The main theme of the Tocheva lab is to understand the ultrastructure of bacteria and how it determines their physiology. The work in her lab focuses on determining the structure, composition and 3D function of two main systems: 1) the bacterial cell envelope and 2) the Type VII secretion system (T7SS) of Mycobacteria. The bacterial cell envelope maintains the shape of the cell and provides the first line of defense from the environment. In addition, it is a major target for antibiotics. The T7SS is employed by Mycobacteria to escape the phagosome of macrophages and thus persist alive in human cells. The Tocheva lab is working towards characterizing both systems structurally and functionally in order to understand how bacteria overcome external factors and are able to cause disease. The long-term goal of this research is to identify novel antimicrobial targets in the bacterial cell envelope and the T7SS that can help in our fight against bacterial infections. The results of this research will have practical health benefits for Canadians and major impact on several key industries, such as biotechnology, food and agriculture, and thus the economy of the country.
Please visit: www.tochevalab.org
For decades bacteria were thought of as “bags” of enzymes lacking a cytoskeleton, in contrast to eukaryotes cells where intracellular compartmentalization and establishment of large-scale order has been known for a long time. The development of cryo-preservation of biological samples sparked a new era for Bacterial Cell Biology. In combination with 3-dimensional data acquisition (ECT) we have been able to preserve and image bacterial ultrastructure and elucidate important new mechanistic insights. New super-resolution fLM methods (such as photoactivation localization microscopy, PALM) are being developed that take advantage of single-molecule activation events and deliver resolution beyond the diffraction limit of light. Today we know that bacterial cytoskeletal proteins polymerize into surprisingly diverse superstructures, such as rods, rings, twisted pairs, tubes, sheets, spirals, moving patches or meshes. The vast majority of bacterial filaments and nanomachines, however, remain unknown. These ultrastructures are the driving force in essential cellular processes including cell division, cell shape
determination, DNA segregation, secretion or motility. Today a major task is to understand the diverse processes by which bacteria generate intracellular order and perform tasks.
Dr. Tocheva’s work focuses on bridging the scales between individual proteins, macromolecular assemblies and neighboring cells. By applying new correlative fLM and ECT techniques we aim to generate much needed insight into the structure and function of two major macromolecular assemblies: the bacterial cell envelope and DNA segregation machines.
1. The bacterial cell envelope
Historically, bacterial cells have been classified on the basis of their ability to retain Gram stain. Gram-negative cells typically have two membranes surrounding a thin layer of peptidoglycan. Gram-positive cells have one membrane and a thicker layer of peptidoglycan. We gained a surprising insight into the relationship between these seemingly different architectures from our ECT studies of sporulation. By imaging a rare endospore-forming Gram-negative bacterium, we found that the inner membrane of the mother cell is transformed into the outer membrane of the germinating spore. This interconversion, and the ability of thick peptidoglycan to be transformed into thin (and vice versa), suggests an evolutionary source of the Gram-negative outer membrane and reveals that monoderm and diderm cell plans may not be so different after all.
2. DNA segregation mechanisms
Bacterial and eukaryotic actins share functional properties that have been conserved through evolution. Both polymerize into dynamic filaments that can assemble and disassemble in response to regulatory proteins and nucleotide-induced conformational changes. While bacterial and eukaryotic actins share limited sequence similarity, they have a conserved tertiary structure. However, bacterial actins have adapted to explore a much wider range of sequence variation and partner interactions and therefore display greater variation in filament architecture and dynamics. They are intimately involved in numerous activities ranging from the coordination of cell wall synthesis to the positioning of subcellular structures, and they are grouped into distinct protein families based on their phylogeny and function. Bacteria have many actin-like proteins (Alps) that are either encoded on the chromosome or on mobile genetic elements. Studying these proteins provides an opportunity to characterize the plasticity of bacterial actin and to identify novel mechanisms resulting in a conserved evolutionary function such as DNA segregation.
Technologies & Methods
Projects
1. Structure and function of the bacterial cell envelope: The cell envelope in bacteria provides the first line of defense from the hostile environment and antibiotics. Even though the majority of the characterized bacterial phyla to date are Gram-negative, there are no hypotheses as to how the second membrane may have evolved. Using cryo electron tomography (cryo-ET) of sporulating Gram-positive (Tocheva, Mol Microbiol, 2013) and Gram-negative bacteria (Tocheva, Cell, 2011), we showed how sporulation could lead to the generation of double membraned cells. Further biochemical and phylogenetic analyses helped us propose that an ancient sporulation-like event gave rise to the outer membrane in bacteria and that the last common ancestor of all bacteria must have been a double membraned sporulator. Mapping the distribution of cell envelope architectures onto a recent phylogenetic tree of life indicated that loss of either the outer membrane or sporulation could explain all the diversity of the cell envelope architectures observed to date (Tocheva, Nat Rev Microbiol, 2016). We are continuing to characterize the diversity of the bacterial cell envelopes and the ability of bacteria to sporulate.
2. Method development for correlative light and electron microscopy: Cryo-ET produces three-dimensional images of intact cells in a near-native, “frozen-hydrated” state and is therefore a powerful tool for investigating subcellular ultrastructures in vivo and at a macromolecular resolution (3-4nm). Identifying objects within the complex environment of the cell, however, can be challenging. One solution to localize structures subcellularly is to tag the structures of interest with fluorescent tags and to correlate cryo-ET with fluorescent light microscopy (fLM). To minimize any changes to the sample between imaging, the sample can be preserved cryogenically. We implemented a super-resolution light microscopy method under cryogenic conditions and correlated it with cryo-ET to study the type VI secretion system in Myxococcus xanthus (Chang, Nat Methods, 2014). We are continuing to develop methods that will allow us to identify macromolecular assemblies in the complex environment of the cell.
3. Ultrastructure of Bacteria: Over the past years we have lead and been involved in several projects aimed at identifying and characterizing microbial ultrastructure. We were the first to describe the role of polyphosphate in energy storage required for outgrowth during germination in Gram-negative bacteria (Tocheva, J Bacteriol, 2014) and the role of propanediol utilization microcompartments (Tocheva, J Bacteriol, 2014). We further identified a new bacterial structure (the “nanopod”) that is used to secrete outer membrane vesicles (Shetty, PLoS One, 2011). Work on the bacterial chemoreceptor arrays depicted a universal hexagonal architecture (Briegel, PNAS, 2009), where as work on the bacterial flagellar motor showed great structural variability (Chen, EMBO J, 2011). Understanding the ultrastructure of bacteria will provide us with tools for future applications and identify new antimicrobial targets. We are driven by the discovery and characterization of novel bacterial ultrastructure.
