Membrane Insertion for the Detection of Lipopolysaccharides: Exploring the Dynamics of Amphiphile-in-Lipid Assays

Shiga toxin-producing Escherichia coli is an important cause of foodborne illness, with cases attributable to beef, fresh produce and other sources. Many serotypes of the pathogen cause disease, and differentiating one serotype from another requires specific identification of the O antigen located on the lipopolysaccharide (LPS) molecule. The amphiphilic structure of LPS poses a challenge when using classical detection methods, which do not take into account its lipoglycan biochemistry. Typically, detection of LPS requires heat or chemical treatment of samples and relies on bioactivity assays for the conserved lipid A portion of the molecule. Our goal was to develop assays to facilitate the direct and discriminative detection of the entire LPS molecule and its O antigen in complex matrices using minimal sample processing. To perform serogroup identification of LPS, we used a method called membrane insertion on a waveguide biosensor, and tested three serogroups of LPS. The membrane insertion technique allows for the hydrophobic association of LPS with a lipid bilayer, where the exposed O antigen can be targeted for specific detection. Samples of beef lysate were spiked with LPS to perform O antigen specific detection of LPS from E. coli O157. To validate assay performance, we evaluated the biophysical interactions of LPS with lipid bilayers both in- and outside of a flow cell using fluorescence microscopy and fluorescently doped lipids. Our results indicate that membrane insertion allows for the qualitative and reliable identification of amphiphilic LPS in complex samples like beef homogenates. We also demonstrated that LPS-induced hole formation does not occur under the conditions of the membrane insertion assays. Together, these findings describe for the first time the serogroup-specific detection of amphiphilic LPS in complex samples using a membrane insertion assay, and highlight the importance of LPS molecular conformations in detection architectures

The Drosophila Transcription Factors Tinman and Pannier Activate and Collaborate with Myocyte Enhancer Factor-2 to Promote Heart Cell Fate

<div><p>Expression of the MADS domain transcription factor Myocyte Enhancer Factor 2 (MEF2) is regulated by numerous and overlapping enhancers which tightly control its transcription in the mesoderm. To understand how <i>Mef2</i> expression is controlled in the heart, we identified a late stage <i>Mef2</i> cardiac enhancer that is active in all heart cells beginning at stage 14 of embryonic development. This enhancer is regulated by the NK-homeodomain transcription factor Tinman, and the GATA transcription factor Pannier through both direct and indirect interactions with the enhancer. Since Tinman, Pannier and MEF2 are evolutionarily conserved from <i>Drosophila</i> to vertebrates, and since their vertebrate homologs can convert mouse fibroblast cells to cardiomyocytes in different activator cocktails, we tested whether over-expression of these three factors in vivo could ectopically activate known cardiac marker genes. We found that mesodermal over-expression of Tinman and Pannier resulted in approximately 20% of embryos with ectopic <i>Hand</i> and <i>Sulphonylurea receptor (Sur)</i> expression. By adding MEF2 alongside Tinman and Pannier, a dramatic expansion in the expression of <i>Hand</i> and <i>Sur</i> was observed in almost all embryos analyzed. Two additional cardiac markers were also expanded in their expression. Our results demonstrate the ability to initiate ectopic cardiac fate in vivo by the combination of only three members of the conserved <i>Drosophila</i> cardiac transcription network, and provide an opportunity for this genetic model system to be used to dissect the mechanisms of cardiac specification.</p></div

Identification of a proximal <i>Mef2</i> Cardiac Enhancer.

<p>(A)Diagram of the <i>Mef2</i> gene and its cardiac enhancers. The most distal enhancer (-6877/-6388) refers to the Seven-up cell enhancer. -5903/-5667 represents the Tinman-dependent enhancer and the red box (-2775/-2432) is the late stage cardiac and somatic mesodermal enhancer characterized here. (B) Activity of <i>Mef2</i> cardiac enhancers fused to <i>lacZ</i> reporters. The embryos were stained for β-Gal accumulation. Top row, the -5903/-5667 enhancer was active earliest in development, and reporter activity was detected until stage 16. Middle row, the -2775/-2432 enhancer became active at stage 13 in cardiac cells (arrow) and skeletal myoblasts, and was active in all cardiac cells by stage 16. Bottom row, when the two enhancers were fused, there was reporter expression in all cardiac cells during embryogenesis. Arrows indicate heart cells. Bar, 100μm. (C)Alignment of the proximal enhancer sequence with four <i>Drosophila</i> species. A conserved Tinman binding site is marked by a blue box, Pannier sites are marked with red boxes, and the proposed Lame duck binding site identified by Duan et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132965#pone.0132965.ref033" target="_blank">33</a>] is marked with a green box.</p

Ectopic expression of <i>tinman</i> and <i>pannier</i> results in expansion of enhancer activity.

<p>All embryos are stage 13–14 embryos carrying the -2775/-2432 <i>Mef2-lacZ</i> with the Lame duck consensus site mutated. (Left column) accumulation of MEF2; (center column) accumulation of ß-Gal; (right column) merge of prior two channels. Embryos have ectopic ectodermal expression of the following genes: (A-C)no additional genes expressed, note that activity of the enhancer can be seen in all cells of the heart (arrows), but the somatic mesodermal stain is reduced; (D-F)ectopic <i>pnr</i> expression, stains are similar to (A-C); (G-I) ectopic <i>tin</i> expression, note that activity of the enhancer is expanded in the ectoderm and amnioserosa (arrowheads). Arrow indicates cardiac cells. (J-L) ectopic <i>tin</i> and <i>pnr</i> expression, note that the enhancer is more robustly activated in the ectoderm and amnioserosa when compared to (H), which can been seen more dramatically in the inset, and ß-Gal accumulation co-localizes with the MEF2 expression seen in (J). Arrow indicates to cardiac cells. Scale bar, 100μm.</p

<i>Pericardin</i> and <i>H15</i> are activated by the over-expression of <i>tinman</i>, <i>pannier</i> and <i>Mef2</i> in the mesoderm.

<p>(A,C and E) Control embryos. (B,D and F): 24B+twi><i>tin+pnr+Mef2</i> embryos. (A,B) Antibody stain against Fasciclin III which marks the visceral mesoderm at stage 10. (C,D) Antibody stain against Pericardin which marks the pericardial cells. (E,F) Antibody stain against H15 which is a cardiac-specific T box transcription factor. Arrows point to normal expression and arrowheads point to expanded expression. Bar, 100μm.</p

Mutation of the Tinman consensus site in the -2775/-2432 enhancer results in loss of enhancer activity in cardiac cells.

<p>(A-F)-2775/-2432 <i>Mef2-lacZ</i> embryos at stages 14 (A-C) and 16 (D-F). (A,D)Antibody stain against MEF2. MEF2 could be detected in all cells of the heart and throughout the somatic mesoderm. (B,E)Antibody stain against β-Galactosidase. Activity of the late stage enhancer was almost identical to that of <i>Mef2</i> expression. In (B), the enhancer is just becoming active in the heart cells, therefore a few cells lacked activity at this stage. By stage 16 (E) all cardiac cells showed ßGal accumulation. (C)Merge of (A) and (B); (F)Merge of (D) and (E). (G-L)Embryos carrying the -2775/-2432 <i>Mef2-lacZ</i> enhancer with the Tinman consensus site mutated, at stages 14 (G-I) or 16 (J-L). (G,J)Antibody stain against MEF2. MEF2 marks all cells of the heart and the somatic mesoderm. (H,K)Antibody stain against β-Galactosidase. Activity of the mutated enhancer was completely lost from the cardiac cells, and was slightly reduced in the somatic mesoderm. (I)Merge of (G) and (H). (L)Merge of (J) and (K). Arrows point to MEF2 positive cardiac cells, arrowheads point to the same cells lacking β-Galactosidase. (M-R) Embryos carrying the -2775/-2432 <i>Mef2-lacZ</i> enhancer with the three Pannier consensus sites mutated, at stages 14 (M-O) or 16 (P-R). (O)Merge of (M) and (N). (R) Merge of (P) and (Q). Arrows point to cardiac cells, arrow heads point to cells that have lost enhancer activity. Asterisk denotes activity in the amnioserosa. Bar, 100μm.</p

Expression of <i>tinman</i>, <i>pannier</i> and <i>Mef2</i> in the ectoderm results in an expansion of cardiac gene expression.

<p>(A,B) 69B><i>tin+pnr+Mef2</i> embryos at stage 14, stained for <i>Hand</i> expression (A) or <i>Sur</i> expression (B). (A) ~ 50% of embryos had expanded <i>Hand</i> expression (arrowhead) with the rest demonstrating normal expression in the heart (inset, arrow). (B) ~70% of embryos had normal <i>Sur</i> expression in the heart (arrow), with a subset showing expanded <i>Sur</i> expression in the nervous system (inset, arrowhead). Bar, 100μm. (C) Quantification of effects of over-expression of cardiac transcription factors. (D,E) Antibody stain against Fasciclin III. (D) shows normal FasIII accumulation in a stage 9 control embryo. (E) shows similar levels of accumulation in a 69B><i>tin+pnr+Mef2</i> embryo.</p

Sequences of oligonucleotide primers used to amplify cDNA for the indicated genes.

<p>Sequences of oligonucleotide primers used to amplify cDNA for the indicated genes.</p