My GWU research program in the department of Microbiology, Immunology, and Tropical Medicine is dedicated to the long-term goal of alleviating human suffering arising from parasitic diseases. We have developed a new state-of-the-art forward genetics approach for the identification of direct targets of drugs and inhibitory compounds, which is also able to identify pathways that can result in drug resistance in Trypanosomatid parasites. Trypanosomatids, which include African Trypanosomes, American trypanosomes, and Leishmania species, threaten the health of over 1 billon of the most socioeconomically challenged people on earth.
Anti-Trypanosomatid therapies are commonly selected for their efficacy without knowledge of their mode-of-action or molecular targets. Most classical therapies are burdened with high host toxicity, complicated treatment regimens, and emerging drug resistance. Our forward genetic tool now enables the identification of drug targets to promote drug optimizations that will promote improved therapeutic strategies and ultimately reduce human suffering.
During 10 years of training as a bacterial geneticist, I learned how to unlock the awesome power of genetics to identify genes and pathways required for pathogenesis. In my doctoral thesis, awarded with distinction from Columbia University in 2009, I discovered regulatory networks required for the intracellular growth of the human lung pathogen Legionella pneumophila. Using an innovative combination of whole-genome transcriptomics, transcription factor mutagenesis, and biosynthetic chemistry I delineated how nutrient starvation pathways affected gene expression during intracellular growth. My thesis work was the culmination of a more than a decade investigating microbial gene regulation and inspired me toward my next challenge.
Trypanosomatids are early branching eukaryotes that have evolved novel strategies to accomplish their essential biological functions. They provide a snapshot into alternative biological mechanisms for diverse functions such as RNA editing, a modified histone code, cell division by binary fission, andpost-transcriptional regulation of gene expression to name a few. A classic example is antigenic variation in the African trypanosome, in which the expression of the primary surface antigen is switched from one gene to the next through DNA recombination based chromosomal shuffling. The outcome of this unusual mechanism of expression regulation is evasion of host immunity, chronic infection of the host, and, ultimately, death. During my postdoctoral research at Rockefeller University, I discovered how specific genetic elements (telomere length and a specific repetitive DNA sequence) control antigenic variation in Trypanosoma brucei, the causative agent of African Sleeping Sickness. Despite these advances in understanding, I found that the field faced an ongoing limitation in functional genomics arising from the lack of genetic conservation between Trypanosomatids and model eukaryotes. In response, I have sought to generate high quality genetic tools that would expand our understating of parasite gene functions.
I assembled a team of postdoctoral researchers to undertake the herculean endeavor of generating a T. brucei ORFeome toward the production of a gold standard Gain-of-Function library. In an NIH funded project that spanned the end of my postdoc and the formation of my GWU laboratory, we amplified and cloned more than 7000 genes (generating a versatile ORFeome), introduced them into T. brucei in the form of a parasite library useful for nearly limitless genetic screens, validated these tools using next-generation sequencing, and proved their usefulness in a critical drug resistance screen (published in mSphere 2020). Gain-of-Function genetic screens allow us to identify the genes that promote a specific phenotype; for instance, overexpression of a drug target can increase resistance to that drug.
Thus, I have now contributed a tool to the Trypanosomatid research community that will broaden research horizons and result in transformative discoveries. Since the completion of this tool in 2018 we have presented at 5 internationally recognized conferences, including an invitation to the British Society of Parasitology meeting in Spain 2020, and launched more than 10 collaborations from the Pacific Northwest to the Czech Republic.
In a genetic screen to identify genes that promote survival during treatment with melarsoprol, the critical drug of last resort against African Sleeping Sickness, we discovered new aspects of its mode-of-action and mechanisms of drug resistance. One gene identified in our screen is required for the biosynthesis of the primary thiol redox carrier in Trypanosomatids (trypanothione), whose inhibition has complex cellular effects on redox biology, ROS stress, and the synthesis of nucleotides required to replicated DNA. We use this information to ask if melarsoprol itself inhibits DNA synthesis using a cutting-edge flow cytometry-based approach. We have now proven that DNA replication is inhibited by melarsoprol, inhibiting cell division, and that this is likely the drugs mode of cell killing, which was a long-standing mystery in the field (published in PARASITOLOGY in 2021). In addition to this pathway, we identified a number of genes that encode mitochondrial proteins and are investigating the role of mitochondrial redox in drug resistance (the subject of a submitted NIH proposal).
In future studies, these tools and discoveries will be the foundation of improved drug design and development against Trypanosomatids. We are also preparing to use the same forward genetic approach to identify the drug targets and pathways of resistance for both established therapeutics and novel inhibitors making their way through clinical trials (the subject of multiple grant applications). Because the genetic tool can be used to ask diverse biological questions, we are also applying to discover new aspects of genome stability and DNA break repair (the subject of a scored NIH grant application with a planned resubmission).
My MITM research program is uniquely positioned to have a transformative impact on the field of Trypanosomatid research that will improve drug design and development, contribute new understanding to basic biology, and directly reduce human suffering from parasitic diseases over the next 10 -20 years. I’m proud to be a member of the GWU research community and excited to bring prestige to my program and SMHS.