Impact/Purpose:

This particular task is comprised of 4 subtasks: 1.) Characterization of Potential Viral Biomarkers by Mass Spectrometry; 2.) Characterization of Parasites by Mass Spectrometric Techniques;

3.) Rapid Discrimination of Bacterial Indicators of Fecal Contamination and Bacterial Pathogens by Mass Spectrometric Techniques; and 4.) Investigation of Aeromonas Virulence Factors Using Mass Spectrometry.

The purpose of this research project is to use mass spectrometric techniques, such as electrospray ionization (ESI), capillary electrophoresis (CE) and matrix assisted laser desorption ionization (MALDI) mass spectrometry, to provide "protein mass fingerprinting" and protein sequencing information for viruses, bacteria and protozoa that cause waterborne disease. These protein mass fingerprinting libraries will be evaluated to determine whether mass spectrometric techniques can identify protein fingerprints related to the infectivity/viability of selected microorganisms and whether they can differentiate between infective / non-infective genus and strains of the selected microorganisms. The characteristic proteins identified by mass spectrometry as markers of infectivity/viability or strain differentiation can then be used to develop more sensitive microbiological drinking water methods.

Description:

Over the last three decades, the Centers for Disease Control and Prevention (CDC) and the U.S. EPA have collected and reported data relating to occurrences and causes of waterborne-disease outbreaks in the United States. During 1997 through 1998, 13 states reported 17 outbreaks associated with drinking water and 10 of these outbreaks were attributed to parasitic and bacterial contamination. A number of these microorganisms are now listed on the 1998 Contaminant Candidate List (CCL) as research needs. The purpose of this research project is to use state-of-the-art mass spectrometric techniques, such as electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) mass spectrometry (MS), to provide "protein mass fingerprinting" and protein sequencing information for viruses, bacteria and protozoa that cause waterborne disease. Although mass spectrometry currently is not sensitive enough to detect single cells in drinking water, MALDI and ESI-MS have been used increasingly over the past decade as proteomic tools to provide crucial protein sequencing information. The vast majority of published research involves proteomics of bacteria with very few references related to viruses or protozoa. Thus, this project can make a significant impact on the microbiological field with respect to proteomics of viruses and protozoa. These protein mass fingerprinting libraries will be evaluated to determine whether mass spectrometric techniques can identify protein fingerprints related to the infectivity/viability of selected microorganisms and whether they can differentiate between genus and strains of the selected microorganisms. Further analysis, using MALDI-MS or liquid chromatography/ electrospray/ mass spectrometry (LC/ESI-MS), can yield the peptide sequence information for proteins which are found to be specific or unique to genus/strain and infectivity/viability. This type of basic proteomic information, that will be delivered in research articles by this project, can be used to develop more sensitive and precise immunological techniques, such as ELISA and IFA, that focus in on these unique microbiological proteins in drinking water samples. These conventional microbiological methods can then be used to gather the occurrence data that will be used to create better EPA regulations for protecting humans from microbiological contaminants in U.S. drinking water supplies.

Record Details:

Record Type:PROJECT

Start Date:12/01/2000

Projected Completion Date:09/01/2007

OMB Category:Other

Record ID: 18338

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Project Information:

Progress :This is 1 of 5 tasks associated with MCEARD's research to assess virulence factors and their use as a predictor of potential hazard to humans. The progress to date of each subtask is as follows:

Subtask 1 (on hold): Potential viral biomarkers have been identified using MS which may differentiate the strains of coxsackievirus and may indicate whether the virus is infective or non-infective. Specifically, the ability to differentiate between coxsackie A20, B3, B5 and B6 strains using viral capsid protein 4 (VP4) as a biomarker has been demonstrated using MALDI-MS (based on their VP4 molecular weight). In addition, a unique peak was observed in the MALDI mass spectra of an infectious coxsackievirus, but was not observed in the mass spectra of non-infectious coxsackievirus. This unique peak corresponds to the addition of 5 myristic acid groups to VP4 and may be responsible for viral infectivity; thus, be a potential biomarker of infectivity. This effort is on-hold while MCEARD recruits an ORISE postdoctoral candidate to continue this research. A manuscript is in preparation.

Subtask 2: MALDI has been used to analyze C. parvum, both in an intact form, as well as oocysts that have been rendered nonviable (through techniques such as heating, freezing and thawing). Excystations of the C. parvum oocysts were performed and the cell walls separated from the sporozoites. The analysis of the walls was inconclusive, as the excystation broke them down into random sized pieces, but MALDI analysis of the sporozoites yielded reproducible spectra. Several attempts were made to analyze C. muris as well. Mass spectra of the intact oocysts were more difficult to obtain, as compared to C. parvum, and no successful analysis of the sporozoites was completed. Work continues to focus on interpretation of the data. Formulas in Excel spreadsheets have been developed to extract the data from the MALDI peak lists and sort it to allow comparisons between multiple samples. A manuscript will be submitted to a peer reviewed journal in FY05.

Subtask 3: Seven enterococci species were studied by MALDI, namely, E. faecalis, E. faecium, E. durans, E. gallinarum, E. avium, E. mundtii, and E. casseliflavus. The mass spectra for E. faecalis and E. durans were nearly identical, but the other species produced protein fingerprints that, while the overall pattern was similar, had differences in the masses of the predominant peaks that can be used the identify the organism. To test this, 20 environmental isolates were analyzed and compared to the identity determined by 16S rDNA analysis. In 75% of the samples, the assignment between the two techniques matched, which further illustrated the utility of the MALDI in the analysis of bacteria. This work was presented at the 2003 ASMS Annual Meeting.

Subtask 4: Virulence factors are being investigated using MS to evaluate the potential to: 1) speciate isolates of Aeromonas and 2) determine an isolate's potential pathogenicity (virulent or avirulent) using MS. Thus far, Aeromonas isolates have been speciated, based on their protein mass fingerprints, by analyzing the intact cells of the organism using MALDI-MS. To date, ~34 well-characterized strains of Aeromonas and ~45 environmental isolates from water distribution systems that were obtained for EPA method 1605 were speciated. Two manuscripts are planned for FY05 (a paper entitled "Development of a Matrix-Assisted Laser Desorption/Ionization-based Method for Phyloproteomic Classification of Aeromonas Species" and a second on the application of MALDI-MS to speciation of environmental isolates). The second objective of this task (virulent vs. avirulent) will begin in FY05. Information is needed from "in-house" mouse studies to identify those isolates that are pathogenic. As this information is obtained, it will be used to examine virulent and avirulent isolates to ascertain if any mass spectral information is available to determine pathogenicity.

Relevance :This is a basic research project which may provide valuable information regarding microorganisms identified on the 1998 CCL (and future CCLs) or other important microbiological contaminants in drinking water. Sensitive and rapid methods, as well as occurrence data, are needed for a number of microorganisms listed on the 1998 CCL. The type of data generated from this research can provide microbiologists with information regarding specific proteins contained within infective/viable microorganisms and these identified proteins can then be used to develop rapid, sensitive methods for microorganisms in drinking water. The vast majority of existing published research involves proteomics of bacteria with very few references related to viruses or protozoa. Therefore, this project can make a significant impact on the field with respect to proteomics of viruses and protozoa. This will be a collaborative basic effort with NERL/MCEARD microbiologists and chemists and the results of our combined efforts will periodically be presented to EPA's Office of Ground Water and Drinking Water (OGWDW). Any unique markers of microbial exposure characterized by these mass spectrometric techniques can then be used to develop a sensitive method(s) for microbes identified on the 1998 CCL and any future CCLs. Thus, this project will provide information needed to develop better microbial methods. These microbial methods will gather occurrence data which will provide critical information to be used in creating better EPA regulations and policies for protecting humans from CCL microorganisms in U.S. drinking water supplies. The PI(s) will establish contacts in the scientific community through attendance/presentation at national conferences as well as through correspondence with researchers in the proteomic field.

Clients :Office of Ground Water and Drinking Water (Hiba Shukairy, Valerie Blank, Kesha Forrest), NERL Microbiologists

Research Component :CCL (MICROBIAL)

Risk Paradigm :EXPOSURE

Project IDs:

ID Code :none

Project type :ORD-DW Plan

ID Code :9420

Project type :OMIS