Fundamental biology and translational research
Our research focuses on the signaling and transcriptional control mechanisms that govern antigenic variation. We are also interested in the development of new therapeutics such as drugs and vaccines.
Model organism: - Why trypanosomes?
Trypanosoma brucei is a single-celled protozoan pathogen and model organism. It causes sleeping sickness in humans and related animal diseases in Africa. We study T. brucei to understand fundamental biology with a focus on cell signaling, transcriptional control, telomeric silencing, and nuclear spatial organization.
Trypanosoma cruzi causes Chagas disease, a chronic disease often manifested as a cardiac disease that affects primarily the Americas, but also Europe and Asia. There are no effective treatments for chronic Chagas disease. We study T. cruzi for the development of therapeutics with a focus on vaccines.
T. brucei, T. cruzi, and Leishmania spp. are parasites from the order Kinetoplastidae. They affect over 2 billion people worldwide and cause devastating health, economic and agricultural impact. The available drugs have limited efficacy especially at chronic disease stages, are often highly toxic, and no vaccines are available.
Signaling and regulation of antigenic variation
Antigenic variation is the mechanism by which pathogens periodically change their surface coat to evade host immune recognition during infection. Antigenic variation is the primary strategy of host antibody evasion by many viruses and unicellular pathogens. In T. brucei, antigenic variation entails the expression of a single Variant Surface Glycoprotein (VSG) gene at a time from a repertoire of over 2,500 VSG genes and pseudogenes and the periodic change in the VSG gene expressed. The VSG gene is transcribed from a telomeric expression site (ES), and antigenic variation occurs by transcriptional switching between ESs or by VSG gene recombination (Cestari and Stuart, 2018; Curr Genomics).
Our laboratory is interested in the signaling and regulatory processes that control VSG expression and switching in T. brucei. Our data indicate that phosphoinositide (PI) signaling plays a role in controlling VSG expression and switching. The knockdown of the plasma membrane-localized phosphatidylinositol phosphate 5-kinase (PIP5K) or overexpression of phospholipase C (PLC) results in the loss of VSG monogenic expression, i.e. expression of multiple VSG genes simultaneously. Genetic perturbation of these enzymes also results in high rates of VSG switching (Cestari and Stuart, 2015; PNAS). The data suggest that switching of VSG genes is a regulated process and involves PI signaling. We are interested in the mechanisms underlying the control of PI signaling activation and the signal transduction processes that result in VSG transcriptional switching or recombination.
Transcriptional control of telomeric expression sites
We identified a phosphoinositide-regulated Telomeric Expression Site protein Complex (TESC) that function in telomeric VSG gene silencing. The TESC consists of ~24 proteins, including the DNA-binding protein repressor activator protein 1 (RAP1) and the regulatory protein phosphatidylinositol phosphate 5-phosphatase (PIP5Pase) (Cestari et al., 2019; Mol Cell Biol). PIP5Pase associates with RAP1 and controls its binding to expression site (ES) DNA sequences via dephosphorylation of PI(3,4,5)P3. The inactivation of PIP5Pase results in PI(3,4,5)P3 association with RAP1, which affects RAP1 binding to the ES chromatin and results in VSG transcription (Cestari et al., 2019; Mol Cell Biol). The TESC includes other regulatory proteins such as kinases and phosphatases, DNA binding proteins, and nuclear lamina proteins. In our model, the TESC provides a link between PI signaling and the nuclear processes that control VSG expression and switching (Cestari; 2020; PLOS Pathog). We are studying the TESC protein composition and topology, its role in regulating ES chromatin structure, and its function in the maintenance of silent and active telomeric VSG genes.
Regulation of nuclear architecture
The mechanisms that control nuclear spatial organization and function remain poorly understood. In trypanosomes, the active VSG gene is transcribed by RNA polymerase I from an extranucleolar site termed expression site body (ESB), whereas silent ESs appears clustered at the nuclear periphery. The ESB is replicated in each cell cycle, and transcription of the active VSG gene maintained in daughter cells. Notably, this organization changes during VSG switching via transcriptional or recombination mechanisms. This genomic organization between active and silent nuclear regions is not atypical in eukaryotes. Moreover, it is non-random, dynamic, and involves regulatory molecules and biophysical forces (e.g., phase separation) to compartmentalize and coordinate transcription, RNA processing, DNA replication, and recombination. We are interested in the molecular and biophysical mechanisms that control the nuclear spatial organization and genome compartmentalization into transcriptionally active and silent regions, with a focus on the active and silent telomeric ESs. Moreover, we are interested in understanding the mechanisms underlying the biogenesis of nuclear compartments, such as the ESB, and the function of such structures in gene regulation and recombination.
Vaccine discovery for Chagas disease
There are limited preventive or therapeutic vaccines for diseases caused by protozoan pathogens. The pathogen T. cruzi causes a chronic and lethal illness that affects millions of people, primarily in the Americas. The Chagas disease develops over decades, often as a debilitating cardiac or gastrointestinal disease. There are no drugs that cure chronic Chagas disease, and no vaccines are available. Hence, there is an urgent need to develop new drugs or vaccines. We are interested in developing therapeutic or preventive vaccines for Chagas disease. We use genomic tools, such as yeast surface display, to express genome-wide libraries of T. cruzi to identify new epitopes for vaccine discovery. Moreover, we use immunology, genomics, and single-cell biology to understand the human and animal immune response to infection, with a particular focus on potential vaccine protection mechanisms. Moreover, we explore the tools that we have developed for target-based drug discovery to advance the therapeutic pipeline against this devastating disease.