UC Santa Cruz

The discovery of ATP-dependent proteolysis began with Lon protease in the1960s. Since that time, Lon (or LonA) has been identified as a key regulator of protein quality control and diverse cellular processes in archaea, bacteria, as well as in the mitochondria of eukaryotic cells. Despite nearly 60 years of research, the substrates of Lon and the mechanisms that dictate Lon proteolysis remain poorly understood. The work presented here focuses on understanding the mechanisms and consequences of Lon proteolysis in V. cholerae. Vibrio cholerae is the Gram-negative facultative pathogen responsible for the diarrheal disease cholera. V. cholerae remains a threat to global public health. There are estimated to be 1.3-4.0 million cases of cholera and 21,000-143,000 deaths worldwide each year. Lon plays a critical role in regulating processes important for V. cholerae's pathogenic cycle. For example, Lon regulates virulence factor production, type VI secretion system (T6SS)-dependent killing, biofilm formation, motility, c-di-GMP levels, cell division, and stress adaptation. Furthermore, V. cholerae mutants defective in Lon poorly colonize the host intestinal tract. Previous work performed by the Yildiz lab identified Lon as a repressor of the T6SS, which was a novel function attributed to prokaryotic Lon. To better understand how Lon might regulate the T6SS, I compiled the known functional and regulatory networks governing activation of the T6SS (Chapter 1). We then used whole proteome analysis to identify potential Lon targets that might explain Lon repression of the T6SS (Chapter 2). We identified TfoY as a Lon substrate and showed that Lon-dependent proteolysis of TfoY represses T6SS-dependent killing and motility. In addition, we used a combination of genetic and biochemical approaches to demonstrate that Lon binds to c-di-GMP and that c-di-GMP inhibits Lon-dependent proteolysis of TfoY. Most analyses on Lon have focused on Lon-dependent regulation in planktonic grown cells. Thus, relatively little is known regarding how Lon regulates processes important for biofilm formation. We performed whole proteome and whole transcriptome analyses on WT and ∆lon biofilms to identify potential Lon substrates and Lon regulated pathways in biofilms (Chapter 3). Our analyses indicates that Lon is important regulator of biofilm matrix production, virulence factor production, nucleotide pool homeostasis, iron homeostasis, and DNA repair pathways during the biofilm growth mode.The work outlined here provides valuable insights into how regulated proteolysis functions to control processes important for V. cholerae pathogenesis.