Examination of the regulatory role of the CpxRA two-component system and its relation to virulence in Legionella pneumophila
Tanner, Jennifer Rose
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Legionella pneumophila is a natural parasite of aquatic protozoa, and due to similarities in uptake mechanisms, is also an opportunistic intracellular pathogen of human pulmonary macrophages that can lead to pneumonia termed Legionnaires’ disease. In protozoa, L. pneumophila features a biphasic intracellular lifestyle that alternates between vegetative replicative forms and resilient infectious cyst forms, that when released ensure transmissibility to fresh protozoan hosts. Legionnaires’ disease outbreaks are often sourced to anthropogenic water systems that promote generation of cyst-laden aerosols that are inadvertently inhaled by susceptible individuals. Successful infection of protozoan and macrophage cells is enabled by the presence of a bacterial Dot/Icm Type IV Secretion System that facilitates the delivery of ~300 effector molecules to establish a replicative niche. The CpxRA two-component system has been shown elsewhere to regulate expression of several effector proteins as well as components of the Dot/Icm apparatus; yet inactivation of cpxRA via marked deletions did not affect intracellular growth in protozoa or human macrophages. To investigate this discrepancy, generated ∆cpxR, ∆cpxA, and ∆cpxRA in-frame null mutant strains were assessed for alterations in virulence. Intracellular growth kinetics of ∆cpxR and ∆cpxRA mutant strains was significantly reduced in the protozoan host Acanthamoeba castellanii, but unaffected in human U937-derived macrophages. In corroboration, ∆cpxR and ∆cpxRA mutant strains demonstrated sodium resistance; a phenotype strongly associated with avirulence. Transcriptome analysis of the cpxRA mutant strain revealed a global regulatory impact, with hundreds of genes including those encoding Dot/Icm effector proteins as well as Type II secreted substrates differentially regulated in the two growth phases examined. Expression of cpxRA located within the five-gene (lpg1441-cpxA) operon is controlled in a negative manner by the transcription regulator OxyR. Overexpression, but not lack, of OxyR limited the growth of L. pneumophila in A. castellanii but not in U937-derived macrophages cells or during broth culture. Recombinant CpxR bound a conserved binding site within the oxyR promoter region; however, expression control of OxyR remains uncertain as the promoter activity was unaffected in cpx mutant strains. Taken together, this study has conclusively determined that the essentiality of the CpxRA system to L. pneumophila virulence is host specific.