K U eeping Our Drinking Water Safe song Faster Cutting Edge TechnoDogy <5> Maura J. Donohue US EPA, Office of Research and Development (ORD) National Exposure Research Laboratory (NERL), Cincinnati OH Contact Info: donohue.maura@epa.gov • 513-569-7634 Toxins: Able to cause damage to the host cell Modulins: Components which stimulate the cytokine synthesis Impedin: Factors allowing the bacteria to overcome host defenses Adhesion: Responsible for adhesion of the organism to the host tissue 0) o Q. Over the last three decades, the Centers for Disease Control and Prevention (CDC) and the US EPA have collected and reported data relating to occurrences and causes of waterborne-disease outbreaks in the United States. From 2000 through 2002,13 states reported 17 outbreaks associated with drinking water and 10 of these outbreaks were attributed to para- sitic and bacterial contamination. A number of these microorganisms are now listed on the 2003 Contami- nant Candidate List (CCL) because of the need for more exposure research. In addition, the National Research Council (NRC) subcommittee has sug- gested that virulence factor activity relationships (VFARs) may provide a more rapid means of identify- ing waterborne pathogens than the current process that relies on exposure and health effects as the two primary categories for screening potential microbial drinking water contaminants. The purpose of this research project is to use state-of-the-art mass spec- trometric techniques, such as electrospray ionization tandem mass spectrometry (ESI-MS/MS) and matrix- assisted laser desorption/ ionization-mass spectrom- etry (MALDI- MS), to identify virulence factors that enable the CCL microorganisms to cause disease. The goal of this research is to use this proteomic in- formation to develop more sensitive and precise methods in order to gather occurrence data that will be used to create better EPA regulations for protect- ing humans from microbiological contaminants in U.S. drinking water supplies. Figure 1. Virulence Factors O m o 2= Q. Q. < Virulence factors are identified using a multidis- cipline approach. Classical protein isolation and purification techniques are used to isolate the desired targets. Biological assays are used to help identify the protein of interest based on a specific biological function (i.e., adhesion, tox- icity, or cellular modification). Next, the isolated protein (that has a biological function) will be identified either by peptide mass fingerprinting (products of a protein digest) or by sequencing the amino acid chain for each peptide using either MALDI-MS or ESI-MS/MS. The spectrum achieved for each type of analysis will be com- pared against the National Center for Biotech- nology Information (NCBI) protein databases to determined the identity of the protein. Table 1. The sample preparation and biological assay will differ depending on which class of virulence factors is being studied: Virulence Factor Location Biological Assay Adhesion Supernatant and cell membrane Adhesion assays Toxins (Exotoxins) Supernatant Cell toxicity Invasion Cytosol Cell based assay Impedins Supernatant *ROS and RNS Modulins Cytosol and cell membrane *TNF analysis * ROS - Reactive Oxygen Species, RNS - Reactive Nitrogen Species, and TNF - Tumor Necrosis Factor Figure 5: Protein Indentification Search Engine Results. Data mining results from query (mass values observed in spectra). ~ Current work is focused on identifying virulence factors for the CCL microorganism Aeromonas hydrophila. A virulent and avirulent pair of strains has been identified for two of the pathogenic species of Aeromonas (A. hydrophila, and A. veronii bv sobria). • 2D gels of the virulent and avirulent strain of A. veronii bv sobria have been done (Figure 9 & 10). • There are 12 proteins that are expressed by the virulent strain and not by the avirulent strain. • Work is currently in progress to identify these proteins. • Thus far, an adhesion protein and a few unique enzymes have been identified. Figure 9 & 10 2D gels of supernatant (Exotoxins and Adhesion Factors) taken from A.) Virulent and B.) Avirulent strain of Aeromonas. Virulent proteins of the CCL microorganism Aeromonas are currently being identified by comparing the protein expression of a virulent strain against an avirulent strain, using 2D gel electrophoresis. Note: The virulent and the avirulent strains were chosen based on infectious dosage (l-D.) information. epascienceforum Your Health • Your Environment • Your Future ------- |