Implication of the VirD4 coupling protein of the Lvh type 4 secretion system in virulence phenotypes of Legionella pneumophila

Abstract

The genome of the Philadelphia-1 strain of Legionella pneumophila, the causative organism of Legionnaires' disease, encodes two virulence-associated type 4 secretion systems (T4SSs), the Dot/Icm type 4B (T4BSS) and the Lvh type 4A (T4ASS). Broth stationary-phase cultures of most dot/icm mutants are defective in entry and evasion of phagosome acidification. However, those virulence defects can be reversed by incubating broth cultures of dot/icm mutants in water, termed water stress (WS). WS reversal requires the lvh T4ASS locus, suggesting an interaction between the two T4SSs in producing Legionella virulence phenotypes. In the current work, the loss of WS reversal in a dotA Δlvh mutant of strain JR32 was shown to be attributable to loss of the lvh virD4 gene, encoding the putative coupling protein of the T4ASS. Transformation of a dotA Δlvh mutant with virD4 also reversed entry and phagosome acidification defects in broth cultures. In addition, broth cultures of Δlvh and ΔvirD4 mutants, which were dot/icm(+), showed 5-fold and >6-fold increases in translocation of the Dot/Icm translocation substrates, proteins RalF and SidD, respectively. These data demonstrate that the Lvh T4ASS functions in both broth stationary-phase cultures conventionally used for infection and cultures exposed to WS treatment. Our studies in a dotA Δlvh mutant and in a dot/icm(+) background establish that VirD4 and the Lvh T4ASS contribute to virulence phenotypes and are consistent with independent functioning of Dot/Icm and Lvh T4SSs or functional substitution of the Lvh VirD4 protein for a component(s) of the Dot/Icm T4BSS.

Fig 1

Translocation of cyclase fusions. J774 macrophages were infected with parental strain JR32 or the indicated mutant strain containing plasmid cyclase fusions to the N terminus of the listed effector protein. Results are the ratio of cAMP for the mutant strain to cAMP for strain JR32, from the mean of 12 to 30 determinations in 2 to 4 independent experiments. **, P < 0.0001.

Fig 2

Intracellular multiplication phenotypes. (A) Deletion mutants for virD4 and ankQ are not defective for intracellular multiplication in J774 macrophages. J774 macrophages were infected at 37°C at an MOI of 1 with stationary-phase JR32 bacteria or the indicated single or double JR32 mutant. At the indicated times, aliquots from the infection medium had titers determined for numbers of CFU. Mean and standard deviation are plotted. (B) Deletion mutants for virD4 and ankQ are not defective for intracellular multiplication in A. castellanii amoebae. A. castellanii amoebae were infected at 30°C at an MOI of 1 with stationary-phase JR32 bacteria or the indicated single or double JR32 mutant. At the indicated times, aliquots from the infection medium had titers determined for numbers of CFU. Mean and standard deviation are plotted.

Fig 3

Entry phenotypes. (A and B) virD4 and ankQ deletion mutants are not defective for entry. (A) Entry into J774 macrophages. J774 macrophages were infected at 37°C at an MOI of 100 with stationary-phase (Stat) or water stress (WS)-treated JR32 bacteria or the indicated single or double JR32 mutant expressing GFP. Mean and standard deviation are plotted. *, P = 0.04. (B) Entry into A. castellanii amoebae. A. castellanii trophozoites were infected at 28°C at an MOI of 5 with stationary-phase (Stat) or water stress (WS)-treated JR32 bacteria or the indicated single or double JR32 mutant expressing GFP. Mean and standard deviation are plotted. *, P = 0.01. (C and D) VirD4 reverses the entry defect of a dotA Δlvh double mutant. (C) Entry into J774 macrophages. J774 macrophages were infected at 37°C at an MOI of 100 with stationary-phase (Stat) or water stress (WS)-treated JR32 bacteria or the indicated single or double JR32 mutant expressing GFP and, where indicated, containing plasmid pMMB207C (pMMB) or the virD4 complementation plasmid (pvirD4). Mean and standard deviation are plotted. *, P = 0.04. (D) Entry into A. castellanii amoebae. A. castellanii trophozoites were infected at 28°C at an MOI of 5 with stationary-phase (Stat) or water stress (WS)-treated JR32 bacteria or the indicated single or double JR32 mutant expressing GFP and, where indicated, containing plasmid pMMB207C (pMMB) or the virD4 complementation plasmid (pvirD4). Mean and standard deviation are plotted. *, P = 0.06; **, P = 0.04.

Fig 4

Vacuole acidification phenotype in macrophages. J774 macrophages were infected at 37°C at an MOI of 50 with stationary-phase (Stat) or water stress (WS)-treated JR32 bacteria or the indicated single or double JR32 mutant expressing GFP and, where indicated, containing plasmid pMMB207C (pMMB) or the virD4 complementation plasmid (pvirD4). Colocalization with LysoTracker Red was calculated as a percentage of internalized bacteria. Mean and standard deviation are plotted. (A) virD4 and ankQ deletion mutants are not defective for the acidification phenotype. (B) VirD4 reverses the defective acidification phenotype of a dotA Δlvh double mutant. **, P = 0.001.