Claretta J. Sullivan

AFM imaging of bacteria in liquid media immobilized on gelatin coated mica surfaces

Immobilization of particulates, especially biomolecules and cells, onto surfaces is critical for imaging with the atomic force microscope (AFM). In this paper, gelatin coated mica surfaces are shown to be suitable for immobilizing and imaging both gram positive, Staphylococcus aureus, and gram negative, Escherichia coli, bacteria in both air and liquid environments. Gelatin coated surfaces are shown to be superior to poly-L-lysine coated surfaces that are commonly used for the immobilization of cells. This cell immobilization technique is being developed primarily for live cell imaging of Rhodopseudomonas palustris. The genome of R. palustris has been sequenced and the organism is the target of intensive studies aimed at understanding genome function. Images of R. palustris grown both aerobically and anaerobically in liquid media are presented. Images in liquid media show the bacteria is rod shaped and smooth while images in air show marked irregularity and folding of the surface. Significant differences in the vertical dimension are also apparent with the height of the bacteria in liquid being substantially greater than images taken in air. In air immobilized bacterial flagella are clearly seen while in liquid this structure is not visible. Additionally, significant morphological differences are observed that depend on the method of bacterial growth.

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.

Circulating bacterial membrane vesicles cause sepsis in rats.

ABSTRACT 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 &agr; 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.

Immobilizing live Escherichia coli for AFM studies of surface dynamics

Atomic force microscopy (AFM) is a probe-based technique that permits high resolution imaging of live bacterial cells. However, stably immobilizing cells to withstand the probe-based lateral forces remains an obstacle in AFM mediated studies, especially those of live, rod shaped bacteria in nutrient media. Consequently, AFM has been under-utilized in the research of bacterial surface dynamics. The aim of the current study was to immobilize a less adherent Escherichia coli strain in a method that both facilitates AFM imaging in nutrient broth and preserves overall cell viability. Immobilization reagents and buffers were systematically evaluated and the cell membrane integrity was monitored in all sample preparations. As expected, the biocompatible gelatin coated surfaces facilitated stable cell attachment in lower ionic strength buffers, yet poorly immobilized cells in higher ionic strength buffers. In comparison, poly-l-lysine surfaces bound cells in both low and high ionic strength buffers. The benefit of the poly-l-lysine binding capacity was offset by the compromised membrane integrity exhibited by cells on poly-l-lysine surfaces. However, the addition of divalent cations and glucose to the immobilization buffer was found to mitigate this unfavorable effect. Ultimately, immobilization of E. coli cells on poly-l-lysine surfaces in a lower ionic strength buffer supplemented with Mg(2+) and Ca(2+) was determined to provide optimal cell attachment without compromising the overall cell viability. Cells immobilized in this method were stably imaged in media through multiple division cycles. Furthermore, permeability assays indicated that E. coli cells recover from the hypoosmotic stress caused by immobilization in low ionic strength buffers. Taken together, this data suggests that stable immobilization of viable cells on poly-l-lysine surfaces can be accomplished in lower ionic strength buffers that are supplemented with divalent cations for membrane stabilization while minimizing binding interference. The data also indicates that monitoring cell viability as a function of sample preparation is important and should be an integral part of the work flow for determining immobilization parameters. A method for immobilizing a less adherent E. coli mutant for AFM imaging in nutrient broth is presented here in addition to a proposed work flow for developing and optimizing immobilization strategies.

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.

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.