A Cyclic di-GMP Two-Component System Contributes to Legionella pneumophila Differentiation and Resilience

Abstract

Legionella pneumophila is a waterborne pathogen and the causative agent of the severe pneumonia Legionnaires’ Disease. This bacterium is an intracellular parasite of both human lung macrophages, as well as amoeba and ciliated protists, its natural host. Within the environment, L. pneumophila exists as either an intracellular parasite of protozoa, within biofilms, or as free-living, planktonic cells. Unfortunately, remediation of contaminated water systems has proven an ongoing challenge, mostly due to the ability of L. pneumophila to differentiate into an environmentally resistant, highly infectious but spore-like cell type called a Mature Infectious Form (MIF). The genetic pathways and regulatory mechanisms that promote MIF cell development remain largely unknown. A few proteins have been identified as abundant in MIF cells, however, including Lpg0279. In this dissertation, I explore the impact of Lpg0279 on L. pneumophila cellular differentiation and its interaction with a two-component system encoded by lpg0278-lpg0277. I show that lpg0279 is co-transcribed with lpg0278 and lpg0277, expression is controlled by the stationary phase sigma factor RpoS, and that transcription is enhanced in nutrient-limited conditions.

The two-component system encoded by lpg0278-lpg0277 modulates the turnover of the second messenger molecule cyclic-di-GMP. This ubiquitous molecule directs lifestyle changes in multiple bacterial species. The L. pneumophila genome codes for multiple proteins that make and break cyclic-di-GMP, but few studies have been conducted examining the impact of this second messenger molecule on the L. pneumophila life cycle. Lpg0277 is a bifunctional enzyme and the response regulator of the two-component system. Using isogenic mutants and a series of phenotypic assays, I show that the diguanylate cyclase activity of Lpg0277 (and therefore an increase in c-di-GMP) promotes transition of L. pneumophila to the stationary phase of growth, the generation of pyomelanin pigment and poly-3-hydroxybutyrate granules, and contributes to long-term survival in low-nutrient conditions. In addition, I provide evidence to support a model in which Lpg0279 initially acts as a negative regulator of this enzymatic activity. This research provides important insight into how c-di-GMP contributes both to L. pneumophila differentiation, and to persistence of this bacterium in low-nutrient environments. This information can inform future surveillance methods, detection of MIF cells, and remediation efforts to control spread of this pathogen in the built environment.