I have spent my entire 30+ year academic career studying parasitic nematodes, especially the hookworms. I have extensive training in hookworm biology and molecular biology, and have done pioneering research in these areas. In graduate school, I devised an in vitro system for the analysis of the hookworm infective process. As a post-doc, I used this system to investigate the molecular biology of infection, including cloning the first molecules from hookworms. Since my post-doc, I have continued studying hookworms, leading projects investigating hookworm population genetics in China and the molecular biology of hookworm infection. My group is one of the first to study mechanistic aspects of signaling during hookworm infection. I have maintained all of the common hookworm model systems in my career.
My interest in nematodes emerged while working with C. elegans as a research assistant in the laboratory of Lew Jacobson at the University of Pittsburgh. When I began graduate school, my goal was to investigate the molecular and biochemical mechanisms by which parasitic nematodes, particularly hookworms, infect their hosts. However, it soon became apparent that an in vitro model for this part of the life cycle was required, as the obligate requirement for a host made the isolation of sufficient parasitic stages for investigation problematic. Reasoning that developmentally arrested infective larvae (iL3) would resume feeding upon infection, I developed and in vitro system that used the resumption of feeding as a marker for activation to the parasitic lifestyle. I characterized the stimulus, the feeding response, the culture conditions, and the response for several hookworm species. This activation assay provided a tractable system to investigate early events in in nematode parasitism, and has subsequently been adapted by several labs (e.g. Lok, Selkirk, Schneider, Loukas, Hotez), and as an anthelmintic screening assay. I was the major intellectual driver behind this system and conducted all of the experiments.
Using my newly developed activation assay, I next examined the molecules that L3 release early in infection. As a post doc, I cloned the first cDNA from a hookworm (the catalytic subunit of protein kinase A) and described the first excretory/secretory molecule from hookworms, an astacin metalloproteinase. I also discovered and was first to report the activation associated secreted proteins (ASPs), an enigmatic family of secreted nematode proteins that subsequently have been identified in essentially every other nematode. I identified the 2 forms, a single domain form I named ASP-2, and a two domain form I called ASP-1. Also known as venom allergen proteins (VAPs) because of their resemblance to Hymenopteran venom components, they represent the most abundant secreted proteins in many nematodes, and their abundance and profile change across the life cycle of the worm. ASP-1 was the lead antigen in the Human Hookworm Vaccine Initiative, and ASP-2 was the first recombinant hookworm vaccine antigen to be tested in humans. I was the driving intellectual source and performed most of the experiments.
I continued my investigation of the hookworm infective process by next studying the signaling pathways that initiate activation. The hookworm L3 is analogous to the dauer stage of the model nematode C. elegans. I and others have reasoned that the pathways regulating dauer exit would also control resumption of development that occurs when hookworms enter a permissive host. Using dauer as a paradigm and activation as an output, I dissected the molecular pathway leading to feeding by activated hookworm L3. I demonstrated that cGMP and insulin/insulin-like growth factor signaling (IIS) pathways were involved inactivation, and cloned several members of these signaling pathways, including the FOXO transcription factor DAF-16, 14-3-3, and the TGF-β ligand DAF-7.
Once I identified the relevant pathways, my lab began concentrating on the mechanism of hookworm DAF-16 action, its role as a central mediator of development, and its downstream targets that mediate development in the host. We found that DAF-16 binding elements from C. elegans were bound by hookworm DAF-16, and that the interaction of hookworm DAF-16 with the shuttling protein 14-3-3 required phosphorylation of conserved AKT binding sites in DAF-16. We also, for the first time, made transgenic C. elegans expressing hookworm wild type and phospho-null hookworm DAF-16 mutants to investigate the behavior and cell localization of hookworm DAF-16 during dauer exit. We found that hookworm daf-16 rescued the dauer defective phenotype of C. elegans daf-2/daf-16 mutants, and that DAF-16 was regulated by both phosphorylation dependent and independent mechanisms. Finally, we characterized two DAF-16 target genes, snr-3 and lpp-1 by mapping their genomic location and gene structure, and showing that a DAF-16 binding element in the 3’UTR of snr-2 could function as a promoter and an enhancer. This was the first reported detailed investigation of a hookworm genomic locus. I was the PI for these studies.
I have also been involved in the sequencing of four hookworm genomes, both through intellectual contributions and provision of hookworm nucleic acids. During my collaboration with Makedonka Mitreva at Washington University, we have published 4 manuscripts examining hookworm genomes and transcriptomes, including the draft genome of the human hookworm Necator americanus. Additionally, we have published a manuscript comparing the draft genomes of three Ancylostoma hookworm species.
Previous projects in the lab include developing transgenic hookworms to use as biodelivery systems to treat cancer and HIV and investigating pharyngeal function in nematodes using microfluidic-based electrical recordings of living nematodes with my collaborator Janis Weeks at the University of Oregon. Current projects in the lab include investigating the molecular mechanism of anthelmintic resistance in strains of drug resistant hookworms maintained in my lab, characterization of anthelmintic activity in the bitter melon for use as treatment for hookworm and other parasitic nematodes, development of the entomopathogenic nematode Heterorhabditis bacteriophora as a model for nematode parasitism, the molecular basis for host specificity, and using human hookworm and whipworm as therapy for autoimmune diseases with my collaborators Jeff Bethony and David Diemert.