1. Molecular Mechanisms of Virulence
A. Apicomplexan Parasites: Parasites of phylum Apicomplexa cause globally devastating diseases such as toxoplasmosis and malaria. T. gondii, the causative agent of toxoplasmosis, has a high global prevalence and infections can not only lead to serious ocular disease, and potentially lethal outcomes in immunocompromised AIDS, cancer and organ transplant patients, but are also correlated with brain cancer and neurological impairments such as schizophrenia. Nearly half the human population is at risk for P. falciparum malaria with at least 500 million cases yearly and half a million deaths, mostly young children. The widespread success of these parasites is dependent on their ability to actively invade a host cell and sequester within the immunoprotective intracellular environment. Intriguingly, apicomplexan parasite invasion requires assembly of a unique invasion pore that functions as a molecular doorway to the host cell.
i. Invasion Pore: Apicomplexan parasites provide both ligand (RON2) and receptor (AMA1) to form the invasion pore. We were the first to structurally characterize the pore, with the T. gondii AMA1-RON2 complex reported first (Science 2011) and followed by the complex from P. falciparum (PLoS Pathogens 2012). We have also recently defined the molecular plasticity of the AMA1-RON2 invasion pore at a structural and functional level in collaboration with Maryse Lebrun’s Lab at Université Montpellier 2 and John Boothroyd‘s Lab at Stanford University. Based on our AMA1-RON2 structures, we were part of a ground-breaking collaborative effort that resulted in a novel engineered malaria vaccine that led to sterilizing immunity in a rodent model (PNAS 2014). We are currently involved in the ongoing design and development of AMA1-RON2 based vaccination strategies with Louis Miller‘s Lab at NIH, and small molecule inhibitors targeting the AMA1-RON2 invasion complex.
Our work describing the structural aspects of the apicomplexan invasion pore has been highlighted by the Canadian Light Source in three major media releases: A new take on tackling malaria (World Malaria Day 2015), In the bank (CLS crystallography scientists celebrate 500 protein structures), and Health: Illuminating a parasite invasion. Our original AMA1-RON2 co-structure was also highlighted in a Science Perspectives article: Revealing a Parasite’s Invasive Trick.
AMA-RON2 complexes of apicomplexan parasites (LtoR: PfAMA1-PfRON2D3, TgAMA1-TgRON2D3, TgAMA4-TgRON2L1D3):
ii. Motility: Building on our elucidation of the structure and function of the apicomplexan invasion pore, the crucial next step is to establish how these parasites are able to exploit this molecular doorway and drive sequestration. Addressing this fundamental question requires dissecting the motility complex and its core Class XIV unconventional myosin, MyoA. Importantly, no detailed information exists describing the architecture or assembly of the full MyoA complex and thus the mechanism of motility is poorly understood. Therefore, our goal is to establish the first detailed molecular blueprint of the core motor complex from an apicomplexan parasite. Pursuant to this goal, we will employ a powerful synergy of structural and functional approaches supported by foremost experts in parasitology (Gary Ward‘s Lab at the University of Vermont) and structural mass spectrometry (John Burke‘s Lab at the University of Victoria).
B. Treponema pallidum: Syphilis infections have been continuously high in resource-limited countries, and over the last decade significant disease outbreaks have re-emerged across Europe, England, the United States, Canada and China. T. pallidum, the etiological agent of Syphilis, is among the most invasive human pathogens and its virulence depends on endothelial transmigration and tissue invasion. In collaboration with Caroline Cameron‘s Lab at the University of Victoria, we are investigating the molecular details of how T. pallidum surface proteins mediate adhesion to vascular receptors, induce host signalling cascades, promote migration across the vasculature, and enable tissue invasion and dissemination.
Crystals of T. pallidum surface proteins:
2. Molecular Basis of Transmission
Microorganisms that survive in specialized environmental niches employ a sophisticated molecular arsenal to promote nutrient acquisition, metabolism, physical protection and colonization. A particularly important yet largely understudied niche is the complex environment experienced by vector-borne microbes during transmission by insect vectors. Deciphering the molecular cross talk between microbe and vector has broad implications, not only for human health but also for the agricultural industry where vector-borne diseases of crops and livestock are a major source of economic loss with global impacts measured in the billions of dollars per annum. The mechanisms by which microorganisms are transmitted by insects range from relatively simple mechanical events, as in the cases of many vector-borne viruses, through complex biological processes that enable microbial growth within the vector. Our two model systems for investigating the latter case are Tsetse-Trypanosome and Anopheles-Plasmodium.
A. Tsetse-Trypanosome: The tsetse fly transmitted kinetoplastid parasites of the genus Trypanosoma are known for their role in causing sleeping sickness in humans and chronic wasting disease in animals. However, very little is known about the molecular interactions that enable the parasite to survive within the fly. Towards addressing this knowledge gap, we have recently characterized the structure of Glutamic acid/alanine rich protein (GARP) from T. congolense and mapped the surface epitopes (JBC 2011). We are currently working to define the molecular architecture of the trypanosome surface by characterizing the most abundant surface proteins from the insect stages of the parasite, and deciphering the functions of these surface proteins by identifying and characterizing molecular partners within tsetse. These data will promote a greater understanding of trypanosome dissemination and provide valuable insight into other less tractable pathogen-vector systems.
Tsetse dissection: GARP epitopes and immunofluorescent localization:
B. Anopheles-Plasmodium: As noted above, P. falciparum malaria is a major global human health concern. Understanding P. falciparum transit through the mosquito vector is essential for building a detailed model of transmission of this human pathogen and for informing the development of transmission blocking vaccines. A predominant family of proteins presented on the surface of P. falciparum in all life cycle stages is the 6-Cys family. We have structurally characterized two 6-Cys proteins, Pf12 and Pf41 of blood stage malaria parasites, providing the first full-length architectural blueprints for this important family. We are now using our established expertise with these proteins to investigate 6-Cys antigens that are upregulated and presented during transit through the Anopheles mosquito vector.
3. Structure-based Drug Design
Building on Dr. Boulanger’s experience in the pharmaceutical industry as a Senior Scientist specializing in structure-guided drug design, the Boulanger Lab is involved in several collaborations with cancer biologists and medicinal chemists, in both academia and industry, to identify and develop small molecule inhibitors for a variety of protein-based cancer and viral targets. Each project incorporates structure-based drug design to iteratively refine the small molecule molecular scaffold.
For example, recognition of a lysine trimethylation on histone 3 by a methyllysine reader protein, chromobox homolog 7 (CBX7), is implicated in silencing of multiple tumor repressors. We were recently involved in a peptide-driven approach to create the first inhibitors of CBX7 (J Med Chem 2014).
Boulanger Lab research was recently the focus of an International Innovation Healthcare article.