Proteomic Analysis of Hookworm Larval Activation

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DESCRIPTION (provided by the applicant): Hookworm infection continues to rank among the world's most important infectious diseases, with over 800 million people infected worldwide. Current control strategies are of limited success, and new vaccine antigens and drug targets are needed. During infection of the definitive host, the third-stage infective larva (L3) of hookworms encounter a host-mediated signal that re-activates its arrested developmental programs. Execution of these developmental programs culminates in the establishment of adult worms in the small intestine. This transition from the free-living L3 to the parasitic L3 is a critically important event in parasitism. Despite its central role, very little is known about the molecular biology of this "transition to parasitism". An in vitro assay employing the resumption of feeding as a marker for activation was used to demonstrate that mRNA and protein synthesis are required for activation. Two-dimensional-gel electrophoresis of non-activated, activated, and cycloheximide inhibited L3 revealed approximately 25 proteins that undergo expression changes during the first 2 hrs of activation. In this study, 2D-gel electrophoresis and liquid chromatography tandem mass spectroscopy (LC/MS/MS) will be used to identify these developmentally regulated proteins. Proteins excised from the gels will be digested with trypsin and sequenced by LC/MS/MS. Protein sequence data will be used to search hookworm expressed sequence tagged databases, and the corresponding cDNAs cloned by PCR or library screening. In the absence of a hookworm EST sequence, degenerate primers designed from the back-translated protein sequence will be used to amplify the cDNA by low stringency PCR. Once the cDNA sequences have been isolated, the expression pattern of the proteins will be determined by quantitative reverse transcription PCR (Q-RT-PCR). TaqMan assays for each protein will be developed and used to determine the expression pattern of each gene during the activation process by real-time quantitative PCR. The identification and characterization of the proteins associated with the transition to parasitism will provide the first information about the molecular processes involved in parasitism, including proteins critical for infection. These proteins represent potential vaccine antigens and drug targets for the prevention of hookworm infection and the control of hookworm disease.