Detection of pathogens.We have developed a simple and fast bacteriophage-based system for detection of small number of bacterial pathogens in clinical or environmental samples. This was achieved by tagging tester phage capsids with biotinylating peptides. This way phage gets biotinylated after infection and growth in specimen bacterial samples. Biotinylated output phage particles are then monitored by specific quantum dots conjugation and then measuring fluorescence in microscope.Capsular surface is a barrier to bacteriophage infection.Escherichia coli strains that produce the K1 polysaccharide capsule have long been associated with pathogenesis. This capsule is believed to increase the cell's invasiveness, allowing the bacteria to avoid phagocytosis and inactivation by complement. It is also recognized as a receptor by some phages, such as K1F and K1-5, which have virion-associated enzymes that degrade the polysaccharide. In this report we show that expression of the K1 capsule is E. coli physically blocks infection by T7, a phage that recognizes lipopolysaccharide as the primary receptor. Enzymatic removal of the K1 antigen from the cell allows T7 to adsorb and replicate. This observation suggests that the capsule plays an important role as a defense against some phages that recognize structure beneath it and the K1-specific phages evolved to counter this physical barrier.A two-hybrid nanotechnology for proteomics.In light of the continued realization of full genome annotations, a comprehensive description of protein-protein interactions is a growing challenge of modern research. A genetic two-hybrid system has been developed for studying protein-protein interactions that is based on bacteriophage lambda display and simple antibiotic resistance selection. The validity of this approach was demonstrated by analyzing the specific interaction and competition of both the protein sorting signal ubiquitin with the CUE domain of Vsp9p, and of artificial acidic and basic aptamers. In contrast to the yeast 2-Hybrid system, peptide immobilization-based panning procedures, and T7 and M13 phage display systems, the approach described here is carried out in the absence of cellular proteins (ex vivo), there is no membrane passage, no bias towards small domains and no harsh chemical treatment, and a high density of display can be achieved. This lambda 9-hybrid system provides a simple way for independent verification of protein interactions obtained through other means, and is a attractive complement for studying protein-protein interactions that aim to identify the large number of protein interactions with lowest amount of background. It should also provide a flexible platform to perform library panning with singular or multiple bait(s), to study protein-DNA association in the absence of high concentration of cellular protein components, drug discovery and to select for and analyze macromolecular interactions that are dependent upon specific modulators.Construction and test of a live commensal bacteria secreting an anti-HIV peptide (collaboration with Dean Hamer).Most HIV transmission occurs on the mucosal surfaces of the gastrointestinal and cervicovaginal tracts, both of which are normally coated by a biofilm of nonpathogenic commensal bacteria. We propose to genetically engineer such naturally occurring bacteria to protect against HIV infection by secreting antiviral peptides. Here we describe the development and characterization of Nissle 1917, a highly colonizing probiotic strain of Escherichia coli, secreting HIV-gp41-hemolysin A hybrid peptides that block HIV fusion and entry into target cells. By using an appropriate combination of cis- and transacting secretory and regulatory signals, micromolar secretion levels of the anti-HIV peptides are achieved.
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| Edgar, Rotem; McKinstry, Michael; Hwang, Jeeseong et al. (2006) High-sensitivity bacterial detection using biotin-tagged phage and quantum-dot nanocomplexes. Proc Natl Acad Sci U S A 103:4841-5 |
| Vitiello, Christal L; Merril, Carl R; Adhya, Sankar (2005) An amino acid substitution in a capsid protein enhances phage survival in mouse circulatory system more than a 1000-fold. Virus Res 114:101-3 |
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| Rao, Srinivas; Hu, Stella; McHugh, Louise et al. (2005) Toward a live microbial microbicide for HIV: commensal bacteria secreting an HIV fusion inhibitor peptide. Proc Natl Acad Sci U S A 102:11993-8 |
| Scholl, Dean; Adhya, Sankar; Merril, Carl (2005) Escherichia coli K1's capsule is a barrier to bacteriophage T7. Appl Environ Microbiol 71:4872-4 |
| Scholl, D; Kieleczawa, J; Kemp, P et al. (2004) Genomic analysis of bacteriophages SP6 and K1-5, an estranged subgroup of the T7 supergroup. J Mol Biol 335:1151-71 |
| Merril, Carl R; Scholl, Dean; Adhya, Sankar L (2003) The prospect for bacteriophage therapy in Western medicine. Nat Rev Drug Discov 2:489-97 |
| Gupta, Amita; Onda, Masanori; Pastan, Ira et al. (2003) High-density functional display of proteins on bacteriophage lambda. J Mol Biol 334:241-54 |
