Implication of Proteins Containing Tetratricopeptide Repeats in Conditional Virulence Phenotypes of Legionella pneumophila

Legionella pneumophila, the causative agent of Legionnaires ’ disease, is a ubiquitous freshwater bacterium whose virulence phe-notypes require a type IV secretion system (T4SS). L. pneumophila strain JR32 contains two virulence-associated T4SSs, the Dot/ Icm and Lvh T4SSs. Defective entry and phagosome acidification phenotypes of dot/icm mutants are conditional and reversed by incubating broth-grown stationary-phase cultures in water (WS treatment) prior to infection, as a mimic of the aquatic environ-ment of Legionella. Reversal of dot/icm virulence defects requires the Lvh T4SS and is associated with a>10-fold induction of LpnE, a tetratricopeptide repeat (TPR)-containing protein. In the current study, we demonstrated that defective entry and phagosome acidification phenotypes of mutants with changes in LpnE and EnhC, another TPR-containing protein, were simi-larly reversed by WS treatment. In contrast to dot/icm mutants for which the Lvh T4SS was required, reversal for thelpnE or theenhC mutant required that the other TPR-containing protein be present. The single and doublelpnE andenhC mutants showed a hypersensitivity to sodium ion, a phenotype associated with dysfunction of the Dot/Icm T4SS. ThelpnE single and thelpnEenhC double mutant showed 3- to 9-fold increases in translocation of Dot/Icm T4SS substrates, LegS2/SplY and LepB. Taken together, these data identify TPR-containing proteins in a second mechanism by which the WS mimic of a Legion-ella environmental niche can reverse virulence defects of broth-grown cultures and implicate LpnE and EnhC directly or indi-rectly in translocation of Dot/Icm T4SS protein substrates. Legionella pneumophila is a ubiquitous freshwater bacteriumand the causative agent of Legionnaires ’ disease, a potentiall