Increases in the prevalence of multidrug resistant bacterial pathogens such as enterococci resistant to vancomycin (VRE) have produced a need for alternatives to antibiotic therapies. The primary goals of the Laboratory's studies were to develop phage that can be used to diagnose and treat bacterial infections. In this regard we recently demonstrated that we could engineer phage by changing a single amino acid in a major capsid protein such that the resulting phage can remain in the mammalian circulatory system with an efficacy of more than 1000 fold when compared to the wild type parental virus. Such long-circulating phage were previously shown by our laboratory to be more effective anti-infectious agents. In addition to the above effort, we are completing the 3rd yr. of our NIAID biodefense proposal for intramural biodefense research that was funded to study bacteriophage therapy for Y. pestis, the causative agent of plague in humans, in collaboration with Dr. Adhya of the NCI. We are currently working on the development of reporter phage for the rapid diagnosis of Y. pestis for use in the clinical setting. In recent years plague has been epidemic in countries such as India and Mozambique but antibiotics and changes in public health policies have greatly reduced the chances of pandemics of historical proportions, and plague is now a rare occurrence in developed countries. However the increased risk of deliberate spread of the disease by terrorists and the recent discovery of antibiotic resistant strains has garnered support for new alternatives. Given the importance of rapidly determining whether a particular phage will be effective as an antibacterial therapeutic agent, we are currently developing phage that express reporter genes. Prior to this, determination of susceptibility to phage strains was performed by plaque assays that can take from 12 hrs to several days to develop. We are currently developing methods for incorporating reporter genes, such as b-galactosidase or luciferase genes into phages that may be of clinical use as antibacterial agents. The reporter phage work has also been aided by our collaboration with researchers at National Institute of Standard and Technology (NIST). With their help we have adapted the fluorescent semiconductor nanocrystals or quantum dots (qdots) as reporters for phage. Prior to the introduction of Q-dots the fluorophores used as reporters had two major problems: the auto-fluorescence background and the low photo-stability of organic fluors. The use of qdots may overcome these limitations. Single QDs can be observed which is possibly one of the most exciting new capabilities offered to biologist. In our studies to use qdots as reporters we combined qdots with a surface-coated with streptavidin with a genetically modified bacteriophage engineered to express a biotin binding motif. Once biotin (made by most bacterial strains) is bound to the phage surface these phage can then bind the streptavidin coated qdots. The highly specific linkage of qdots to the engineered phage and to the biological target such as bacteria (qdot-phage-bacteria complex) provides a unique fluorescent signal at the single copy sensitivity. The procedure we have developed provides a general method which could be applied to potentially any phage ?host system. Qdot labeled phages provide a rapid and sensitive method for bacterial detection without loss of signal associated with conventional flours. A manuscript is in preparation regarding this work and a patent has been filed. In addition the laboratory is studying phage of interest by characterizing their lytic capabilities by measuring burst size and eclipse time, stability of the phage is determined by decay studies in various physical environment and most importantly by sequencing their genomes. These studies will provide information on the biology of phages, if they encode any toxin genes, or are able to form lysogens, and insight into their mechanisms for host specificity. These studies have already shown that phage tail enzymes are the main determinant in host specificity and that some phages are able to replicate on any strain once they introduce their genetic material. Our studies of phage tail enzymes provides an approach to enhance the therapeutic efficacy of phages extending their host range. We have also isolated a novel phage, M59-2, which is specific for E. coli strains that overproduce colanic acid, an important component in biofilm formation. We have found that M59-2 encodes a tail protein that degrades colonic acid. This phage and its colanic acid hydrolase are potential tools for control of biofilms (which play a significant role in infectious disease ranging from lung infections to infections in the urinary tract) and we are conducting experiments to examine this. We have completed a draft sequence of the genome of M59-2 and a manuscript ?M59-2, A novel phage specific for colanic acid-producing strains of E. coli.? (Scholl, Adhya, McKinstry, and Merril) is in preparation. We are also sequencing phages to determine the mode of host specificity and have completed the sequence of phage K1-F, a novel K1 specific phage, and two manuscripts regarding this work have been produced (3,4). In addition, a recent observation in our Laboratory suggesting that phage may affect mammalian host gene transcription has renewed our interest in phage-mammalian interactions. We used the human microarray system developed by the LOG to search for human gene expression in Hela cells that might be perturbed by a highly purified (including CsCl density centrifugation) preparation of the E. coli phage T7. In these preliminary studies we found a number of mRNAs that were apparently upregulated and others that were down regulated when compared with control Hela cells treated either with UV inactivated T7 phage or Hela cells treated with the buffer solution used to store the phage.
