Iridescent biofilms of Cellulophaga lytica are tunable platforms for scalable, ordered materials.

Nature offers many examples of materials which exhibit exceptional properties due to hierarchical assembly of their constituents. In well-studied multi-cellular systems, such as the morpho butterfly, a visible indication of having ordered submicron features is given by the display of structural color. Detailed investigations of nature's designs have yielded mechanistic insights and led to the development of biomimetic materials at laboratory scales. However, the manufacturing of hierarchical assemblies at industrial scales remains difficult. Biomanufacturing aims to leverage the autonomy of biological systems to produce materials at lower cost and with fewer carbon emissions. Earlier reports documented that some bacteria, particularly those with gliding motility, self-assemble into biofilms with polycrystalline structures and exhibit glittery, iridescent colors. The current study demonstrates the potential of using one of these bacteria, Cellulophaga lytica, as a platform for the large scale biomanufacturing of ordered materials. Specific approaches for controlling C. lytica biofilm optical, spatial and temporal properties are reported. Complementary microscopy-based studies reveal that biofilm color variations are attributed to changes in morphology induced by cellular responses to the local environment. Incorporation of C. lytica biofilms into materials is also demonstrated, thereby facilitating their handling and downstream processing, as would be needed during manufacturing processes. Finally, the utility of C. lytica as a self-printing, photonic ink is established by this study. In summary, autonomous surface assembly of C. lytica under ambient conditions and across multiple length scales circumvent challenges that currently hinder production of ordered materials in industrial settings.

Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles.

Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

Isolation and characterization of Lactobacillus-derived membrane vesicles.

Bacterial membrane vesicles have been implicated in a broad range of functions in microbial communities from pathogenesis to gene transfer. Though first thought to be a phenomenon associated with Gram-negative bacteria, vesicle production in Staphylococcus aureus, Lactobacillus plantarum, and other Gram-positives has recently been described. Given that many Lactobacillus species are Generally Regarded as Safe and often employed as probiotics, the engineering of Lactobacillus membrane vesicles presents a new avenue for the development of therapeutics and vaccines. Here we characterize and compare the membrane vesicles (MVs) from three different Lactobacillus species (L. acidophilus ATCC 53544, L. casei ATCC 393, and L. reuteri ATCC 23272), with the aim of developing future strategies for vesicle engineering. We characterize the vesicles from each Lactobacillus species comparing the physiochemical properties and protein composition of each. More than 80 protein components from Lactobacillus-derived MVs were identified, including some that were enriched in the vesicles themselves suggesting vesicles as a vehicle for antimicrobial delivery. Additionally, for each species vesicular proteins were categorized based on biological pathway and examined for subcellular localization signals in an effort to identify possible sorting mechanisms for MV proteins.

Outer membrane vesicles alter inflammation and coagulation mediators.

INTRODUCTION: Outer membrane vesicles (OMVs) were previously shown to be capable of initiating the inflammatory response seen in the transition of an infection to sepsis. However, another tenet of sepsis is the development of a hypercoagulable state and the role of OMVs in the development of this hypercoagulability has not been evaluated. The objective of this study was to evaluate the ability of OMVs to elicit endothelial mediators of coagulation and inflammation and induce platelet activation. METHODS: Human umbilical vein endothelial cells (HUVECs) were incubated with OMVs and were analyzed for the expression of tissue factor (TF), thrombomodulin, and the adhesion molecules P-selectin and E-selectin. Supernatants of OMV-treated HUVECs were mixed with whole blood and assessed for prothrombotic monocyte-platelet aggregates (MPA). RESULTS: OMVs induce significantly increased expression of TF, E-selectin, and P-selectin, whereas, the expression of thrombomodulin by HUVECs is significantly decreased (P < 0.05). The lipopolysaccharide inhibitor clearly inhibited the expression of E-selectin following incubation with OMVs, although its impact on TF and thrombomodulin expression was nominal. Incubation of whole blood with supernatant from HUVECs exposed to OVMs resulted in increased MPAs. CONCLUSIONS: This study demonstrates that, at the cellular level, OMVs from pathogenic bacteria play a complex role in endothelial activation. Although OMV-bound lipopolysaccharide modulates inflammatory proteins, including E-selectin, it has a negligible effect on the tested coagulation mediators. Additionally, endothelial activation by OMVs facilitates platelet activation as indicated by increased MPAs. By influencing the inflammatory and coagulation cascades, OMVs may contribute to the hypercoagulable response seen in sepsis.

Outer membrane vesicles from pathogenic bacteria initiate an inflammatory response in human endothelial cells.

INTRODUCTION: Gram-negative bacteria release outer membrane vesicles (OMVs) during growth that contain various membrane components involved in eliciting an inflammatory response, including lipopolysaccharide and virulence factors. However, little is known about the role of OMVs in sepsis. The objective of this study was to determine how OMVs, derived from Escherichia (E.) coli, elicit the cellular responses involved in activating the inflammatory cascade, and to determine whether additional virulence factors in pathogenic OMVs augment the inflammatory response. METHODS: Human umbilical endothelial cells were inoculated with OMVs from non-pathogenic E. coli (npOMV) or pathogenic E. coli (pOMV) and analyzed for adhesion protein synthesis, cytokine production, and necrosis factor (NF)-κB translocation. RESULTS: Flow cytometry demonstrated that human umbilical vein endothelial cells exposed to npOMV or pOMV significantly increased expression of E-selectin and intercellular adhesion molecule, with a large population of cells demonstrating increased expression of both proteins. Interleukin-6 levels were significantly elevated by 4 h after exposure to npOMV and pOMVs. NF-κB translocation to the nucleus was shown to be induced by npOMV and pOMVs. However, the role of additional virulence factors associated with pOMVs remains undefined. CONCLUSIONS: Both npOMVs and pOMVs are capable of initiating the inflammatory cascade in endothelial cells. OMVs trigger NF-κB translocation to the nucleus, resulting in up-regulation of adhesion molecules and cytokines, presumably for the recruitment of leukocytes. By eliciting an inflammatory response, OMVs could facilitate the transition from a localized infection to a systemic response, and ultimately sepsis.

Envelope control of outer membrane vesicle production in Gram-negative bacteria.

All Gram-negative bacteria studied to date have been shown to produce outer membrane vesicles (OMVs), which are budded, released spheres of outer membrane with periplasmic content. OMVs have been implicated in the delivery of virulence factors in pathogenesis. However, OMVs also benefit nonpathogenic species by delivering degradative enzymes to defend an ecological niche against competing bacterial species, and they can serve as an envelope stress response. Despite these important roles, very little is known about the mechanism of production of OMVs. Here we review the advantage of vesiculation, particularly in a nonpathogenic context, as well as the hurdles that have to be overcome in Gram-negative envelope architecture before a vesicle can form and bud. Lastly, we address the question of whether OMV production is a stochastic or regulated process.

Circulating bacterial membrane vesicles cause sepsis in rats.

Gram-negative bacteria remain the leading cause of sepsis, a disease that is consistently in the top 10 causes of death internationally. Curing bacteremia alone does not necessarily end the disease process as other factors may cause inflammatory damage. Bacterial outer membrane vesicles (OMVs) are naturally produced blebs from the outer membrane of gram-negative bacteria, which contain various proteins and lipopolysaccharide (LPS). We hypothesize that these vesicles initiate an inflammatory response independent of the parent bacteria. Outer membrane vesicles were isolated from cultures of Escherichia coli, and the concentration of LPS in the OMVs was measured. Adult male Sprague-Dawley rats were separated into five treatment groups: OMV, 2xOMV, LPS, lactated Ringer's, and sham. Our findings show that infused OMVs elicit physiological, histological, and molecular changes in rats that are consistent with sepsis. Hyperdynamic changes in heart rates and mean arterial pressures are observed as well as the elevation of the proinflammatory cytokines tumor necrosis factor α and interleukin 6. Downstream events such as the recruitment of neutrophils into tissues due to the presentation of vascular adhesion molecules also occur in OMV-treated animals. Although soluble LPS elicits stronger responses than did OMVs, responses to the latter consistently exceeded those associated with lactated Ringer's infusion. These results indicate OMVs, independent of the parent bacteria, do initiate an inflammatory response; however, further studies are required to better characterize the temporal biomolecular interactions involved.

Bacterial immobilization for imaging by atomic force microscopy.

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Atomic force microscopy of biological samples.

The ability to evaluate structural-functional relationships in real time has allowed scanning probe microscopy (SPM) to assume a prominent role in post genomic biological research. In this mini-review, we highlight the development of imaging and ancillary techniques that have allowed SPM to permeate many key areas of contemporary research. We begin by examining the invention of the scanning tunneling microscope (STM) by Binnig and Rohrer in 1982 and discuss how it served to team biologists with physicists to integrate high-resolution microscopy into biological science. We point to the problems of imaging nonconductive biological samples with the STM and relate how this led to the evolution of the atomic force microscope (AFM) developed by Binnig, Quate, and Gerber, in 1986. Commercialization in the late 1980s established SPM as a powerful research tool in the biological research community. Contact mode AFM imaging was soon complemented by the development of non-contact imaging modes. These non-contact modes eventually became the primary focus for further new applications including the development of fast scanning methods. The extreme sensitivity of the AFM cantilever was recognized and has been developed into applications for measuring forces required for indenting biological surfaces and breaking bonds between biomolecules. Further functional augmentation to the cantilever tip allowed development of new and emerging techniques including scanning ion-conductance microscopy (SICM), scanning electrochemical microscope (SECM), Kelvin force microscopy (KFM) and scanning near field ultrasonic holography (SNFUH).

Effects of colistin on surface ultrastructure and nanomechanics of Pseudomonas aeruginosa cells.

Chronic lung infections in cystic fibrosis patients are primarily caused by Pseudomonas aeruginosa. Though difficult to counteract effectively, colistin, an antimicrobial peptide, is proving useful. However, the exact mechanism of action of colistin is not fully understood. In this study, atomic force microscopy (AFM) was used to evaluate, in a liquid environment, the changes in P. aeruginosa morphology and nanomechanical properties due to exposure to colistin. The results of this work revealed that after 1 h of colistin exposure the ratio of individual bacteria to those found to be arrested in the process of division changed from 1.9 to 0.4 and the length of the cells decreased significantly. Morphologically, it was observed that the bacterial surface changed from a smooth to a wrinkled phenotype after 3 h exposure to colistin. Nanomechanically, in untreated bacteria, the cantilever indented the bacterial surface significantly more than it did after 1 h of colistin treatment (P-value = 0.015). Concurrently, after 2 h of exposure to colistin, a significant increase in the bacterial spring constant was also observed. These results indicate that the antimicrobial peptide colistin prevents bacterial proliferation by repressing cell division. We also found that treatment with colistin caused an increase in the rigidity of the bacterial cell wall while morphologically the cell surface changed from smooth to wrinkled, perhaps due to loss of lipopolysaccharides (LPS) or surface proteins.