modify the expression of antioxidant repair enzymes and a potassium channel in the septohippocampal system.

Alzheimer's disease (AD) is a progressive, neurodegenerative brain disorder characterized by extracellular accumulations of amyloid ß (Aß) peptides, intracellular accumulation of abnormal proteins, and early loss of basal forebrain neurons. Recent studies have indicated that the conformation of Aß is crucial for neuronal toxicity, with intermediate misfolded forms such as oligomers being more toxic than the final fibrillar forms. Our previous work shows that Aß blocks the potassium (K(+)) currents IM and IA in septal neurons, increasing firing rates, diminishing rhythmicity and firing coherence. Evidence also suggests that oxidative stress (OS) plays a role in AD pathogenesis. Thus we wished to determine the effect of oligomeric and fibrillar forms of Aß1₋42 on septohippocampal damage, oxidative damage, and dysfunction in AD. Oligomeric and fibrillar forms of Aß1₋42 were injected into the CA1 region of the hippocampus in live rats. The rats were sacrificed 24 hours and 1 month after Aß or sham injection to additionally evaluate the temporal effects. The expression levels of the K(+) voltage-gated channel, KQT-like subfamily, member 2 (KCNQ2) and the OS-related genes superoxide dismutase 1, 8-oxoguanine DNA glycosylase, and monamine oxidase A, were analyzed in the hippocampus, medial, and lateral septum. Our results show that both forms of Aß exhibit time-dependent differential modulation of OS and K(+) channel genes in the analyzed regions. Importantly, we demonstrate that Aß injected into the hippocampus triggered changes in gene expression in anatomical regions distant from the injection site. Thus the Aß effect was transmitted to anatomically separate sites, because of the functional coupling of the brain structures.

Intrahippocampal amyloid-ß (1-40) injections injure medial septal neurons in rats.

Alzheimer's disease (AD) is a devastating disorder that leads to memory loss and dementia. Neurodegeneration of cholinergic neurons in the septum and other basal forebrain areas is evident in early stages of AD. Glutamatergic neurons are also affected early in AD. In these stages, amyloid-ß-peptide (Aß) plaques are present in the hippocampus and other cortices but not in the basal forebrain, which includes the septum. We postulate that early deposition of hippocampal Aß damages the axon terminals of cholinergic and glutamatergic septo-hippocampal neurons, leading to their degeneration. To determine the mechanisms underlying septal degeneration, fibrillar Aß1-40 was injected into the Cornu Ammonis (CA1) hippocampal region of rats. Controls were injected with reverse peptide Aß40-1. A 16% reduction in NeuN+ cells was observed around the injection sites when compared to controls (p < 0.05) one week after injections. Stereology was used to estimate the number of choline acetyl transferase (ChAT), glutamate and glutamic acid decarboxylase 67 (GAD67) immunoreactive septal neurons. Medial septal ChAT and glutamate immunoreactive neurons were reduced 38% and 26%, respectively by hippocampal injections of Aß1-40 peptide in relation to controls. In contrast, the number of GAD67 inmunoreactive neurons was not significantly reduced. Apoptotic cells were detected in the medial septal region of Aß1-40 treated animals but not in controls. These results indicate that limited Aß-induced hippocampal lesions lead to an overall damage of vulnerable septal neuronal populations, most likely by Aß interaction with septo-hippocampal axon terminals. Thus, axon terminals constitute an important target for novel therapeutics dedicated to control Aß-induced toxicity.

Subcellular Min oscillations as a single-cell reporter of the action of polycations, protamine, and gentamicin on Escherichia coli.

BACKGROUND: In Escherichia coli, MinD-GFP fusion proteins show rapid pole to pole oscillations. The objective was to investigate the effects of extracellular cations on the subcellular oscillation of cytoplasmic MinD within Escherichia coli. METHODOLOGY/PRINCIPAL FINDINGS: We exposed bacteria to the extracellular cations Ca(++), Mg(++), the cationic antimicrobial peptide (CAP) protamine, and the cationic aminoglycoside gentamicin. We found rapid and substantial increases in the average MinD oscillation periods in the presence of any of these polyvalent cations. For Ca(++) and Mg(++) the increases in period were transient, even with a constant extracellular concentration, while increases in period for protamine or gentamicin were apparently irreversible. We also found striking interdependence in the action of the small cations with protamine or gentamicin, distorted oscillations under the action of intermediate levels of gentamicin and Ca(++), and reversible freezing of the Min oscillation at high cationic concentrations. CONCLUSIONS/SIGNIFICANCE: Intracellular Min oscillations provide a fast single-cell reporter of bacterial response to extracellular polycations, which can be explained by the penetration of polycations into cells.

Contribution of type IV pili to the virulence of Aeromonas salmonicida subsp. salmonicida in Atlantic salmon (Salmo salar L.).

Aeromonas salmonicida subsp. salmonicida, a bacterial pathogen of Atlantic salmon, has no visible pili, yet its genome contains genes for three type IV pilus systems. One system, Tap, is similar to the Pseudomonas aeruginosa Pil system, and a second, Flp, resembles the Actinobacillus actinomycetemcomitans Flp pilus, while the third has homology to the mannose-sensitive hemagglutinin pilus of Vibrio cholerae. The latter system is likely nonfunctional since eight genes, including the gene encoding the main pilin subunit, are deleted compared with the orthologous V. cholerae locus. The first two systems were characterized to investigate their expression and role in pathogenesis. The pili of A. salmonicida subsp. salmonicida were imaged using atomic force microscopy and Tap- and Flp-overexpressing strains. The Tap pili appeared to be polar, while the Flp pili appeared to be peritrichous. Strains deficient in tap and/or flp were used in live bacterial challenges of Atlantic salmon, which showed that the Tap pilus made a moderate contribution to virulence, while the Flp pilus made little or no contribution. Delivery of the tap mutant by immersion resulted in reduced cumulative morbidity compared with the cumulative morbidity observed with the wild-type strain; however, delivery by intraperitoneal injection resulted in cumulative morbidity similar to that of the wild type. Unlike the pili of other piliated bacterial pathogens, A. salmonicida subsp. salmonicida type IV pili are not absolutely required for virulence in Atlantic salmon. Significant differences in the behavior of the two mutant strains indicated that the two pilus systems are not redundant.

Molecular recognition forces between immunoglobulin G and a surface protein adhesin on living Staphylococcus aureus.

We report AFM measurements of binding events between immunoglobulin G (IgG) and protein A (PA) on the surface of live Staphylococcus aureus bacteria. The experiments were carried out with IgG molecules tethered via CM-amylose linkers to thiol SAMs on gold-coated AFM tips. For comparison, a model system consisting of protein A molecules tethered to thiol SAMs on gold-coated silicon substrates was also investigated. Histograms of binding forces for the PA-IgG bond showed comparable rupture forces of 59 and 64 pN for the model system and live bacteria, respectively. We suggest that linker molecules with a length comparable to the AFM tip radius should make it possible to detect specific binding events on the surface of live bacteria with a lateral resolution of a few tens of nanometers. Furthermore, because S. aureus is an important human pathogen, especially methicillin-resistant strains (MRSA), it is possible that additional virulence factors beyond PA can be probed using this technique.

Temperature dependence of MinD oscillation in Escherichia coli: running hot and fast.

We observed that the oscillation period of MinD within rod-like and filamentous cells of Escherichia coli varied by a factor of 4 in the temperature range from 20 degrees C to 40 degrees C. The detailed dependence was Arrhenius, with a slope similar to the overall temperature-dependent growth curve of E. coli. The detailed pattern of oscillation, including the characteristic wavelength in filamentous cells, remained independent of temperature. A quantitative model of MinDE oscillation exhibited similar behavior, with an activated temperature dependence of the MinE-stimulated MinD-ATPase rate.

The p14 fusion-associated small transmembrane (FAST) protein effects membrane fusion from a subset of membrane microdomains.

The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WX- and Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)-resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipid-anchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.

Nanoscale characterization and determination of adhesion forces of Pseudomonas aeruginosa pili by using atomic force microscopy.

Type IV pili play an important role in bacterial adhesion, motility, and biofilm formation. Here we present high-resolution atomic force microscopy (AFM) images of type IV pili from Pseudomonas aeruginosa bacteria. An individual pilus ranges in length from 0.5 to 7 microm and has a diameter from 4 to 6 nm, although often, pili bundles in which the individual filaments differed in both length and diameter were seen. By attaching bacteria to AFM tips, it was possible to fasten the bacteria to mica surfaces by pili tethers. Force spectra of tethered pili gave rupture forces of 95 pN. The slopes of force curves close to the rupture force were nearly linear but showed little variation with pilus length. Furthermore, force curves could not be fitted with wormlike-chain polymer stretch models when using realistic persistence lengths for pili. The observation that the slopes near rupture did not depend on the pili length suggests that they do not represent elastic properties of the pili. It is possible that this region of the force curves is determined by an elastic element that is part of the bacterial wall, although further experiments are needed to confirm this.

Liposome reconstitution of a minimal protein-mediated membrane fusion machine.

Biological membrane fusion is dependent on protein catalysts to mediate localized restructuring of lipid bilayers. A central theme in current models of protein-mediated membrane fusion involves the sequential refolding of complex homomeric or heteromeric protein fusion machines. The structural features of a new family of fusion-associated small transmembrane (FAST) proteins appear incompatible with existing models of membrane fusion protein function. While the FAST proteins function to induce efficient cell-cell fusion when expressed in transfected cells, it was unclear whether they function on their own to mediate membrane fusion or are dependent on cellular protein cofactors. Using proteoliposomes containing the purified p14 FAST protein of reptilian reovirus, we now show via liposome-cell and liposome-liposome fusion assays that p14 is both necessary and sufficient for membrane fusion. Stoichiometric and kinetic analyses suggest that the relative efficiency of p14-mediated membrane fusion rivals that of the more complex cellular and viral fusion proteins, making the FAST proteins the simplest known membrane fusion machines.

Atomic force microscopy of cell growth and division in Staphylococcus aureus.

The growth and division of Staphylococcus aureus was monitored by atomic force microscopy (AFM) and thin-section transmission electron microscopy (TEM). A good correlation of the structural events of division was found using the two microscopies, and AFM was able to provide new additional information. AFM was performed under water, ensuring that all structures were in the hydrated condition. Sequential images on the same structure revealed progressive changes to surfaces, suggesting the cells were growing while images were being taken. Using AFM small depressions were seen around the septal annulus at the onset of division that could be attributed to so-called murosomes (Giesbrecht et al., Arch. Microbiol. 141:315-324, 1985). The new cell wall formed from the cross wall (i.e., completed septum) after cell separation and possessed concentric surface rings and a central depression; these structures could be correlated to a midline of reactive material in the developing septum that was seen by TEM. The older wall, that which was not derived from a newly formed cross wall, was partitioned into two different surface zones, smooth and gel-like zones, with different adhesive properties that could be attributed to cell wall turnover. The new and old wall topographies are equated to possible peptidoglycan arrangements, but no conclusion can be made regarding the planar or scaffolding models.

Aggregation of yeast cells: direct measurement of discrete lectin-carbohydrate interactions.

Aggregation of microbial cells mediated by specific interactions plays a pivotal role in the natural environment, in medicine and in biotechnological processes. Here we used atomic force microscopy (AFM) to measure individual lectin-carbohydrate interactions involved in the flocculation of yeast cells, an aggregation event of crucial importance in fermentation technology. AFM probes functionalized with oligoglucose carbohydrates were used to record force-distance curves on living yeast cells at a rate of 0.5 micro m s(-1). Flocculating cells showed adhesion forces of 121+/-53 pN, reflecting the specific interaction between individual cell-surface lectins and glucose residues. Similar adhesion forces, 117+/-41 pN, were measured using probes functionalized with the lectin concanavalin A and attributed to specific binding to cell-surface mannose residues. By contrast, specific interaction forces were not observed in non-flocculating conditions, i.e. in the presence of mannose or when using non-flocculating cells, pointing to their involvement in yeast flocculation. The single molecule force spectroscopy measurements presented here provide a means to study a variety of cellular interactions at the molecular level, such as the adhesion of bacteria to animal and plant tissues.

Real-time imaging of the surface topography of living yeast cells by atomic force microscopy.

Atomic force microscopy (AFM) was used to image the surface topography of living Saccharomyces cerevisiae cells at high resolution and to monitor enzyme digestion of the cell wall in real time. Apart from the presence of bud scars, the surface of native cells imaged in aqueous solution was homogeneous and smooth. Topographic images of the surface were recorded to a lateral resolution of 2 nm without significant modification of the surface morphology. Successive images of single cells were collected at fixed time intervals following addition of protease and amyloglucosidase solutions. Protease caused a progressive increase of surface roughness. Large depressions surrounded by protruding edges, approximately 50 nm in height, were formed and attributed to the erosion of the mannoprotein outer layer. By contrast, no modification of the cell surface was noted upon addition of amyloglucosidase, which was consistent with the cell wall biochemical composition. These results indicate that AFM is a complementary tool to electron microscopy in that it allows the surface of living cells to be explored directly in real time.