Vernita Gordon

The Pel Polysaccharide Can Serve a Structural and Protective Role in the Biofilm Matrix of Pseudomonas aeruginosa

Bacterial extracellular polysaccharides are a key constituent of the extracellular matrix material of biofilms. Pseudomonas aeruginosa is a model organism for biofilm studies and produces three extracellular polysaccharides that have been implicated in biofilm development, alginate, Psl and Pel. Significant work has been conducted on the roles of alginate and Psl in biofilm development, however we know little regarding Pel. In this study, we demonstrate that Pel can serve two functions in biofilms. Using a novel assay involving optical tweezers, we demonstrate that Pel is crucial for maintaining cell-to-cell interactions in a PA14 biofilm, serving as a primary structural scaffold for the community. Deletion of pelB resulted in a severe biofilm deficiency. Interestingly, this effect is strain-specific. Loss of Pel production in the laboratory strain PAO1 resulted in no difference in attachment or biofilm development; instead Psl proved to be the primary structural polysaccharide for biofilm maturity. Furthermore, we demonstrate that Pel plays a second role by enhancing resistance to aminoglycoside antibiotics. This protection occurs only in biofilm populations. We show that expression of the pel gene cluster and PelF protein levels are enhanced during biofilm growth compared to liquid cultures. Thus, we propose that Pel is capable of playing both a structural and a protective role in P. aeruginosa biofilms.

HIV TAT Forms Pores in Membranes by Inducing Saddle‐Splay Curvature: Potential Role of Bidentate Hydrogen Bonding

The TAT protein transduction domain (PTD) of the human immunodeficiency virus (HIV-1) can cross cell membranes with unusual efficiency and has many potential biotechnological applications. Extant work has provided important clues to the molecular mechanism underlying the activity of this peptide, which consists of 11 amino acids, 8 of which are cationic and 6 of these are arginines. TAT PTD synthesized with d-amino acids enters cells as efficiently as the native form, thereby indicating that the mechanism of transduction is receptor independent; this conclusion is consistent with recent results that suggest that the TAT PTD may enter cells through receptor-independent macropinocytosis. Substitution of any of the PTD1s cationic residues with neutral alanine decreases activity, while substitution of neutral residues has no effect. This indicates the importance of electrostatic interactions between cationic TAT PTD and anionic phospholipid membranes. Recent work has shown that the physics of electrostatic interactions can drive a rich polymorphism of self-assembled structures that depend on parameters such as charge density and intrinsic membrane curvature. However, although arginine-rich polycations can enter cells, cationic polylysine cannot. This shows that electrostatic interactions alone are insufficient for PTD activity and that the arginine residues play a specific, essential role. We use confocal microscopy and synchrotron X-ray scattering (SAXS) to study the interaction of the TAT PTD with model membranes at room temperature. We find that the transduction activity correlates with induction of negative Gaussian (“saddle-splay”) membrane curvature, which is topologically required for pore formation. Moreover, we show that the TAT PTD can drastically remodel vesicles into a porous bicontinuous phase with analogues in block-copolymer systems, and we propose a geometric mechanism facilitated by both electrostatics and bidentate hydrogen bonding. The latter is possible for the TAT PTD but not for similarly cationic, nonarginated polypeptides. Cell membranes are composed of lipids that have fundamentally different interactions with cationic macroions such as TAT PTD. We examine representative model membranes composed of lipids with different charges and intrinsic curvatures: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) have zwitterionic headgroups, while 1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] (sodium salt) (DOPS) and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG) have anionic headgroups; all have zero intrinsic curvature (C0 = 0, “cylinder-shaped”) except for DOPE, which has negative intrinsic curvature (C0< 0, “cone-shaped”). When rhodamine-tagged TAT PTD (Rh-PTD) is applied to the exterior of giant unilamellar vesicles (GUVs, diameters of 5–30 mm) with low DOPE content (0 and 20%), rhodamine fluorescence is seen only outside the GUVs (Figure 1a), thereby indicating that the Rh-PTD has not crossed these membranes. However, when Rh-PTD is applied to GUVs with 40% DOPE content, the rhodamine intensity equilibrates across the membrane over tens of seconds (Figure 1b and c; see also the movie in the Supporting Information). This shows that Rh-PTD has crossed the GUV membranes, which remain intact (Figure 1c). Thus, we see that the membrane transduction activity of Rh-PTD requires the presence of a threshold amount of DOPE in the membrane.

Bacteria Use Type IV Pili to Walk Upright and Detach from Surfaces

A searchable database of images allows detailed analysis of bacterial motility. Bacterial biofilms are structured multicellular communities involved in a broad range of infections. Knowing how free-swimming bacteria adapt their motility mechanisms near surfaces is crucial for understanding the transition between planktonic and biofilm phenotypes. By translating microscopy movies into searchable databases of bacterial behavior, we identified fundamental type IV pili–driven mechanisms for Pseudomonas aeruginosa surface motility involved in distinct foraging strategies. Bacteria stood upright and “walked” with trajectories optimized for two-dimensional surface exploration. Vertical orientation facilitated surface detachment and could influence biofilm morphology.

Flagella and Pili-Mediated Near-Surface Single-Cell Motility Mechanisms in P. aeruginosa

Bacterial biofilms are structured multicellular communities that are responsible for a broad range of infections. Knowing how free-swimming bacteria adapt their motility mechanisms near a surface is crucial for understanding the transition from the planktonic to the biofilm phenotype. By translating microscopy movies into searchable databases of bacterial behavior and developing image-based search engines, we were able to identify fundamental appendage-specific mechanisms for the surface motility of Pseudomonas aeruginosa. Type IV pili mediate two surface motility mechanisms: horizontally oriented crawling, by which the bacterium moves lengthwise with high directional persistence, and vertically oriented walking, by which the bacterium moves with low directional persistence and high instantaneous velocity, allowing it to rapidly explore microenvironments. The flagellum mediates two additional motility mechanisms: near-surface swimming and surface-anchored spinning, which often precedes detachment from a surface. Flagella and pili interact cooperatively in a launch sequence whereby bacteria change orientation from horizontal to vertical and then detach. Vertical orientation facilitates detachment from surfaces and thereby influences biofilm morphology.

Measuring the mechanical stress induced by an expanding multicellular tumor system: a case study

Rapid volumetric growth and extensive invasion into brain parenchyma are hallmarks of malignant neuroepithelial tumors in vivo. Little is known, however, about the mechanical impact of the growing brain tumor on its microenvironment. To better understand the environmental mechanical response, we used multiparticle tracking methods to probe the environment of a dynamically expanding, multicellular brain tumor spheroid that grew for 6 days in a three-dimensional Matrigel-based in vitro assay containing 1.0-microm latex beads. These beads act as reference markers for the gel, allowing us to image the spatial displacement of the tumor environment using high-resolution time-lapse video microscopy. The results show that the volumetrically expanding tumor spheroid pushes the gel outward and that this tumor-generated pressure propagates to a distance greater than the initial radius of the tumor spheroid. Intriguingly, beads near the tips of invasive cells are displaced inward, toward the advancing invasive cells. Furthermore, this localized cell traction correlates with a marked increase in total invasion area over the observation period. This case study presents evidence that an expanding microscopic tumor system exerts both significant mechanical pressure and significant traction on its microenvironment.

Mechanism of a Prototypical Synthetic Membrane-Active Antimicrobial: Efficient Hole-Punching Via Interaction With Negative Intrinsic Curvature Lipids

Phenylene ethynylenes comprise a prototypical class of synthetic antimicrobial compounds that mimic antimicrobial peptides produced by eukaryotes and have broad-spectrum antimicrobial activity. We show unambiguously that bacterial membrane permeation by these antimicrobials depends on the presence of negative intrinsic curvature lipids, such as phosphatidylethanolamine (PE) lipids, found in high concentrations within bacterial membranes. Plate-killing assays indicate that a PE-knockout mutant strain of Escherichia coli drastically out-survives the wild type against the membrane-active phenylene ethynylene antimicrobials, whereas the opposite is true when challenged with traditional metabolic antibiotics. That the PE deletion is a lethal mutation in normative environments suggests that resistant bacterial strains do not evolve because a lethal mutation is required to gain immunity. PE lipids allow efficient generation of negative curvature required for the circumferential barrel of an induced membrane pore; an inverted hexagonal HII phase, which consists of arrays of water channels, is induced by a small number of antimicrobial molecules. The estimated antimicrobial occupation in these water channels is nonlinear and jumps from ≈1 to 3 per 4 nm of induced water channel length as the global antimicrobial concentration is increased. By comparing to exactly solvable 1D spin models for magnetic systems, we quantify the cooperativity of these antimicrobials.

Mechanism of A Prototypical Synthetic Membrane-Active Antimicrobial: Efficient Hole-Punching by Targeting Lipids With Negative Spontaneous Curvature Lipids

a. Department of Materials Science and Engineering, d. Departments of Microbiology and Biochemistry, b. Department of Physics, e.Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801; and c. Department of Polymer Science and Engineering, University of Massachusetts, Amherst, MA 01003

Role of Multicellular Aggregates in Biofilm Formation

ABSTRACT In traditional models of in vitro biofilm development, individual bacterial cells seed a surface, multiply, and mature into multicellular, three-dimensional structures. Much research has been devoted to elucidating the mechanisms governing the initial attachment of single cells to surfaces. However, in natural environments and during infection, bacterial cells tend to clump as multicellular aggregates, and biofilms can also slough off aggregates as a part of the dispersal process. This makes it likely that biofilms are often seeded by aggregates and single cells, yet how these aggregates impact biofilm initiation and development is not known. Here we use a combination of experimental and computational approaches to determine the relative fitness of single cells and preformed aggregates during early development of Pseudomonas aeruginosa biofilms. We find that the relative fitness of aggregates depends markedly on the density of surrounding single cells, i.e., the level of competition for growth resources. When competition between aggregates and single cells is low, an aggregate has a growth disadvantage because the aggregate interior has poor access to growth resources. However, if competition is high, aggregates exhibit higher fitness, because extending vertically above the surface gives cells at the top of aggregates better access to growth resources. Other advantages of seeding by aggregates, such as earlier switching to a biofilm-like phenotype and enhanced resilience toward antibiotics and immune response, may add to this ecological benefit. Our findings suggest that current models of biofilm formation should be reconsidered to incorporate the role of aggregates in biofilm initiation. IMPORTANCE During the past decades, there has been a consensus around the model of development of a biofilm, involving attachment of single planktonic bacterial cells to a surface and the subsequent development of a mature biofilm. This study presents results that call for a modification of this rigorous model. We show how free floating biofilm aggregates can have a profound local effect on biofilm development when attaching to a surface. Our findings show that an aggregate landing on a surface will eventually outcompete the biofilm population arising from single cells attached around the aggregate and dominate the local biofilm development. These results point to a regime where preformed biofilm aggregates may have a fitness advantage over planktonic cells when it comes to accessing nutrients. Our findings add to the increasingly prominent comprehension that biofilm lifestyle is the default for bacteria and that planktonic single cells may be only a transition state at the most. During the past decades, there has been a consensus around the model of development of a biofilm, involving attachment of single planktonic bacterial cells to a surface and the subsequent development of a mature biofilm. This study presents results that call for a modification of this rigorous model. We show how free floating biofilm aggregates can have a profound local effect on biofilm development when attaching to a surface. Our findings show that an aggregate landing on a surface will eventually outcompete the biofilm population arising from single cells attached around the aggregate and dominate the local biofilm development. These results point to a regime where preformed biofilm aggregates may have a fitness advantage over planktonic cells when it comes to accessing nutrients. Our findings add to the increasingly prominent comprehension that biofilm lifestyle is the default for bacteria and that planktonic single cells may be only a transition state at the most.

Structures of the linear silicon carbides SiC4 and SiC6: Isotopic substitution and Ab Initio theory

The structures of two linear silicon carbides, SiC4 and SiC6, have been determined by a combination of isotopic substitution and large-scale coupled-cluster ab initio calculations, following detection of all of the singly substituted isotopic species in a supersonic molecular beam with a Fourier transform microwave spectrometer. Rotational constants obtained by least-squares fitting transition frequencies were used to derive experimental structures; except for those nearest the center of mass, individual bond lengths for both chains have an error of less than 0.008 A. Accurate equilibrium structures were derived by converting the experimental rotational constants to equilibrium constants using the vibration–rotation coupling constants from coupled-cluster calculations, including connected triple substitutions. Equilibrium dipole moments and harmonic vibrational frequencies were also calculated for both chains. On the basis of the calculated vibration–rotation and l-type doubling constants, weak rotational...

Adhesion promotes phase separation in mixed-lipid membranes

We investigate the interplay of domain formation and adhesion in mixed-lipid membranes. Giant unilamellar vesicles consisting of two- and three-component lipid mixtures are studied using confocal fluorescence microscopy. Upon driving the system towards the demixing transition, phase separation is invariably found to occur first in regions where membranes adhere to one another, despite identical lipid headgroups and negligible curvature effects. We propose a simple generic mechanism based on the suppression of thermal shape fluctuations to explain these observations. Our findings suggest novel possibilities by which biomembranes can create and utilize lateral lipid heterogeneities.