Molecular cell biology of Leishmania
In the Gluenz Lab we study the biology of protozoan parasites called trypanosomatids, which cause diseases in humans and animals in many of the poorest countries in the world.
Leishmania are transmitted by the bite of the sand fly and cause a spectrum of diseases, with symptoms ranging from mild but potentially disfiguring cutaneous lesions to life threatening visceral infections. A population of 350 million people globally are at risk of leishmaniasis, and the WHO estimates that 12 million people are currently infected. In the human body, Leishmania parasites are taken up by macrophages and proliferate within the harsh conditions of the phagolysosome.
Much remains unclear about the molecular and cellular mechanisms that enable Leishmania to subvert macrophage defense mechanisms and cause disease.
Development of genetic tools
To understand the biology of the parasite we use a range of genetic tools enabling us to track proteins within the cell and study the effect of mutations and loss of gene function. We have recently developed new protocols and plasmids for rapid gene tagging (Dean et al., 2015) and high-throughput CRISPR-Cas9 gene editing tools (Beneke, Madden et al., 2017) to harness the information from genomic analyses to answer questions about the function of the Leishmania flagellum, the cell biology of amastigotes and the mechanisms through which these parasites cause disease. Our website LeishGEdit.net is an online resource for detailed information about our CRISPR tools and allows download of plasmid sequences and primer sequences for tagging and/or knockout in diverse kinetoplastid species.
Structure and function of the Leishmania flagellum
Cilia and flagella are cellular projections built around a microtubule axoneme whose molecular architecture is highly conserved across eukaryotic groups. Cilia and flagella serve two main functions: motility and sensory perception. Many cell types (ranging from single celled organisms such as Leishmania to mammalian sperm cells) use their motile flagellum for locomotion. Sensory cilia serve a wide range of functions. In the human body, cilia are important for the detection of developmental signals, and the perception of chemical and mechanical stimuli. Single-celled organisms also use cilia and flagella for detection of signals from the environment.
When the Leishmania parasite is engulfed by a macrophage, it changes shape and we discovered that its flagellum is remodelled from a device built for swimming (with a 9+2 arrangement of axoneme microtubules), into a structure resembling a sensory cilium (with a 9+0 axoneme) (Gluenz et al., 2010; Wheeler et al., 2015). We aim to dissect the mechanisms that govern this change in flagellar structure and test our hypothesis that the amastigote flagellum serves as a sensory organelle in host-parasite interactions. We found that the tip of many amastigote flagella associates closely with the membrane of the host cell vacuole. The flagellar membrane forms a distinct surface, and we are now studying the molecular composition of this domain to discover what receptors or transporters are expressed in the amastigote form and how they contribute to the parasite’s ability to sense its environment and survive in the macrophage.
Changes in gene expression during the life cycle
We used RNA-sequencing to map for the first time L. mexicana transcript boundaries on a genome-wide level (Fiebig et al., 2015). We discovered several hundred transcripts that did not correspond to any previously annotated genes and predict that some of these encode novel small proteins whose function awaits clarification. We also compared gene expression patterns in the insect- and mammalian-infective forms of L. mexicana and future work aims to link changes in gene expression patterns with the changes in cell morphology (e.g. flagellum restructuring) and identify genes and pathways important for survival in a macrophage.
Gluenz et al., (2010) FASEBJ 24, 3117-21
Joining the Lab