Barbara J. Panessa-Warren

Exosporial membrane plasticity of Clostridium sporogenes and Clostridium difficile

This investigation examines the morphological alterations of the exosporial membranes of Clostridium sporogenes ATCC 3584 and Clostridium difficile ATCC 43594 and 9689 endospores in relation to their possible function during germination in the attachment/colonization process of these pathogenic bacteria. There is no reported function for the exosporial membrane, nor exosporial appendages, of clostridial endospores. Advances in high resolution, scanning electron microscopy (SEM) permit the examination of these delicate, morphological projections on intact spores in the process of attachment. The morphological plasticity of the exosporial membrane projections during activation and germination was examined to determine whether the appearance of these exosporial projections coincided with attachment of the spores to the nutritive substrate, and whether this attachment could be altered by physical agitation, cation competition with Ba2+, chelation with EDTA, or treatment with colchicine. Following incubation, activated spores could not be removed from the agar surface by agitation in water (pH 7.2 or 9.1), nor by agitation in buffer or colchicine, indicating that some form of adherence or attachment to the agar had taken place. When agitated in the presence of Ba2+ or EDTA in phosphate buffered saline or EDTA in water, all activated spores detached from the agar and exhibited decreased exosporial projections and minimal, if any, attachment structures to the agar surface. Activated clostridial spores were found to attach to agar by delicate extensions of the exosporium that could be disrupted by EDTA or Ba2+ exposure, but were unchanged when shaken in buffer or water.

Human epithelial cell processing of carbon and gold nanoparticles

This paper describes some early cellular and intracellular interactions of human polarised lung and colon epithelial cells (representative of two portals of entry, inhalation and ingestion), following exposure to specific carbon and gold engineered nanoparticles in vitro. Cells were incubated with functionalised and non-functionalised carbon nanotube-derived nanoloops (∼28?60 nm diameter), or gold nanoparticles (2 nm and 10 nm Au-core) which were either non-functionalised, or functionalised with biological proteins or ssDNA and analysed using viability staining, transmission electron microscopy (TEM) and field emission scanning (FESEM) electron microscopy. Even with such diverse nanoparticles and functionalisations, we found that the surface properties and size of the nanoparticles determined their cellular binding, incorporation and/or cytotoxicity. However the cells responded to the different types of nanoparticles using various intracellular routes which differed with the cell type, but all of the nanoparticles ultimately were consolidated into aggregates and transported to the basal cell surface. Nanoparticles that were completely covered with biological macromolecules (i.e., recombinant gClq-R protein, non-immune IgGk, monoclonal antibody to gClq-R, or ssDNA) did not cause ultrastructural damage or changes in the cell monolayers. Monoclonal antibody (mAb)-functionalised carbon nanoloops and ssDNA 100% covered Au-nanodots were incorporated and transported within the colon cells using different cellular pathways than those used by the lung cells. Citrate-capped Au-nanoparticles (2 nm and 10 nm) and 20% DNA covered Au-nanoparticles passed into the colon and lung cells through small holes in the apical cell membrane, which could possibly be produced by lipid peroxidation. Serious forms of cell damage were observed with citrate capped 2 nm and 10 nm Au-nanoparticles (i.e., nuclear localisation (2 nm-Au); intracellular membrane damage (10 nm-Au)). Vital staining used to identify cellular necrosis following nanoparticle exposure, was sometimes misleading showing cell necrosis statistics similar to normal controls, when TEM analysis revealed intracellular and organellar damage in identically treated cells.

The Exosporium of B.cereus Contains a Binding Site for gC1qR/p33: Implication in Spore Attachment and/or Entry

B. cereus, is a member of a genus of aerobic, gram-positive, spore-forming rod-like bacilli, which includes the deadly, B. anthracis. Preliminary experiments have shown that gC1qR binds to B. cereus spores that have been attached to microtiter plates. The present studies were therefore undertaken, to examine if cell surface gC1qR plays a role in B. cereus spore attachment and/or entry. Monolayers of human colon carcinoma (Caco-2) and lung cells were grown to confluency on 6 mm coverslips in shell vials with gentle swirling in a shaker incubator. Then, 2 microl of a suspension of strain SB460 B. cereus spores (3x10(8)/ml, in sterile water), were added and incubated (1-4 h; 36 degrees C) in the presence or absence of anti-gC1qR mAb-carbon nanoloops. Examination of these cells by EM revealed that: (1) When B. cereus endospores contacted the apical Caco-2 cell surface, or lung cells, gC1qR was simultaneously detectable, indicating upregulation of the molecule. (2) In areas showing spore contact with the cell surface, gC1qR expression was often adjacent to the spores in association with microvilli (Caco-2 cells) or cytoskeletal projections (lung cells). (3) Furthermore, the exosporia of the activated and germinating spores were often decorated with mAb-nanoloops. These observations were further corroborated by experiments in which B.cereus spores were readily taken up by monocytes and neutrophils, and this uptake was partially inhibited by mAb 60.11, which recognizes the C1q binding site on gC1qR. Taken together, the data suggest a role, for gC1qR at least in the initial stages of spore attachment and/or entry.

High resolution FESEM and TEM reveal bacterial spore attachment.

Transmission electron microscopy (TEM) studies in the 1960s and early 1970s using conventional thin section and freeze fracture methodologies revealed ultrastructural bacterial spore appendages. However, the limited technology at that time necessitated the time-consuming process of imaging serial sections and reconstructing each structure. Consequently, the distribution and function of these appendages and their possible role in colonization or pathogenesis remained unknown. By combining high resolution field emission electron microscopy with TEM images of identical bacterial spore preparations, we have been able to obtain images of intact and sectioned Bacillus and Clostridial spores to clearly visualize the appearance, distribution, resistance (to trypsin, chloramphenicol, and heat), and participation of these structures to facilitate attachment of the spores to glass, agar, and human cell substrates. Current user-friendly commercial field emission scanning electron microscopes (FESEMs), permit high resolution imaging, with high brightness guns at lower accelerating voltages for beam sensitive intact biological samples, providing surface images at TEM magnifications for making direct comparisons. For the first time, attachment structures used by pathogenic, environmental, and thermophile bacterial spores could be readily visualized on intact spores to reveal how specific appendages and outer spore coats participated in spore attachment, colonization, and invasion.

DETERMINING BIOLOGICAL FINE STRUCTURE BY DIFFERENTIAL ABSORPTION OF SOFT X‐RAYS

The use of soft x-ray contact microscopy in examining histochemically treated human tissue embedded in plastic and exposed as unstained thin sections is demonstrated. When our preliminary data revealed that we could clearly image not only the histochemical reaction product, but the unstained biological fine structure of the surrounding tissues, we decided to test our hypothesis further and see if we could image unstained biological molecular aggregates as well. For this part of the investigation, we chose to examine hydrated proteoglycan aggregates. Proteoglycans are an essential component of the organic matrix of cartilage, and play a primary role in the retention and maintenance of extracellular water. To avoid any artifacts due to the introduction of exogeneous materials, and examine the proteoglycan aggregates in their hydrated, natural configuration, we made contact x-ray images of isolated proteoglycan aggregates in water.

Absorption edge imaging of bacterial endospores with synchrotron radiation

This article describes a new method of viewing biological specimens by taking advantage of the absorptive characteristics of monochromatic X-rays above and below the absorption edge of a specific element. Bacterial endospores were imaged before and after treatment with an experimental vanadium-containing sporocide using monochromatic synchrotron radiation at the nitrogen absorption edge, and above and below the vanadium LIII absorption edge. This morphological study demonstrates a rapid, easy-to-use method of soft X-ray absorption edge imaging that can be used by the biologist to obtain morphological and elemental information that is not readily accessible using conventional microscopic and analytic techniques.

Contact microscopy with synchrotron radiation

Soft X-ray contact microscopy with synchrotron radiation offers the biologist, and especially the microscopist, a way to morphologically study specimens that could not be imaged by conventional TEM, STEM, or SEM methods (i.e., hydrated samples, samples easily damaged by an electron beam, electron-dense samples, thick specimens, unstained, low-contrast specimens) at spatial resolutions approaching those of the TEM, with the additional possibility to obtain compositional (elemental) information about the sample as well. Although flash X-ray sources offer faster exposure times, synchrotron radiation provides a highly collimated, intense radiation that can be tuned to select specific discrete ranges of X-ray wavelengths or specific individual wavelengths that optimize imaging or microanalysis of a specific sample. This paper presents an overview of the applications of X-ray contact microscopy to biological research and some current research results using monochromatic synchrotron radiation to image biological samples.

Cation shifts in human retina-choroid.

Human ocular tissues from 50 donor eyes were elementally and morphologically analyzed in order to correlate the elemental content and distribution of Ca, Ba, Cr, Cu, Zn, and Se with ocular morphology, sex, race, irideal pigmentation, age, time of death, birth weight, presence and severity of diabetes, and other pathologies noted at autopsy. Initially, to facilitate the transport of donor tissue to the laboratory, the eyes were fixed in glutaraldehyde. Because our preliminary data revealed alterations in elemental content following chemical fixation of ocular tissues, all of the subsequent samples were analyzed in their fresh, hydrated (unfixed) condition as soon after enucleation as possible. Samples were elementally analyzed by X-ray fluorescence spectrometry (XRF) and proton-induced X-ray emission spectrometry (PIXE) using high resolution Si(Li) X-ray detectors. Tissue was morphologiscally examined by scanning electron microscopy and light microscopy histoichemistry.