CHARACTERIZATION OF A PROTEIN INVOLVED IN CELL MORPHOLOGY AND PYOMELANIN PRODUCTION IN LEGIONELLA PNEUMOPHILA
thesisposted on 14.12.2020, 21:29 by Victor A RomanVictor A Roman
Legionella pneumophila is an intracellular pathogen and the etiological agent of Legionnaires’ disease, a severe atypical pneumonia. This bacterium is ubiquitous to freshwater ecosystems where it spreads in the planktonic form but is primarily found associated with protozoa. Protozoa serve as a niche for its replication because the extracellular environment often does not offer sufficient nutrients to support the growth of this bacterium. L. pneumophila is an opportunistic pathogen in humans and utilizes an arsenal of virulence factors to colonize hosts and cause Legionnaires’ disease. The transition between extracellular and intracellular milieus triggers a series of metabolic, morphological and genetic changes that define two developmental stages in this bacterium: replicative and transmissive. Relatively high concentration of nutrients triggers the replicative stage of growth, where the bacterium has the appearance of a thin, elongated rod without the presence of flagella. In addition, is characterized by active metabolism and expression of genes required for productive replication. In contrast, once nutrient levels are relatively low, L. pneumophila switches to its transmissive form. In this form, the bacterium activates a genetic program that includes the expression of many traits associated with the transmissive stage, such as coccoid cell shape, motility, pigmentation and important virulence factors. These multifaceted changes in gene expression leading the differentiation from replicative to the transmissive form, are controlled by two-component regulatory systems. Specifically, the LetAS two-component system plays a key role in the regulation of cell morphology and in the production of the pigment pyomelanin. Here we report the identification of a LetAS-regulated protein, Lpg0586 (designated as Larp1), capable of inducing changes in cell morphology and pigment production. We found that Larp1 expression was accompanied by accumulation of the RecA protein, but evaluation of recA deletion mutants indicated that RecA is not involved in cell morphology changes in L. pneumophila. The specific reason as to why RecA accumulates upon Larp1 expression remains to be elucidated. However, we show that upon synthetic HGA treatment, L. pneumophila cultures display cell elongation and increased RecA levels. Lastly, Larp1 expression restored pyomelanin production in an un-pigmented mutant and increased the transcription of important genes involved in the pyomelanin production pathway. Based on these findings, Larp1 is the first LetAS-regulated protein reported to be involved in pyomelanin production.