Dual Control of Host Actin Polymerization by a Legionella Effector Pair

Figure 3

LegK2 and VipA effectors are produced and secreted at the early stage of infection. legK2 and vipA genes are weakly expressed, mostly in the exponential growth phase for vipA (a) and in postexponential and stationary phases for legK2 (b). Three cultures of L. pneumophila Paris WT were grown in liquid medium AYE (37°C). At the desired growth phases (, postexponential/start of mobility , and stationary collected 2 hours after the postexponential sample), samples were collected, and their RNA content was analyzed by RNAseq. The graphs show normalized read counts of legK2 or vipA mRNAs for each sample at the different growth phases. (c, d) Production of VipA-Luc and LegK2-Luc fusion proteins is maximum at the onset of the stationary growth phase. Luminescence emission from chromosomal translational fusions of vipA::luc (c) and legK2::luc (d) in L. pneumophila Paris strain grown in AYE medium at 37°C measured by OD₆₀₀ (dashed red lines). The data shown are representative of 3 independent clones of each fusion. (e) VipA accumulates in L. pneumophila up to . Three independent cultures of L. pneumophila Paris WT were grown in liquid medium AYE (at 30°C). At the desired OD₆₀₀ (1–5), samples were collected, and their protein content was analyzed by mass spectrometry after sample-specific labelling (see Materials and Methods for details). The graphs show the normalized abundance of protein detected at each OD₆₀₀. The LegK2 protein has not been detected in our conditions. The RocC protein is used as a control as it is detected at the same range of quantity as VipA, and its production during growth was previously studied by Western blot [23]. Of note, the RocC pattern of production detected by mass spectrometry corresponds to the one previously obtained by Western blot [23]. (f) legK2 is expressed during A. castellanii infection at the onset of the transmissive phase. A chromosomal legK2::gfp translational fusion was constructed in a mCherry expressing Paris strain. After infection of A. castellanii amoebae, the mCherry fluorescence was used to monitor the bacteria intracellular multiplication (dashed red line), and the GFP fluorescence was a read out of the legK2 gene expression (blue line). The data shown are representative of 3 independent clones of each fusion. (g) Translocation kinetics of TEM-LegK2 and TEM-VipA fusion proteins. U937 cells were infected () with wild-type (WT) Paris strains expressing plasmidic TEM-fusion proteins, TEM-LegK2 or TEM-VipA. Induction of fusion protein expression by 0.5 mM IPTG has been monitored by Western blot analysis. Results are obtained from 3 independent experiments made in triplicates and are presented as .

(a)

(b)

(c)

(d)

(e)

(f)

(g)