1a. Objectives (from AD-416)
Vibrio parahaemolyticus is a serious threat to the consumer of molluscan shellfish in the United States and has led to a significant number of illnesses and closures of shellfish harvesting areas over the past two summers. We will identify virulence mechanisms in V. parahaemolyticus in order to better understand the regulation of V. parahaemolyticus pathogenicity and to differentiate pathogenic strains from nonpathogenic strains. To date, two toxin-producing genes have been identified in V. parahaemolyticus; however, analyses have shown that strains lacking these toxins and genomic regions can still be pathogenic, indicating that important virulence factors remain to be identified. One of the key virulence genes in V. parahaemolyticus and V. cholerae is the toxRS gene which regulates at least one of the toxins produced in most pathogenic strains. We will determine the range of genes that are regulated by the toxRS gene in V. parahaemolyticus by examining a toxRS deletion mutant already developed by our collaborator, Dr. Fidelma Boyd. We will examine the transcriptome (the full complement of unique RNA molecules with coded transcripts) and proteome (the set of proteins specified by genes within an organism). We will perform expression analyses using a V. parahaemolyticus whole genome microarray to determine the range of genes up and down regulated in the toxRS mutant strain compared to the wild-type strain. The toxRS gene will also be evaluated as a possible regulator of a potential virulence enhancer identified by ARS research as phosphoglucose isomerase containing a lysyl aminopeptidase (PGI-LysAP) activity. The virulence of the toxRS mutant will be assayed by in vitro and in vivo invasion assays. Overall, we will identify potential virulence mechanisms associated with V. parahaemolyticus and will determine if PGI-LysAP transcription is regulated by the toxRS gene. New assay methods for pathogenic strains of V. parahaemolyticus, including the recently emerged O3:K6 pandemic strain of V. parahaemolyticus, will be developed with the ultimate goal of reducing outbreaks of illness among shellfish consumers and the impediments that such outbreaks have on the industry and other stakeholders.
1b. Approach (from AD-416)
Under the ARS portion of this research, ARS will characterize the expression of phosphoglucose isomerase with a lysyl aminopeptidase (PGI-LysAP) activity in wild-type V. parahaemolyticus, and in toxRS, rpoS, and pathogenicity island deletion mutants to determine the effects of the deleted genes on PGI-LysAP expression. This will be performed in several stages. First, the growth rates of the wild-type and mutant strains will be determined spectrophotometrically at OD600 at 2-hr intervals over a 24-h period. Enzymatic (LysAP) activity of the cultures will be screened in triplicate using the synthetic fluorogenic substrate L-lysyl-7-amino-4-methylcoumarin as previously published in our laboratory. The expression levels of LysAP activity will be compared among the wild-type and deletion mutants. Then, enzyme purification and quantification will be performed on each bacterial culture. Cell lysates from the wild-type and deletion mutants, will be purified by anion exchange chromatography. Fractions containing enzyme activity will be pooled for each Vibrio strain and concentrated. Concentrates will be analyzed separately for total PGI activity and for total LysAP activity. The relative ability of each enzyme to process vasoactive substrates (bradykinins) will be determined using an ARS-developed fluorescent ninhydrin assay. Results will be compared with the results from microarray and whole genomic analyses (objective 1) and mouse infectivity studies (objective 2) in order to determine the up- or down-regulation of PGI or its LysAP activity by ToxRS, RpoS, or pathogenicity islands. Research will then proceed to characterize the isolated enzymes in terms of mass, substrate specificity, inhibition by protease inhibitors, and optimal pH. Their mass will be determined by SDS-polyacrylamide gel electrophoresis under reducing conditions, substrate specificities will be determined using synthetic fluorogenic substrates, while the inhibition of LysAP activity will be monitored in the presence of serine, cysteine, aspartic acid, and metallo- protease inhibitors. All the methods are commonly used in our laboratory and all equipment needed to perform these studies is currently available.
3. Progress Report
This research combines a variety of approaches to assess the likely mechanisms of virulence (infectivity) for the pandemic strain of Vibrio parahaemolyticus and in mutants lacking two proposed virulence genes. Vibrio parahaemolyticus is a bacterium that infects humans and is the primary cause of seafood-related bacterial illness in the U.S. and many parts of Asia. Studies were undertaken to identify the presence of a proposed virulence enzyme in wild-type and mutant strains of V. parahaemolyticus. Current research has shown no significant differences in the levels or composition of enzymes produced between the strains. Studies to address the uptake and growth of these strains in Eastern oysters showed that this pathogen did accumulate in oysters over the short term (within 24 h after inoculation), but readily died off between 24 and 72 h. Vibrio die-offs were similar when tests were performed in natural seawater obtained from Delaware and the Gulf Coast (from the U.S. FDA laboratory at Dauphin Island, AL). Virtually all seawater and shellfish collected throughout this past year contained some agent capable of killing V. parahaemolyticus. The cause of this inhibition is being investigated in a companion study with the University of Delaware entitled Comparison of the Survival and Proliferation of Virulent & Avirulent Strains of vulnificus & parahaemolyticus in Seawater & Shellfish (Project no. 1935-42000-065-04S), which also evaluates the growth and persistence of another important species of vibrio (Vibrio vulnificus) in seawater and oysters.