A Two-Component System that Modulates Cyclic-di-GMP Metabolism Promotes Legionella pneumophila Differentiation and Viability in Low-Nutrient Conditions

Abstract

During its life cycle, the environmental pathogen Legionella pneumophila alternates between a replicative and a transmissive cell type when cultured in broth, macrophages, or amoebae. Within a protozoan host, L. pneumophila further differentiates into the hardy cell type known as the Mature Infectious Form (MIF). The second messenger cyclic-di-GMP coordinates lifestyle changes in many bacterial species, but its role in the L. pneumophila life cycle is less understood. Using an in vitro broth culture model that approximates the intracellular transition from the replicative to transmissive form, here we investigate the contribution to L. pneumophila differentiation of a two-component system (TCS) that regulates cyclic-di-GMP metabolism. The TCS is encoded by lpg0278-lpg0277 and is co-transcribed with lpg0279, which encodes a protein upregulated in MIF cells. Using a gfp-reporter, we demonstrate that the promoter for this operon is RpoS-dependent and induced in nutrient-limiting conditions that do not support replication. The response regulator of the TCS (Lpg0277) is a bifunctional enzyme that both synthesizes and degrades cyclic-di-GMP. Using a panel of site-directed point mutants, we show that cyclic-di-GMP synthesis mediated by a conserved GGDEF domain promotes growth arrest of replicative L. pneumophila, production of pigment and poly-3-hydroxybutyrate storage granules, and viability in nutrient-limiting conditions. Genetic epistasis tests predict that the MIF protein Lpg0279 acts upstream of the TCS as a negative regulator. Thus, L. pneumophila is equipped with a regulatory network in which cyclic-di-GMP stimulates the switch from a replicative to a resilient state equipped to survive in low-nutrient environments.\n\nIMPORTANCEAlthough an intracellular pathogen, L. pneumophila has developed mechanisms to ensure long-term survival in low-nutrient aqueous conditions. Eradication of L. pneumophila from contaminated water supplies has proven challenging, as outbreaks have been traced to previously remediated systems. Understanding the genetic determinants that support L. pneumophila persistence in low-nutrient environments can inform design of remediation methods. Here we characterize a genetic locus that encodes a two-component signaling system (lpg0278-lpg0277) and a putative regulator protein (lpg0279) that modulates production of the messenger molecule cyclic-di-GMP. We show that this locus promotes both L. pneumophila cell differentiation and survival in nutrient-limiting conditions, thus advancing our understanding of the mechanisms that contribute to L. pneumophila environmental resilience.