1. Bacteriophage therapy. In the past, we have been developing bacteriophages for their potential uses in the treatment of several bacterial infections in food, animals and humans. We developed several such phages as detailed below. We selected specific phages to treat (i) vancomycin resistant Enterococeus faecalis infections, (ii) E. coli K1 and K-5 infections, and (iii) Salmonella infections. We characterized each phage to be of virulent type (non-lysogenizing), does not carry any toxin genes, gives high burst size within a short time (like E. coli phage T7) and have broad host specificity. Specifically, the phages are phiENB6 for VRE, phiK-7 and phiK1-5 for E. coli, phiSP6 and a newly isolated phage phi111 for food borne Salmonella pathogens. Except for the Salmonella phage phi111, all of the research accomplished in this area during the last 4 years, including the experiments to show the rescue of animals with experimental bacterimia have been published. The animal experiments involving phi111 of Salmonella is now being carried out, by Dr. Sangryeol Ryu, who left the laboratory and is at Seoul National University, South Korea. Despite our success to isolate or identify and characterize proper phages and their successful use in rescue of animals in experimental bacterimia, we have indefinitely postponed our own efforts to take the projects to clinical levels. In the past, these trials were being conducted, in consultation with us, by two phage technology companies, Exponential Biotherapies, Inc. and Gangagen, Inc. However, we could not pursue such joint efforts because of more recent NIH guidelines, which discouraged the commercial enterprises to continue interactions with us. Nonetheless, we extended our interest to develop engineered bacteriophages for easy detection of specific bacteria in clinical and environmental samples. We also successfully developed methods to use bacteriophage lambda as a protein display agents. Moreover, we constructed using the lambda-display phage system, a protein 2 hybrid system, called 2lambda system, to study protein-protein interactions in vitro. The phage technology research accomplishments in the last four years are summarized below. (i) Characterization of a lambda phage mutant that survives mammalian circulatory system. In experiments with germ free mice, free from adaptive antibodies to bacteriophage lambda, phage titers in the circulatory system decrease by more than 10(9)pfu within 48 h of intraperitoneal intravenous or oral administration. Based on these observations, we previously used serial passage techniques to select lambda phage mutants, with 13,000-16,000-fold greater capacity to remain in the mouse circulatory system 24h after intraperitoneal injection. These long-circulating phages, had at least three mutations in the original isolates. We have now demonstrated that one of the changes is in the major phage capsid (E) protein, which resulted in the change of glutamic acid to a lysine at residue 158, and is sufficient to confer the long-circulating phenotype that presumably evade the innate immunity. (ii). Characterization of Salmonella and E. coli phages of therapeutic values. We have determined the genome sequences of two lytic bacteriophages, SP6 phi111, which infect Salmonella typhimurium LT2, and phiK1-5, which infects E. coli serotypes K1 and K5. The genome organization of the phages except phi111 is almost identical with the notable exception of the tail fiber genes that confer the different host specificities. It appears that the SP6 and phiK1-5 have diverged extensively at the nucleotide level but they are still more closely related to each other than either is to any other phage currently characterized. The SP6 and K1-5 genomes contain, respectively, 43,769 bp and 44,385 bp, with 174 bp and 234 bp direct terminal repeats. About half of the 105 putative open reading frames in the two genomes combined show no significant similarity to database proteins with a known or predicted function that is obviously beneficial for growth of a bacteriophage. The overall genome organization of SP6 and K1-5 is comparable to that of the T7 group of phages, although the specific order of genes coding for DNA metabolism functions has not been conserved. (iii) Capsular polysaccharides may be a barrier to phage invasion. E. coli strains that produce the K1 polysaccharide capsule have long been associated with pathogenesis. This capsule is believed to increase the cells 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. We have shown that expression of the K1 capsule in E. coli physically blocks infections by T7, a phage that recognizes lipopolysaccharide as the primary receptor.

2. Highly sensitive detection of bacteria using phage and Quantum dots. With current concerns of antibiotic-resistant bacteria and biodefence, it has become important to rapidly identify infectious bacteria. We developed a rapid and simple method that combines in vivo biotinylation of engineered host-specific bacteriophage and conjugation of the phage to streptavidin-coated quantum dots. The method, provides specific detection of 10 bacterial cells or less per milliliter in experimental and environmental samples, with an approximately 100-fold amplification of the signal over background in one h. The potential for simultaneous detection of different bacterial species in a single sample and applications in the study of phage biology are now feasible since one can label different phages with Quantum dots of different colors.

3. A lambda-based genetic system to study protein-protein interaction in vitro: 2lambda system. Analyzing protein-protein interactions is critical in proteomics and drug discovery. The usage of 2-Hybrid systems is limited to an in vivo environment. We have developed lambda 2-Hybrid system for studying protein interactions in vitro. Bait and prey are displayed as fusions to the surface protein D of phage lambda that are marked with different selectable drug resistant markers. An interaction of phages in vitro through displayed proteins allows bacterial infection by two phages resulting lysogenic cells, which are detected on double drug resistant bacterial colonies at a very low moi of infection under which coincidental double infection is practically zero. We demonstrated interaction of the protein sorting signal Ubiquitin with the Vps9-CUE, a Ubiquitin binding domain, and by the interaction of [Gly-Glu]4 and [Gly-Arg]4 peptides. Interruptions of the phage interactions by non-fused (free) bait or prey molecules show how robust and unique our approach is. We also showed the use of Ubiquitin and CUE display phages to find binding partners in a lambda-display library.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010017-12
Application #
7592622
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
12
Fiscal Year
2007
Total Cost
$516,709
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Bair, Catherine L; Oppenheim, Amos; Trostel, Andrei et al. (2008) A phage display system designed to detect and study protein-protein interactions. Mol Microbiol 67:719-28
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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