A Novel Fluorescent Protein-Based Biosensor for Gram-Negative Bacteria

Abstract

Increasing concern regarding the microbiological safety of food, water, dairy products, industrial waste, and pharmaceutical preparations has provided an urgency for detection methods that are fast, sensitive, specific, reliable, and quantitative for quality assurance in order to prevent infections and epidemics (10). There are a large number of detection methods for microorganisms, including immunomagnetic separation and flow cytometry (18), flexural plate wave (16), quartz crystal (8), and surface acoustic wave (9). In addition, due to the ubiquity and lethality of endotoxin (or lipopolysaccharide) from the outer cell-wall of gram-negative bacteria, many pharmaceutical products are rigorously tested for the presence of contaminating lipopolysaccharide or gram-negative bacteria before the products are sold for human use. Both the United States Pharmacopoeia and the European Pharmacopoeia specify the rabbit pyrogen test and the Limulus amoebocyte lysate test as the quality control tests for the presence of endotoxin in injectables and medical devices.A number of new approaches to pyrogen testing have been reported. These are mainly based on an in vitro pyrogen test involving the use of human cells such as leukocyte cell lines, isolated primary blood, and whole blood (4, 20). Recently, genetic engineering of an endotoxin-sensitive Limulus amoebocyte lysate protein, recombinant factor C expressed in a baculovirus system, produced an enzyme with remarkable sensitivity to endotoxin, at 0.001 endotoxin unit (EU)/ml (2).The green fluorescent protein (GFP) has been a popular choice for development of reporter-biosensors to detect various environmentally hazardous compounds (7,11,19).Through computer-aided simulation and rational design, we have recently developed a fluorescent biosensor for lipopolysaccharide and lipid A (the bioactive moiety of lipopolysaccharide) with enhanced green fluorescent protein (EGFP) as a scaffold protein (6). Previously, we have shown (5) that lipopolysaccharide or lipid A can interact strongly with short cationic amphipathic sequences of five alternating basic (B) and hydrophobic (H) residues (BHBHB). Thus, such sequence motifs were introduced into the ␤-sheets located on the surface of the EGFP barrel in the vicinity of the chromophore (6). The EGFP mutants (EGFP i ) showed a range of lipid A binding affinities (26.12 to 0.13 M), resulting in concentration-dependent fluorescence quenching (6).The high level of endogenous lipopolysaccharide, which is the ligand that binds EGFP i , and a serious host incompatibility problem that may result in its growth inhibition or even cell death during the expression of recombinant EGFP i in Escherichia coli are the major challenges facing the expression of EGFP i in a gram-negative bacterial host. However, this problem did not arise in the present study. Furthermore, Schnaitman (17) has demonstrated that treatment of E. coli with the combination of Triton X-114, EDTA, and lysozyme resulted in solubilization of all lipopolysaccharide from the cell wall...

Lipopolysaccharide-binding molecules: transporters, blockers and sensors

“…Another important goal was the design of endotoxin detection reagents. A green fluorescent protein mutant bearing an LPS binding motif was recently created [169], and can now be used to detect low amounts of endotoxin for the evaluation on the safety of water and pharmaceutical preparations. The exciting developments in the field of LPS-binding molecules have thus led to a much better understanding of how the host processes an LPS molecule and respond to it.…”

Analysis of Chemical and Biological Properties

“…Biological material, such as bacteria or microbial toxins, may also be detected by fluorescence or chemiluminescence means, or with biosensors which employ combined techniques of different kinds (Angell et al, 1993;Wittwer et al, 1997;Carlson et al, 2000;Goh et al, 2002;Yacoub-George et al, 2002;Rotman et al, 2003). Most of these advanced methods, originally developed for bacterial detection in the food industry or medicine, are aimed at rapid detection without culturing the organism.…”

Recent developments in rapid multiplexed bioanalytical methods for foodborne pathogenic bacteria detection

“…The detection limits were comprised between 3× 10 3 and 3×10 6 cells mL −1 for the different target bacteria, with a 13-min assay time [46]. Mutagenesis of enhanced green fluorescent protein was performed to develop a fluorescence-based biosensor for gram-negative bacteria, like E. coli and Pseudomonas aeruginosa [65]. However, multiplexed capabilities are still to be demonstrated.…”