pathogenicity island 2-encoded type III secretion system (T3SS-2) that translocates bacterial effector proteins inside host cells. (effectors) from the bacterial to the eukaryotic cytosol. Effectors are capable LAQ824 of influencing host functions. T3SS-1 effectors manipulate the cortical actin cytoskeleton, trigger the formation of membrane ruffles and facilitate the uptake of bacteria by non-phagocytic cells. T3SS-2 translocates more than 20 effectors that are collectively required for intracellular replication and virulence in mice (for review see ref.3). vacuoles have several unique morphological characteristics. A SCV contains a single bacterium and divides when the bacterium undergoes cell division. 4 This trait requires the function of host proteins. Abnormal vacuoles enclosing several bacteria have been observed upon inhibition of dynein function4 or in the absence Plekhm1, a host protein that interacts with the T3SS-2 effector SifA.5 The SCVs are connected to each other by membrane tubules that elongate with the mechanical support of the microtubule cytoskeleton and motors. Tubules were originally described in infected epithelial cells6 but LAQ824 also form in macrophages7-9 though they are more difficult to observe. Different kinds of tubules have been described10 and are together referred to as expressing CFP and PipB2-2HA. Cells were fixed at 16?h p.i., immunostained for LAMP-1 and HA, and imaged by confocal microscopy for CFP (blue), … ICTs do not form between independently infected HeLa cells To define the conditions that lead to the formation of ICTs, we examined if these tubules could arise upon encounter of 2 infected cells. For this, we trypsinized and then co-cultured 2 batches of HeLa cells previously infected for 3? h with strains expressing either GFP or DsRed. ICTs were almost exclusively observed between cells enclosing expressing the same color protein (Fig.?2A). ICTs connecting cells enclosing GFP and DsRed bacteria were very rare (less than 1% of infected cells presenting ICTs) and, in most cases, both cells LAQ824 contained the 2 types of bacteria, strongly suggesting a secondary infection. We conclude that ICTs LAQ824 do not form upon encounter of 2 infected cells. Figure 2. ICTs form between cytokinetic cells. (A) ICTs do not exist between cells that have been infected independently. Schematic representation of the experiment. HeLa cells were infected with GFP- or DsRed-expressing has been reported to perturb the cell cycle,16 we first evaluated the consequences of an infection on the capacity of HeLa cell to divide. We infected cells with GFP-expressing bacteria for 14?h and analyzed by flow cytometry the DNA content of the does not substantially affect the capacity of HeLa cells to go through their regular cell cycle. Next, we examined whether a correlation exists between the cell cycle and the formation of ICTs. Cytokinesis is the last step of the M phase and this process immediately precedes the G1 phase. Therefore, we checked whether entry in G1 phase and the formation of ICTs were concomitant. We treated we investigated the cell cycle and the presence of ICTs in mouse macrophages. RAW 264.7 cells were infected with for 14?h. AIM-1 immunostaining was used to identify dividing cells (Fig.?3A). We found similar percentages of mitotic cells in the infected and non-infected populations of macrophages (5.8 0.7 % and LAQ824 5.2 0.4 %, respectively). It suggests that do not alter the capacity of RAW 264.7 macrophages to go through mitosis. We also analyzed infected macrophages for the presence of tubules after immunostaining for LAMP-1 or PipB2-2HA, but we could not detect the presence of ICTs in these cells. Figure 3. for 14?h. Fixed cells were immunostained … Next we followed the cell cycle of infected non-infected mouse macrophages cells. As compared to non-infected cells, we found 1.63 0.15 and 2.04 0.26?times more infected cells in the G2/M phase 30?min and 2?h p.i., respectively (Fig.?3B), indicating that are preferentially taken-up by pre-mitotic and/or mitotic macrophages. Between 8 and 10?h p.i the fraction of infected G2/M cells returned to a normal level whereas cells in S phase were slightly under-represented. At 14?h p.i., the infected and non-infected populations were not statistically different (Fig.?3C). T3SS-2-expressing bacteria are preferentially taken up by mitotic macrophages A T3SS-1-dependent preferential invasion of G2/M Mouse monoclonal to Cytokeratin 8 HeLa cells has been previously reported.19 In this study, we used grown in minimal medium that favors the expression of T3SS-2 rather than of T3SS-120 in order to decrease the T3SS-1-mediated killing of macrophages.21 Thus, T3SS-1 is probably not involved in the preferential uptake of by G2/M RAW 264.7 cells. To verify this point, we incubated macrophages for 30?min with different types of live or inert particles, analyzed their DNA content by flow cytometry and determined the fractions of cells having taken.