Beverley Lab Research
Our laboratory studies the protozoan parasite Leishmania, a relative of trypanosomes which infects more than 10 million people in tropical regions. We especially wish to understand the infectious cycle of the parasite, which normally consists of an intracellular stage within the phagolysosome of the vertebrate macrophage and an extracellular stage within the gut of a sand fly vector. Studies of these parasites have lead to the discovery of an astonishing array of novel molecular mechanisms that are now paradigms in higher eukaryotes, including RNA editing, trans-splicing, GPI anchors, bent DNA, and the Th1-Th2 T-cell subset paradigm.
Most projects in the lab focus on the use of functional genetic complementation and transfection to identify and dissect new Leishmania genes of interest. These approaches take into account the fact that the parasite is diploid and lacks an experimentally manipulable sexual cycle. We have developed several approaches for creating mutants, using powerful genetic selections or FACS manipulation of parasites selectively expressing the green fluorescent protein (GFP). We have characterized and now routinely use the phenomenon of loss-of-heterozygosity (LOH) for the creation of homozygous mutants.
One powerful system for identifying mutations defective in the synthesis of a surface glycoconjugate, lipophosphoglycan (LPG), which is an essential virulence determinant involved in adhesion and survival. Using a simple combination of lectin selections for or against surface LPG expression, we have identified a large panel of lpg - mutations and thus far rescued 7 different genes. These have identified proteins involved in LPG biosynthesis, compartmentalization within the eukaryotic secretory pathway, and regulation. We are using these mutants and genes to 1) identify novel biochemical targets for chemotherapeutic attack, 2) to identify new cellular pathways, such a pathway for GPI anchor biosynthesis for glycans separate from that for proteins, and 3) to probe the role of LPG in more detail in parasite survival within both the fly and the macrophage. We are especially interested in understanding the role of LPG in manipulating host cell signal transduction, which is radically altered in Leishmania infections.
We have studied the process of drug resistance mediated by gene amplification, using antifolate inhibition as a paradigm. Leishmania are auxotrophic for folates and pteridines, and these studies have revealed many unanticipated complexities about the pteridine biosynthetic pathway, including a bi-functional dihydrofolate reductase-thymidylate synthase and a novel broad spectrum pteridine reductase (PTR1), a folate transporter (FT1) and a pteridine transporter (BT1). All these loci have been implicated in genomic rearrangements in vitro and in vivo, yielding important insights into genome plasticity and chromosome evolution. Significantly, PTR1 can metabolically bypass antifolate inhibition and we have shown recently that combined inhibition of PTR1 and DHFR-TS can result in effective parasite control. Unexpectedly, we showed that PTR1, through the provision of reduced pteridines such as tetrahydrobiopterin, was implicated in the resistance of Leishmania to oxidative stress. We are currently probing the relevance of this in vivo. Since pteridines are essential cofactors for NO synthase which is important in host cell resistance to Leishmania, we are also curious as to whether Leishmania actively manipulates host pteridine levels to survive.
To isolate genes involved in parasite virulence more generally, we have used transfection of avirulent lines (obtained after extensive growth in vitro) to identify several genes which restore virulence in mouse infection tests. These genes specifically affect the ability of the parasite to establish macrophage infections, and act genetically by multi-copy suppression. One of these, UBF1, encodes an ubiquitin-like protein fusion showing homology to several yeast proteins involved in DNA repair. This suggests that as in other pathogens, DNA repair plays an important role in resisting host defense mechanisms.
An exciting development has been the introduction of transposable element systems into the study of parasites. One example of this is shuttle mutagenesis, where we use the yeast Ty1 system to insert mini-transposons into parasite genes in vitro for scoring in vivo following transfection into the parasite. Recently we have been able to engineer Leishmania in which the Drosophila transposable element mariner is expressed and active, yielding parasite populations where nearly all exhibit at least one transposition event. With this system we have selected for inactivation of the DHFR-TS gene and obtained lpg- mutations, and selected for transcriptional gene fusions to a hygromycin resistance marker. We plan to develop this system in several ways: as an insertion element for generating large panels of homozygous mutants that can be screened for effects on virulence, and for making fusions to GFP for FACS selection of stage specific gene expression.
Other interests of the lab include studies of gene structure, transcription and regulation; artificial Leishmania chromosomes and regulatable expression vectors; basic parasite genetics; the use of gene knockouts as potential live vaccines in people; and molecular evolution of parasites and virulence.