Immobilization of Intact Phage and Phage-Derived Proteins for Detection and Biocontrol Purposes.

The natural specificity of bacteriophages toward their hosts represents great potential for the development of platforms for the capture and detection of bacterial pathogens. Whole phage can carry reporter genes to alter the phenotype of the target pathogen. Phage can also act as staining agents or the progeny of the infection process can be detected. Alternatively, using phage components as probes offer advantages over whole phage particles, including smaller probe size and resilience to desiccation. Phage structures can be engineered for improved affinity, specificity, and binding properties. However, such concepts require the ability to anchor phage and phage-components onto mechanical supports such as beads or flat surfaces. The ability to orient the anchoring is desired in order to optimize binding efficiency. This chapter presents various methods that have been employed for the attachment of phage and phage components onto support structures such as beads, filters, and sensor surfaces.

Development of prototypes of bioactive packaging materials based on immobilized bacteriophages for control of growth of bacterial pathogens in foods.

Due to lack of adequate control methods to prevent contamination in fresh produce and growing consumer demand for natural products, the use of bacteriophages has emerged as a promising approach to enhance safety of these foods. This study sought to control Listeria monocytogenes in cantaloupes and RTE meat and Escherichia coli O104:H4 in alfalfa seeds and sprouts under different storage conditions by using specific lytic bacteriophage cocktails applied either free or immobilized. Bacteriophage cocktails were introduced into prototypes of packaging materials using different techniques: i) immobilizing on positively charged modified cellulose membranes, ii) impregnating paper with bacteriophage suspension, and iii) encapsulating in alginate beads followed by application of beads onto the paper. Phage-treated and non-treated samples were stored for various times and at temperatures of 4°C, 12°C or 25°C. In cantaloupe, when free phage cocktail was added, L. monocytogenes counts dropped below the detection limit of the plating technique (<1 log CFU/g) after 5 days of storage at both 4°C and 12°C. However, at 25°C, counts below the detection limit were observed after 3 and 6h and a 2-log CFU/g reduction in cell numbers was seen after 24h. For the immobilized Listeria phage cocktail, around 1-log CFU/g reduction in the Listeria count was observed by the end of the storage period for all tested storage temperatures. For the alfalfa seeds and sprouts, regardless of the type of phage application technique (spraying of free phage suspension, bringing in contact with bacteriophage-based materials (paper coated with encapsulated bacteriophage or impregnated with bacteriophage suspension)), the count of E. coli O104:H4 was below the detection limit (<1 log CFU/g) after 1h in seeds and about a 1-log cycle reduction in E. coli count was observed on the germinated sprouts by day 5. In ready-to-eat (RTE) meat, LISTEX™ P100, a commercial phage product, was able to significantly reduce the growth of L. monocytogenes at both storage temperatures, 4°C and 10°C, for 25 days regardless of bacteriophage application format (immobilized or non-immobilized (free)). In conclusion, the developed phage-based materials demonstrated significant antimicrobial effect, when applied to the artificially contaminated foods, and can be used as prototypes for developing bioactive antimicrobial packaging materials capable of enhancing the safety of fresh produce and RTE meat.

Targeted disinfection of E. coli via bioconjugation to photoreactive TiO2.

The selective control of pathogenic bacteria is an ongoing challenge. A strategy is proposed that combines targeted binding of the bacterium, using antibodies, with their photoactivated oxidative destruction. Photoactive colloidal TiO2 was first derivatized with E. coli antibodies (EA-TiO2). When mixtures of the organisms E. coli and Pseudomonas putida ( P. putida ) were exposed to modified EA-TiO2, the particles preferentially selected E. coli for surface binding. Two consequences arose from surface bioconjugation: bacteria were found to flocculate upon mixing at appropriate ratios of EA-TiO2/ E. coli , and EA-TiO2-bound E. coli underwent cell death after exposure to UV light. In the former case, flocculation of the bacteria was optimal at ~50 EA-TiO2 particles per E. coli . Selective flocculation provides an alternative strategy for pathogen removal. With respect to UV disinfection, as few as 26 EA-TiO2 particles per E. coli gave a 10 000-fold decrease in viable bacteria. Thus, it is possible to selectively target and kill one type of bacteria in a mixture of pathogens. The results give support to the proposal that photocatalytic TiO2 most effectively delivers an oxidizing agent when the titania is bound to the bacterial surface.

Signaling through P2X7 receptor in human T cells involves p56lck, MAP kinases, and transcription factors AP-1 and NF-kappa B.

ATP-gated ion channel P2X receptors are expressed on the surface of most immune cells and can trigger multiple cellular responses, such as membrane permeabilization, cytokine production, and cell proliferation or apoptosis. Despite broad distribution and pleiotropic activities, signaling pathways downstream of these ionotropic receptors are still poorly understood. Here, we describe intracellular signaling events in Jurkat cells treated with millimolar concentrations of extracellular ATP. Within minutes, ATP treatment resulted in the phosphorylation and activation of p56(lck) kinase, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase but not p38 kinase. These effects were wholly dependent upon the presence of extracellular Ca(2+) ions in the culture medium. Nevertheless, calmodulin antagonist calmidazolium and CaM kinase inhibitor KN-93 both had no effect on the activation of p56(lck) and ERK, whereas a pretreatment of Jurkat cells with MAP kinase kinase inhibitor P098059 was able to abrogate phosphorylation of ERK. Further, expression of c-Jun and c-Fos proteins and activator protein (AP-1) DNA binding activity were enhanced in a time-dependent manner. In contrast, DNA binding activity of NF-kappa B was reduced. ATP failed to stimulate the phosphorylation of ERK and c-Jun N-terminal kinase and activation of AP-1 in the p56(lck)-deficient isogenic T cell line JCaM1, suggesting a critical role for p56(lck) kinase in downstream signaling. Regarding the biological significance of the ATP-induced signaling events we show that although extracellular ATP was able to stimulate proliferation of both Jurkat and JCaM1 cells, an increase in interleukin-2 transcription was observed only in Jurkat cells. The nucleotide selectivity and pharmacological profile data supported the evidence that the ATP-induced effects in Jurkat cells were mediated through the P2X7 receptor. Taken together, these results demonstrate the ability of extracellular ATP to activate multiple downstream signaling events in a human T-lymphoblastoid cell line.