Phase-Separating Peptides Recruiting Aggregation-Induced Emission Fluorogen for Rapid E. coli Detection.

Rationally designed biomolecular condensates have found applications primarily as drug-delivery systems, thanks to their ability to self-assemble under physico-chemical triggers (such as temperature, pH, or ionic strength) and to concomitantly trap client molecules with exceptionally high efficiency (>99%). However, their potential in (bio)sensing applications remains unexplored. Here, we describe a simple and rapid assay to detect E. coli by combining phase-separating peptide condensates containing a protease recognition site, within which an aggregation-induced emission (AIE)-fluorogen is recruited. The recruited AIE-fluorogen's fluorescence is easily detected with the naked eye when the samples are viewed under UV-A light. In the presence of E. coli, the bacteria's outer membrane protease (OmpT) cleaves the phase-separating peptides at the encoded protease recognition site, resulting in two shorter peptide fragments incapable of liquid-liquid phase separation. As a result, no condensates are formed and the fluorogen remains non-fluorescent. The assay feasibility was first tested with recombinant OmpT reconstituted in detergent micelles and subsequently confirmed with E. coli K-12. In its current format, the assay can detect E. coli K-12 (108 CFU) within 2 h in spiked water samples and 1-10 CFU/mL with the addition of a 6-7 h pre-culture step. In comparison, most commercially available E. coli detection kits can take anywhere from 8 to 24 h to report their results. Optimizing the peptides for OmpT's catalytic activity can significantly improve the detection limit and assay time. Besides detecting E. coli, the assay can be adapted to detect other Gram-negative bacteria as well as proteases having diagnostic relevance.

Rapid Detection of Escherichia coli: Optimized Peptide-Polythiophene Interactions Help Reduce Assay Time and Improve Naked-Eye Detection.

Recent improvements in methods for rapid detection of microbial contamination in food and water samples have aided in the development of on-site and point-of-care testing. Early detection, made possible via on-site testing, can help limit the spread of food and waterborne illnesses. Recently, we reported a novel fluorescence-based Omptin-Polythiophene assay (the assay) to detect Escherichia coli in contaminated water samples. The assay targets OmpT─an E. coli outer membrane protease─and exploits the protease's ability to cleave at dibasic sites within a peptide. By combining a peptide substrate optimized for OmpT with a conjugated polythiophene reporter molecule whose optical properties vary upon interaction with the intact or cleaved peptide, we demonstrated the detection of 1-10 CFU/mL and 105 CFU/mL E. coli in 5.5 and 1 h, respectively. In comparison, most microbial detection methods that rely on culturing and plating techniques take anywhere between 8 and 24 h to report their results. Herein we report significant improvements in the assay which include reducing the assay time from an already short 1 h to a mere 10 min for detecting E. coli in highly contaminated samples and augmenting the assay with colorimetric sensing capability for naked eye discernment under normal visible light or under UV-A light. These improvements were made possible by characterizing the optical changes resulting from the interaction of the peptide with five carboxylate-functionalized polythiophene variants carrying different 3- side chain carboxylic acids and by identifying preferential peptide substrates via the screening of ten peptide sequence variants for OmpT activity.

Outer-Membrane Protease (OmpT) Based E. coli Sensing with Anionic Polythiophene and Unlabeled Peptide Substrate.

E. coli and Salmonella are two of the most common bacterial pathogens involved in foodborne and waterborne related deaths. Hence, it is critical to develop rapid and sensitive detection strategies for near-outbreak applications. Reported is a simple and specific assay to detect as low as 1 CFU mL-1 of E. coli in water within 6 hours by targeting the bacteria's surface protease activity. The assay relies on polythiophene acetic acid (PTAA) as an optical reporter and a short unlabeled peptide (LL37FRRV ) previously optimized as a substrate for OmpT, an outer-membrane protease on E. coli. LL37FRRV interacts with PTAA to enhance its fluorescence while also inducing the formation of a helical PTAA-LL37FRRV construct, as confirmed by circular dichroism. However, in the presence of E. coli LL37FRRV is cleaved and can no longer affect the conformations and optical properties of PTAA. This ability to distinguish between an intact and cleaved peptide was investigated in detail using LL37FRRV sequence variants.

Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding.

Outer membrane protease (OmpT) is a 33.5 kDa aspartyl protease that cleaves at dibasic sites and is thought to function as a defense mechanism for E. coli against cationic antimicrobial peptides secreted by the host immune system. Despite carrying three dibasic sites in its own sequence, there is no report of OmpT autoproteolysis in vivo. However, recombinant OmpT expressed in vitro as inclusion bodies has been reported to undergo autoproteolysis during the refolding step, thus resulting in an inactive protease. In this study, we monitor and compare levels of in vitro autoproteolysis of folded and unfolded OmpT and examine the role of lipopolysaccharide (LPS) in autoproteolysis. SDS-PAGE data indicate that it is only the unfolded OmpT that undergoes autoproteolysis while the folded OmpT remains protected and resistant to autoproteolysis. This selective susceptibility to autoproteolysis is intriguing. Previous studies suggest that LPS, a co-factor necessary for OmpT activity, may play a protective role in preventing autoproteolysis. However, data presented here confirm that LPS plays no such protective role in the case of unfolded OmpT. Furthermore, OmpT mutants designed to prevent LPS from binding to its putative LPS-binding motif still exhibited excellent protease activity, suggesting that the putative LPS-binding motif is of less importance for OmpT's activity than previously proposed.

A Bottom-Up Proteomic Approach to Identify Substrate Specificity of Outer-Membrane Protease OmpT.

Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity, guides development of inhibitors, and improves assay sensitivity. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer-membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1') and nearest-neighbor positions (P2, P2') and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a greater than 400-fold improvement in OmpT catalytic efficiency, with a kcat /Km value of 6.1×106  L mol-1 s-1 . Wild-type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme.

Controlled Supramolecular Self-Assembly of Super-charged ß-Lactoglobulin A-PEG Conjugates into Nanocapsules.

The synthesis and characterization of a new protein-polymer conjugate composed of ß lactoglobulin A (ßLG A) and poly(ethylene glycol) PEG is described. ßLG A was selectively modified to self-assemble by super-charging via amination or succinylation followed by conjugation with PEG. An equimolar mixture of the oppositely charged protein-polymer conjugates self-assemble into spherical capsules of 80-100 nm in diameter. The self-assembly proceeds by taking simultaneous advantage of the amphiphilicity and polyelectrolyte nature of the protein-polymer conjugate. These protein-polymer capsules or proteinosomes are reminiscent of protein capsids, and are capable of encapsulating solutes in their interior. We envisage this approach to be applicable to other globular proteins.

A cluster of aspartic residues in the extracellular loop II of PAR 4 is important for thrombin interaction and activation of platelets.

Thrombin activates platelets via proteolytic cleavage of protease-activated receptors (PARs) 1 and 4. The two PARs have distinct but complementary roles. The mechanisms responsible for PAR1 activation by thrombin have been extensively studied. However, much less is known regarding thrombin activation of PAR4, especially the potential involvement of regions of PAR4 other than the N-terminal, which is bound to the catalytic site of thrombin. We have studied PAR4 in S. cerevisiae strain MMY12, an expression system in which the GPCR receptors are connected to a Lac Z reporter gene resulting in increased ß-galactosidase activity. This approach was used to assess PAR4 mutants to evaluate the contribution of different aspartic residues in facilitating PAR4 activation. Furthermore, peptides mimicking parts of the PAR4 N-terminal and the second extracellular loop (ECLII) were tested for their ability to inhibit platelet activation by thrombin. Binding of these peptides to γ-thrombin was studied by monitoring the decrease in tryptophan fluorescence intensity of thrombin. We conclude that not only the N-terminal but also the electronegative aspartic residues D224, D230 and D235 (located in ECLII) are be important for PAR4 binding to thrombin. We further suggest that they play a role for the tethered ligand binding to the receptor, as mutations also affected activation in response to a PAR4-activating peptide mimicking the new N-terminal formed after cleavage. This agrees with previous results on PAR1 and thrombin binding. We suggest that the ECLII of PAR4 could be a potential target for antithrombotic drug development.

A review of solute encapsulating nanoparticles used as delivery systems with emphasis on branched amphipathic peptide capsules.

Various strategies are being developed to improve delivery and increase the biological half-lives of pharmacological agents. To address these issues, drug delivery technologies rely on different nano-sized molecules including: lipid vesicles, viral capsids and nano-particles. Peptides are a constituent of many of these nanomaterials and overcome some limitations associated with lipid-based or viral delivery systems, such as tune-ability, stability, specificity, inflammation, and antigenicity. This review focuses on the evolution of bio-based drug delivery nanomaterials that self-assemble forming vesicles/capsules. While lipid vesicles are preeminent among the structures; peptide-based constructs are emerging, in particular peptide bilayer delimited capsules. The novel biomaterial-Branched Amphiphilic Peptide Capsules (BAPCs) display many desirable properties. These nano-spheres are comprised of two branched peptides-bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK, designed to mimic diacyl-phosphoglycerides in molecular architecture. They undergo supramolecular self-assembly and form solvent-filled, bilayer delineated capsules with sizes ranging from 20 nm to 2 µm depending on annealing temperatures and time. They are able to encapsulate different fluorescent dyes, therapeutic drugs, radionuclides and even small proteins. While sharing many properties with lipid vesicles, the BAPCs are much more robust. They have been analyzed for stability, size, cellular uptake and localization, intra-cellular retention and, bio-distribution both in culture and in vivo.

Liposomes as nanoreactors for the photochemical synthesis of gold nanoparticles.

A simple and novel method for the photochemical synthesis of AuNPs in liposomes is described. Gold salt is co-encapsulated with the photoinitiator Irgacure-2959 in POPC liposomes prepared via traditional thin-film hydration technique. UVA irradiation for 15 min results in encapsulated AuNPs of 2.8±1.6 nm in diameter that are primarily dispersed in the aqueous interior of the liposomes.

Branched amphiphilic cationic oligopeptides form peptiplexes with DNA: a study of their biophysical properties and transfection efficiency.

Over the past decade, peptides have emerged as a new family of potential carriers in gene therapy. Peptides are easy to synthesize and quite stable. Additionally, sequences shared by the host proteome are not expected to be immunogenic or trigger inflammatory responses, which are commonly observed with viral approaches. We recently reported on a new class of branched amphiphilic peptide capsules (BAPCs) that self-assemble into extremely stable nanospheres. These capsules are capable of retaining and delivering alpha-emitting radionuclides to cells. Here we report that, in the presence of double stranded plasmid DNA, BAPCs are unable to form. Instead, depending of the peptide/DNA ratios, the peptides either coat the plasmid surface forming nanofibers (high peptide to DNA ratio) or condense the plasmid into nanometer-sized compacted structures (at low peptide to DNA ratios). Different gene delivery efficiencies are observed for the two types of assemblies. The compacted nanometer-sized structures display much higher transfection efficiencies in HeLa cells. This level of transfection is greater than that observed for a lipid-based reagent when the total number of viable transfected cells is taken into account.

Branched oligopeptides form nanocapsules with lipid vesicle characteristics.

In a recent article (Gudlur et al. PLOS ONE, 2012, 7 (9) e45374), we described the special properties of a mixed branched peptide assembly in which equimolar bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK self-associate to form bilayer delimited capsules capable of trapping solutes. These polycationic vesicle-like capsules are readily taken up by epithelial cells in culture, escape or evade the endocytic pathway, and accumulate in the perinuclear region where they persist without any apparent degradation. In this report, we examine the lipidlike properties of this system including initial assembly; solute encapsulation and washing; fusion and resizing by membrane extrusion through polycarbonate filters with defined pore sizes. The resized peptide capsules have uniform diameters in nm size ranges. Once resized, the capsules can be maintained at the new size by storing them at 4 °C. Having the ability to prepare stable uniform nanoscale capsules of desired sizes makes them potentially attractive as biocompatible delivery vehicles for various solutes/drugs.

Peptide nanovesicles formed by the self-assembly of branched amphiphilic peptides.

Peptide-based packaging systems show great potential as safer drug delivery systems. They overcome problems associated with lipid-based or viral delivery systems, vis-a-vis stability, specificity, inflammation, antigenicity, and tune-ability. Here, we describe a set of 15 & 23-residue branched, amphiphilic peptides that mimic phosphoglycerides in molecular architecture. These peptides undergo supramolecular self-assembly and form solvent-filled, bilayer delimited spheres with 50-200 nm diameters as confirmed by TEM, STEM and DLS. Whereas weak hydrophobic forces drive and sustain lipid bilayer assemblies, these all-peptide structures are stabilized potentially by both hydrophobic interactions and hydrogen bonds and remain intact at low micromolar concentrations and higher temperatures. A linear peptide lacking the branch point showed no self-assembly properties. We have observed that these peptide vesicles can trap fluorescent dye molecules within their interior and are taken up by N/N 1003A rabbit lens epithelial cells grown in culture. These assemblies are thus potential drug delivery systems that can overcome some of the key limitations of the current packaging systems.

Human ß2-glycoprotein I attenuates mouse intestinal ischemia/reperfusion induced injury and inflammation.

Intestinal ischemia-reperfusion (IR)-induced injury results from a complex cascade of inflammatory components. In the mouse model of intestinal IR, the serum protein, ß2-glycoprotein I (ß2-GPI) binds to the cell surface early in the cascade. The bound ß2-GPI undergoes a conformational change which exposes a neoantigen recognized by naturally occurring antibodies and initiates the complement cascade. We hypothesized that providing additional antigen with exogenous ß2-GPI would alter IR-induced tissue injury. Administration of human but not mouse ß2-GPI attenuated IR-induced tissue damage and prostaglandin E(2) production indicating a physiological difference between ß2-GPI isolated from the two species. To investigate whether structural features were responsible for this physiological difference, we compared the chemical, physical and biochemical properties of the two proteins. Despite possessing 76% amino acid identity and 86% sequence homology, we found that mouse ß2-GPI differs from the human protein in size, carbohydrate chain location, heterogeneity and secondary structural content. These data suggest that the structural differences result in mouse Ab recognition of soluble human but not mouse ß2-GPI and attenuated IR-induced injury. We conclude that caution should be exercised in interpreting results obtained by using human ß2-GPI in a mouse model.