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CURRENT VFARS ONGOING RESEARCH AND WHAT NEEDS TO BE DONE TO EVALUATE THE VFARS CONCEPT AND DETERMINE ITS FEASIBILITY
Citation:
Stelma Jr., G N., S J. Vesper, S L. Hayes, D J. Lye, M J. Donohue, AND M R. Rodgers. CURRENT VFARS ONGOING RESEARCH AND WHAT NEEDS TO BE DONE TO EVALUATE THE VFARS CONCEPT AND DETERMINE ITS FEASIBILITY. Presented at Water Quality Technology Conference, San Antonio, TX, November 14-18, 2004.
Impact/Purpose:
1. Develop a method, based on microarray analysis, to identify potential waterborne pathogens using the mRNA response in mammalian cells exposed to these pathogens.
2. Develop computational toxicology approach to understanding hemolysins as virulence factors and for predicting virulence of untested microorganisms.
Description:
The VFARs concept predicts that pathogens can be identified using structural similarities among virulence genes from diverse species. This concept is of interest to the EPA for several reasons: the Agency's need to discriminate between virulent and avirulent isolates of pathogen species listed on the Agency's Contaminant Candidate List (CCL), the Agency's desire to identify organisms for future CCLs and the Agency's desire to identify currently unrecognized waterborne pathogens. Several issues have created skepticism among some microbiologists concerning the immediate utility of the VFARs concept. (i) The degree to which the structure-function relationships seen in chemistry carry over into biology is unknown. Some identical or similar virulence genes may have arisen in diverse species due to parallel evolution; however, other virulence genes may express similar biological activities but may be structurally unique to one genus or species. (ii) The expression of an entire array of virulence genes is necessary for an organism to infect and cause illness. This means that gene probes must be available to test for all of the virulence genes, not just one or two critical genes. Many virulence genes are currently unknown; and it is unlikely that the entire arrays of virulence genes of most pathogens will be identified in the immediate future. (iii) nonpathogenic microorganisms occasionally carry key virulence genes but do not express them. This could cause gene probes to identify harmless strains as pathogens. (iv) The concept may not carry over to viruses and protozoan parasites because viruses and protozoa are all obligate intracellular pathogens and are all, therefore, virulent.
EPA is currently using several approaches to ascertain the long-term and short-term utility of the VFARs concept for evaluating CCL organisms and for identifying currently unknown pathogens from the environment. Our STAR Grants program has funded a data mining effort to find common DNA sequences among diverse species of pathogenic bacteria. One approach of our in-house research uses proteomics to examine and compare gene products from virulent and avirulent isolates of Aeromonas that are growing in cell culture. Another approach, which will be accomplished through an Interagency Agreement, will compare gene products and genes in Cryptosporidium strains that cause illness in humans with those from Cryptosporidium isolates that are avirulent in humans. Other protozoan species that infect humans will then be examined for genes that are analogous to those associated with those Cryptosporidium strains that are virulent to humans.
The most creative approach that EPA is currently comparing the responses of mammalian cells to pathogenic strains of Aeromonas to the responses observed with avirulent strains from the same genus. If the responses are significantly different, this approach should eliminate the need to identify the entire array of genes necessary for virulence in order to distinguish between virulent and avirulent isolates. This approach should also allow us to accurately characterize strains that carry one or more virulence genes but do not express them as avirulent. In addition, this approach should allow us to screen newly discovered microorganisms for virulence potential. The preliminary results obtained from these in-house studies are promising. Neonatal mice were challenged with a virulent isolate of A. hydrophila and an avirulent isolate of A. caviae. Five hours later microarrays were used to measure host mRNA responses. The differences were dramatic; over 400 genes were up-regulated after exposure of the host to the virulent strain; whereas, only 28 host genes were up-regulated after exposure to the avirulent strain. Furthermore, many of the genes that were induced by the virulent isolate were genes that are associated with immune responses. These experiments will be repeated using tissue culture and eventually using other genera of pathogens.