Erin D. Goley, Ph.D. - US grants

? DESCRIPTION (provided by applicant): The faithful execution of cytokinesis is required to produce viable offspring. In bacteria, cytokinesis is orchestrated by the cytoskeletal GTPase, FtsZ. FtsZ polymerizes to form a cytokinetic Z-ring that directs assembly of more than two dozen other factors. Together, the division machinery promotes constriction and fission of the membranes and remodeling of the peptidoglycan (PG) cell wall to ultimately split the cell into two. Despite its conserved and essential function in the majority of bacteria, the precise role of FtsZ in cytokinesis remains poorly defined. In addition to acting as a scaffold, FtsZ is hypothesized to generate constrictive force and/or to regulate PG remodeling. If and how it fulfills these functions in the cell is unknown. Like other cytoskeletal proteins, the assembly properties and structure of FtsZ are central to defining and executing its function. Using genetic, cell biological, biochemical, and advanced imaging approaches, this proposal addresses how three distinct modes of regulating of FtsZ polymer and Z-ring assembly alter its functional output in defined ways in the model bacterium Caulobacter crescentus. Aim 1 addresses how FtsZ polymers are physically targeted to the inner membrane by different tethering proteins to direct FtsZ activity to different functions. Aim 2 focuses on how FtsZ protofilament curvature is used to promote envelope constriction. Aim 3 determines how the disordered C-terminal linker of FtsZ affects its assembly and structure in a manner required for the regulation of PG remodeling enzymes. Successful completion of the proposed research will yield broad insights into the mechanisms of FtsZ function across the bacterial kingdom. Given the essentiality of FtsZ in bacteria, understanding the mechanisms by which it directs growth and division will facilitate its development as a target for novel antibiotics.

To impact human health, bacteria must reproduce through successive rounds of growth and cell division. Moreover, bacteria must be able to adapt their growth to changing environmental conditions, including changes in nutrient availability or the presence of antibiotics, to ensure survival. Research in my laboratory focuses on two unanswered questions central to bacterial growth and adaptation. In the first, we ask how do bacterial cells locally regulate growth to achieve cell division? To address this question, we will build on our recent work demonstrating that the conserved, polymerizing GTPase FtsZ is a dynamic regulator of cell wall synthesis and remodeling during cell division. This idea represents a paradigm shift in defining FtsZ as an active regulator, rather than passive scaffold, for cell wall metabolism. We will leverage our expertise in bacterial genetics, imaging, biochemistry, and in vitro reconstitution to map the players and mechanisms in two signaling pathways from FtsZ to cell wall metabolism we identified in our model organism, Caulobacter crescentus. Given the urgent need for new antibiotics and proven efficacy of the cell wall as an antibacterial target, a complete understanding of the mechanisms and regulation of cell wall metabolism is a critical goal. In our second question, we ask how do bacteria adapt to changing nutrient availability and other stresses? We recently described the role of a conserved transcriptional regulator called CdnL in regulating metabolism, specifically in upregulating biosynthetic pathways, in Caulobacter. In addition, we have observed that CdnL is cleared from the cell during nutrient limitation in a manner dependent on the signaling alarmone ppGpp, suggesting a mechanism by which cells may downregulate transcription of proliferative pathways when nutrients are scarce. We will use a combination of genetic, genomic, and biochemical approaches to determine the contributions of CdnL inactivation and ppGpp to reprogramming transcription to ensure bacterial survival during nutrient limitation and other stresses. As both CdnL and ppGpp are implicated in adaptation to a variety of stresses in diverse bacteria, this work will inform our understanding stress and antibiotic resistance mechanisms in important bacterial pathogens.

Members of the Spotted Fever Group (SFG) of the bacterial genus Rickettsia are obligate intracellular pathogens that cause spotted fever disease in humans ranging from mild to life-threatening. Like all species in the order Rickettsiales, SFG bacteria have undergone genome streamlining and are dependent on a eukaryotic host for dozens of essential metabolites. Notably, reductive evolution has led to loss or modification of factors in metabolic and morphogenetic pathways predicted to impact peptidoglycan cell wall metabolism. Peptidoglycan metabolism is essential for growth and viability and is therefore an effective antibiotic target. The impact of genome streamlining on the mechanisms of growth and division of the Rickettsiales, as compared to their free- living relatives, is not clear. This gap in knowledge persists, in part, because we lack tools to investigate growth of intracellular bacteria with sufficient resolution to derive a mechanistic understanding. We hypothesize that reductive evolution of metabolic and morphogenetic pathways necessitates diversification of known mechanisms to support intracellular growth. In this proposal, we aim to develop and apply methods to quantitatively analyze growth and cell wall metabolism of Rickettsia parkeri in eukaryotic host cells. R. parkeri is a SFG organism that causes relatively mild disease. As such, it is a BSL2 pathogen that has been studied as a representative to understand SFG pathogenesis. Through the proposed work, we will expand the study of R. parkeri to investigate its intracellular growth mechanisms. In aim 1, we will develop and apply imaging methods to quantitatively analyze the cell shape of, subcellular protein distribution in, cell cycle status of, and kinetics of growth of individual R. parkeri in the cytoplasm of eukaryotic host cells. In aim 2, we will determine the chemical composition of and spatial patterning of peptidoglycan cell wall metabolism of R. parkeri growing in the host cytoplasm. Completion of the proposed aims will provide foundational knowledge on the growth kinetics and mechanisms of PG metabolism of a tick-borne, obligate intracellular human pathogen and establish broadly applicable quantitative approaches for studying growth of bacteria within a eukaryotic host cell. Ultimately, the information and methods derived from this project will inform development of preventive and therapeutic strategies for limiting Rickettsial disease.