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Title:   Author  item
                                            Richards, Garyitem
                                            Nunez, Alberto
  Submitted to: Journal of Bacteriology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 1/6/2006
                                    Publication  Date: 3/1/2006
                                    Citation: Richards, G.P., Nunez, A. 2006. Specificity of a vibrio vulnificus aminopeptidase toward kinins and other peptidyl substrates. Journal of Bacteriology. Mar. 2006. p. 2056-2062.Interpretive Summary: Vibrio vulnificus is a marine bacterium that causes death among shellfish consumers.  Previously, we identified an enzyme in V. vulnificus that may enhance bacterial invasiveness.  In the current study, we found that this enzyme is capable of digesting seven small proteins (peptides) that are involved in inflammation, dilation of blood vessels, and in the transmission of V. vulnificus directly through blood vessels.  The ability of bacterial enzymes to cleave peptides was detected by three techniques, including a fluorescent assay previously described only in the Chinese literature.  These techniques offer new approaches to discovering the mechanisms of bacterial invasiveness.
                                Technical Abstract: Recently, phosphoglucose isomerase with a lysyl aminopeptidase activity (PGI-LysAP) was identified in V. vulnificus.  In this paper, we demonstrate the proteolytic cleavage of human-derived peptides by PGI-LysAP of V. vulnificus using three approaches: i) a quantitative fluorescent ninhydrin assay for free lysine; ii) matrix-assisted laser desorption/ionization two-stage time of flight mass spectrometry (MALDI-TOF-TOF), and iii) tricine gel electrophoresis.  PGI-LysAP hydrolyzed bradykinin, Lys-bradykinin, Lys-[des-Arg9]-bradykinin, neurokinin A, Met-Lys-bradykinin, histatin 8, and a myosin light chain fragment.  Proteolytic processing of kinins alters their affinities toward specific cellular receptors and initiates signal transduction mechanisms responsible for inflammation, vasodilation, and enhanced vascular permeability.  The hydrolytic release of free lysine from peptide digests was determined by a rapid, simple, sensitive and quantitative fluorescent ninhydrin assay and was confirmed by MALDI-TOF-TOF.  Visualization of peptide hydrolysis was accomplished by tricine gel electrophoresis.  By applying novel approaches to determine the proteolytic potential of bacterial enzymes, we demonstrate that PGI-LysAP has broad exopeptidase activity which may enhance V. vulnificus invasiveness by altering peptides involved in signal transduction pathways.  Use of the fluorescent ninhydrin assay to quantitatively detect free lysine hydrolyzed from peptides is the first application of its kind and serves as a paradigm for future studies.


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