Antibiotic persistence remains one of the most challenging barriers to effective clearance of chronic bacterial infections. Despite the intervening decades since its original discovery, we still lack a basic understanding of how molecular networks implement the defining features of persistence: phenotypic heterogeneity and tolerance to lethal levels of antibiotic exposure. Much of the work on persistence has been phenomenological and the mechanistic studies which, so far, have only focused on a few candidate pathways, have failed to provide an adequate understanding of this complex phenotype. Here, we propose an unbiased systems biology approach to characterize the genetic and regulatory underpinnings of persistence. A primary aim is to precisely define the cellular state of persister cells which typically make up only a small (< 10-4) fraction of the population. We will utilize our previously developed genetic and chemically induced models of E. coli hyper-persistence to trigger persister cells, and to follow the trajectory of their phenotypic diversification by using our recently developed single-cell RNA sequencing technology (PETRI-seq). This will enable us to define the dynamics of the process at single-cell resolution, and identify highly specific persister markers, which we can use to purify them to near- homogeneity. We will then profile the global gene regulatory state of persisters by using our recently optimized technologies for in vivo Protein-DNA and RNA-RNA interactions. Combining the single-cell RNA atlas and global regulatory interactions will enable us to generate causal graphical models of pivotal regulatory events that underlie persister formation. We will utilize our recently developed CRISPR-interference technology (CALM) to systematically determine how knock-down perturbations to all essential and non-essential genes affect quantitative parameters of persistence, including persister-fraction and kill-rates. Finally, by combining the regulatory and genetic maps of persistence, we will identify and validate the most critical vulnerability nodes whose targeting will eliminate persister formation and survival. The massive scale of these observations will reveal the most comprehensive and unbiased global view of persister generation, gene regulation, and physiology, to date. This is a critical foundation upon which we can devise rational strategies for reducing the clinical burden of persisters. Finally, the unique conceptual and technological approaches here will serve as a blueprint for exploring the genetic and regulatory basis of other complex bacterial phenomena, such as biofilms, where phenotypic heterogeneity is a defining hallmark.
The proposed research is a systems biology approach to characterize the genetic and regulatory underpinnings of antibiotic persistence in the bacterium E. coli. The proposed studies will provide critical insights into how small sub-population of bacteria become tolerant to otherwise lethal levels of antibiotics. This phenomenon is highly relevant to emerging health crisis of antibiotic tolerance.
| Khare, Anupama; Tavazoie, Saeed (2015) Multifactorial Competition and Resistance in a Two-Species Bacterial System. PLoS Genet 11:e1005715 |
| Freddolino, Peter L; Goodarzi, Hani; Tavazoie, Saeed (2014) Revealing the genetic basis of natural bacterial phenotypic divergence. J Bacteriol 196:825-39 |
| Hottes, Alison K; Freddolino, Peter L; Khare, Anupama et al. (2013) Bacterial adaptation through loss of function. PLoS Genet 9:e1003617 |
| Freddolino, Peter L; Goodarzi, Hani; Tavazoie, Saeed (2012) Fitness landscape transformation through a single amino acid change in the rho terminator. PLoS Genet 8:e1002744 |
| Freddolino, Peter L; Tavazoie, Saeed (2012) Beyond homeostasis: a predictive-dynamic framework for understanding cellular behavior. Annu Rev Cell Dev Biol 28:363-84 |
| Freddolino, Peter L; Amini, Sasan; Tavazoie, Saeed (2012) Newly identified genetic variations in common Escherichia coli MG1655 stock cultures. J Bacteriol 194:303-6 |
| Freddolino, Peter L; Tavazoie, Saeed (2012) The dawn of virtual cell biology. Cell 150:248-50 |
| Girgis, Hany S; Harris, Kendra; Tavazoie, Saeed (2012) Large mutational target size for rapid emergence of bacterial persistence. Proc Natl Acad Sci U S A 109:12740-5 |
| Hottes, Alison K; Tavazoie, Saeed (2011) Microarray-based genetic footprinting strategy to identify strain improvement genes after competitive selection of transposon libraries. Methods Mol Biol 765:83-97 |
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