Lipopolysaccharide (LPS) is an amphiphilic lipoglycan that is the primary component of the outer membrane of Gramnegative bacteria. Classified as a pathogen associated molecular pattern (PAMPs), LPS is an essential biomarker for identifying pathogen serogroups. Structurally, LPS is comprised of a hydrophobic lipophilic domain that partitions into the outer membrane of Gram-negative bacteria. Previous work by our team explored biophysical interactions of LPS in supported lipid bilayer assemblies (sLBAs), and demonstrated LPS-induced hole formation in DOPC lipid bilayers. Here, we have incorporated cholesterol and sphingomyelin into sLBAs to evaluate the interaction of LPS in a more physiologically relevant system. The goal of this work was to determine whether increasing membrane complexity of sLBAs, and changing physiological parameters such as temperature, affects LPS-induced hole formation. Integrating cholesterol and sphingomyelin into sLBAs decreased LPS-induced hole formation at lower concentrations of LPS, and bacterial serotype contributed to differences in hole formation as a response to changes in temperature. We also investigated the possibility of LPS-induced hole formation in cellular systems using the cytokine response in both TLR4 (+)/(-) murine macrophages. LPS was presented to each cell line in murine serum, delipidated serum, and buffer (i.e. no serum), and the resulting cytokine levels were measured. Results indicate that the method of LPS presentation directly affects cellular cytokine expression. The two model systems presented in this study provide preliminary insight into the interactions of LPS in the host, and suggest the significance of amphiphile-carrier interactions in regulating host-pathogen biology during infection.
Shiga toxin-producing Escherichia coli (STEC) poses a serious threat to human health through the consumption of
contaminated food products, particularly beef and produce. Early detection in the food chain, and discrimination from
other non-pathogenic Escherichia coli (E. coli), is critical to preventing human outbreaks, and meeting current
agricultural screening standards. These pathogens often present in low concentrations in contaminated samples, making
discriminatory detection difficult without the use of costly, time-consuming methods (e.g. culture). Using multiple signal
transduction schemes (including novel optical methods designed for amphiphiles), specific recognition antibodies, and a
waveguide-based optical biosensor developed at Los Alamos National Laboratory, we have developed ultrasensitive
detection methods for lipopolysaccharides (LPS), and protein biomarkers (Shiga toxin) of STEC in complex samples
(e.g. beef lysates). Waveguides functionalized with phospholipid bilayers were used to pull down amphiphilic LPS,
using methods (membrane insertion) developed by our team. The assay format exploits the amphiphilic biochemistry of
lipoglycans, and allows for rapid, sensitive detection with a single fluorescent reporter. We have used a combination of
biophysical methods (atomic force and fluorescence microscopy) to characterize the interaction of amphiphiles with lipid
bilayers, to efficiently design these assays. Sandwich immunoassays were used for detection of protein toxins.
Biomarkers were spiked into homogenated ground beef samples to determine performance and limit of detection. Future
work will focus on the development of discriminatory antibodies for STEC serotypes, and using quantum dots as the
fluorescence reporter to enable multiplex screening of biomarkers.
The increasing need for highly polychromatic approaches to flow cytometry, coupled with rapid technological advances,
have driven the design and implementation of commercial instruments that measure up to 19 parameters using multiple
lasers for excitation, an intricate optical filter/mirror arrangement, and analysis using fluorescence compensation
approaches. Although such conventional multiparameter flow cytometers have proven highly successful, there are
several types of analytical measurements that would benefit from higher density of spectral information and a more
flexible approach to spectral analysis including, but certainly not limited to: spectral deconvolution of overlapping
spectra, fluorescence resonance energy transfer measurements, metachromic dye analysis, cellular autofluorescence
characterization, and flow based Raman spectroscopy. For these purposes, we have developed a high resolution spectral
flow cytometer using an EMCCD camera with 1600 by 200 pixels, which is capable of detecting less than 200
fluorescein molecules with a spectral resolution of less than 3 nm. This instrument will enable high throughput
characterization of single cell or particle emission spectra. For proof of principle instrument operation, we have begun
characterization of intrinsic cellular autofluorescence, which is the major source of background for cell-based
fluorescence assays. Specifically, we will describe recent work on the high resolution spectral characterization of
autofluorescence for several commonly used cell types. Autofluorescence emission is known to cover over almost the
entire spectrum from 300 to nearly 800 nm. These emissions are attributed to flavins, elastin, Indolamine dimers and
trimers, NADH and collagen among other molecules. We will show that several unique autofluorescence spectra arise in
the different cell lines thereby suggesting the possibility of discrimination of cell types based on intrinsic fluorescence.
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