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Part I. Vibrio species present in coastal waters are of worldwide human health concern (such as V. cholerae, V. parahaemolyticus, and V. vulnificus), while other non-pathogenic species (V. harveyi, V. natriegens, V. coralliilyticus ) may be reservoirs for toxin-encoding genes and bacteriophages that transmit them. Many bacterial toxins, including cholera toxin, are encoded by transmissible phages. Vibrio phage KVP40 is a lytic, T4-type bacteriophage whose complete dsDNA genome sequence has been determined1. The 244,835 base pair KVP40 genome includes among its 400 genes those for pyridine nucleotide enzymes that were inferred to encode an NAD+ salvage pathway. NAD+ scavenging has been characterized in bacteria10,11, but this is the first reconstruction of the process from a phage or viral genome. NAD+/NADH conversions are critical for cellular redox control, energy levels, nutrient processing, and NAD+ is an important substrate for covalent ADP-ribosylation of protein, such as that directed by the cholera toxin CT. We hypothesize that when infecting its Vibrio hosts (V. cholerae, V. parahaemolyticus and others), phage KVP40 modulates NAD+ levels via the encoded salvage pathway to provide a developmental advantage to the phage. This suggests that Vibrio species have limiting cellular levels of NAD+ during environmental growth conditions and that this impacts phage development as evinced by the existence of the pyridine scavenging enzymes. <P>
Specific Aims of this study are to: <ol>
<li> Characterize reaction & kinetic properties of the KVP40 pyridine nucleotide scavenging enzymes NadV (NAmPRTase) and NatV (NMNATase/Nudix), including biochemical and genetic confirmation of a potential phosphoenzyme reaction intermediate of NadV. <li> Define the expression pattern of nadV and natV during KVP40 development in V. parahaemolyticus using qRT-PCR and western blot analysis, and determine the affects of disrupting these genes on phage growth. <li> Quantify the intracellular NAD+ levels in KVP40-infected V. parahaemolyticus cells and determine affects of the KVP40 NAD+ salvage pathway on ADP-ribosylation processes. Part II. Bacteriophage systems will be developed to rapidly and specifically identify viable pathogenic Vibrio (V. parahaemolyticus and V. vulnificus of primary concern) present in contaminated seafood and shellfish. </ol> By using this approach during depuration, a tractable, single post-harvest strategy will be available to detect, reduce or eliminate Vibrio spp. in oysters. <P>
The specific aims are: <ol>
<li> Construct Vibrio-specific KVP40 and nt-1 phages containing the xylE and luxAB reporter genes. <li> Deploy existing lytic vibriophages (KVP40, KVP20, nt-1, others) in cell lysis-dependent detection assays for measuring diagnostic cellular enzymes that differentiate clinical strains of V. parahaemolyticus and V. vulnificus from non-clinical environmental isolates. <li> Newly isolated Vibrio phages will be used in the cell detection platforms of Aims 1 & 2 and, most importantly, during oyster depuration for mitigating pathogen load. </ol>
NON-TECHNICAL SUMMARY: This project uses bacteriophage and RNA-binding proteins, to study basic biological processes and to derive biotechnology reagents of interest to agriculture. The purpose of these studies is to identify new targets for anti-bacterial treatments of human and animal pathogens. New reagents for biotechnology applications in food safety, food processing, and environmental quality control can be developed.
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NON-TECHNICAL SUMMARY: This project uses bacteriophage and RNA-binding proteins, to study basic biological processes and to derive biotechnology reagents of interest to agriculture. The purpose of these studies is to identify new targets for anti-bacterial treatments of human and animal pathogens. New reagents for biotechnology applications in food safety, food processing, and environmental quality control can be developed.