Figure 1.
Src is recruited and activated early at Shigella entry sites.
(A,B) Represent two sequences of bacterial entry events of HeLa cells transiently transfected with Src-GFP. The numbers correspond to the elapsed time from the addition of bacteria in seconds. Src-GFP is recruited early at the intimate bacterial–contact site (arrowheads), followed by recruitment in bacterial-induced ruffles. The arrow points at large ruffles that occur at bacterial entry sites without detectable prior recruitment at the intimate bacterial contact site. Scale bar = 5 µm. (C) HeLa cells transfected with Src-GFP (green) were challenged with bacteria for 15 min at 37°C and processed for immunofluorescent staining of F-actin (greyscale) and anti-PY416 (red). Shigella-induced Src activation occurs during bacterial invasion.
Figure 2.
Differential pattern of Src and actin localization at Shigella entry sites.
HeLa cells transfected with Src-GFP were challenged at 37°C with the wild-type strain M90T. (A,B) Reconstructions of deconvolved images from focal planes spaced by 0.2 µm. Samples were fixed and processed for fluorescent staining with rhodamine-phalloidin (actin), anti-bacterial LPS (blue). GFP-fluorescence is shown in green. (A) Cells were challenged with bacteria for 15 min. Src localizes in an inner region of foci, whereas F-actin labels bacterial-induced ruffles (arrowheads). (B) Cells were treated with cytochalasin D at a final concentration of 1 µg/ml for 30 min and challenged with bacteria for 10 min in the absence of cytochalasin D (Materials and Methods). Src-GFP is recruited at the bacterial-cell intimate contact, whereas Shigella-induced actin polymerization is detected as individual patches juxtaposing the bacteria (arrowheads). (C,D) HeLa cells transfected with Src-GFP were challenged for 10 min at 37°C with the ipaC/C1 strain. Samples were fixed and processed for fluorescent staining with rhodamine-phalloidin (red), and anti-IpaC pAb (blue). GFP-fluorescence is shown in green. (C) Cells treated with cytochalasin D at a final concentration of 1 µg/ml for 30 min prior to bacterial challenge. Src, actin, and IpaC: deconvolved images of a single focal plane; IpaC+phase: overlay of the IpaC panel with the corresponding focal plane acquired in phase contrast (Materials and Methods). During early stages of bacterial invasion, IpaC is detected at bacterial-cell contact sites and colocalizes with Src-GFP, in a region where little F-actin is detected. Scale bar = 5 µm.
Figure 3.
Shigella T3S translocated IpgB1 effector is not required for Src recruitment and activation.
(A) HeLa cells were transfected with Src-GFP and challenged at 37°C for 15 min. with wild-type Shigella, the ipaC, or the ipgB1 mutant strains. Samples were fixed and processed for anti-LPS immunofluorescence labelling (blue), and F-actin staining (red). GFP fluorescence is shown in green. The panel corresponds to reconstructions of deconvolved images from focal planes spaced by 0.2 µm. Unlike the ipaC mutant, the wild-type and ipgB1 mutant induces Src recruitment at bacterial entry sites. (B) HeLa cells were challenged with the indicated Shigella strains, and at various time intervals samples were lysed and processed for anti-phosphotyrosine Western-blot analysis (Materials and Methods). The time is indicated above each lane in min. The Shigella mutant strains deficient for the translocated T3S effectors ipaA, ipgD, ipgB1, and ipgB2 induce phosphotyrosine patterns that are similar to that induced by wild-type, whereas the ipaC mutant is defective at inducing tyrosine phosphorylation. The arrows indicate the expected migration of tyrosylphosphorylated cortactin.
Figure 4.
Domain organization of IpaC. Solid black box: secretion signal sequence; checked box: chaperone binding region; empty box: hydrophobic predicted transmembrane domain; hatched box: coiled-coil region; dotted-box: region involved in actin polymerization and bacterial invasion. The name of IpaC derivatives is indicated on the left. The solid triangles represent the insertion of an 11-residue epitope from phage C3 within the ipaC coding sequence [29]; the position of the residue corresponding to the insertion site is indicated. Deletions corresponding to the indicated residues are represented by a dotted line. The myc epitope insertion at the carboxyterminal end is represented by a grey box with horizontal lines.
Table 1.
Effects of ipaC mutations on Shigella-induced contact hemolysis.
Figure 5.
The C351 insertion in the IpaC effector domain does not prevent T3S translocation.
Bacterial strains were grown to exponential phase, and T3S was induced by the addition of Congo Red in the culture medium. Bacterial lysates (Lysate) or supernatants (CRsupe) were analyzed by anti-IpaB and anti-IpaC Western blot analysis. HeLa cells were challenged with the various bacterial strains for 30 min at 37°C. Translocated T3S effectors were subjected to immuno-precipitation and analyzed by Western-blot analysis (Transloc, Materials and Methods) or by anti-actin Western-blot analysis (actin). The corresponding T3S effectors are indicated on the right. Cells challenged with: C1: ipaC/pC1; C1+lat: ipaC/pC1 after latrunculin treatment; mxiD: the non-invasive mxiD strain; C351: ipaC/pC351; SF126: a polar insertion upstream of the ipa locus that leads to decreased expression of Ipa proteins [31].
Figure 6.
The C351 insertion in the IpaC effector domain impairs Src-dependent signaling.
(A) HeLa cells were challenged for 15 min at 37°C with the bacterial strain indicated above each lane. Cell lysates were subjected to anti-phosphotyrosine (top panel) or anti-cortactin (bottom panel) Western blot analysis. The molecular weight markers are indicated. The arrows point at cortactin. As oppposed to C57, C351 do not complement the ipaC mutant strain for induction of the phosphorylation of cortactin. (B) HeLa cells were challenged for 15 min at 37°C with bacteria. Samples were fixed and processed for fluorescent staining with rhodamine-phalloidin (green) and anti-bacterial LPS (red). Arrows indicate actin foci. Scale bar = 10 µm. (C), Actin foci were scored microscopically on at least 4 independent experiments (n>200). Cells challenged with the indicated strains, or with the ipaC mutant complemented with C1, C57, or C351. SF126 harbours a polar insertion upstream of the ipa locus that leads to decreased expression of Ipa proteins [31]. Expression of the C57 construct allows the ipaC mutant to induce actin foci at the same frequency as the wild-type strain, whereas expression of full length IpaC leads to actin foci that are smaller and less numerous than those observed with wild-type Shigella. Very few actin foci could be observed for the ipaC mutant strain expressing the C351 insertion.
Figure 7.
Cell treatment with the actin polymerization inhibitor Latrunculin B leads to decreased T3S translocation.
(A) HeLa cells were challenged with A3F1-expressing bacteria for 30 min at 37°C, and translocated A3F1 was subjected to anti-IpaA immunoprecipitation and analysed by anti-FLAG Western-blot analysis (Transloc). In control experiments, total lysates corresponding to bacteria-infected cells were analyzed by anti-FLAG Western blot (Lysates). A3F1: bacteria expressing the A3F1 construct. WT: wild type Shigella; ipaC/C1: ipaC mutant complemented with full length IpaC; mxiD: a T3S defective isogenic mutant; ipaC/C351: ipaC mutant expressing C351. The arrows point at the A3F1 construct. (B) HeLa cells were challenged with wild-type Shigella expressing A3F1 for 15 min at 37°C in the absence (A3F1) or presence of latrunculin A (A3F1+Latrunc). Samples were fixed and processed for immunofluorescence staining of bacterial LPS (bacteria), FLAG epitope (Flag), and actin (actin). FB: bacteria are in green and the FLAG staining is in red. The panels represent reconstructions of deconvolved images from focal planes spaced by 0.2 µm. The insets in the right panels are shown. Scale bar = 5 µm. Translocation of A3F1 is reduced upon latrunculin treatment.
Figure 8.
The IpaC last 72 carboxyterminal residues fused to the Iota Ia component induces actin foci-like structures.
(A), Schematic construction of IaC. Ia, the Iota Ia component. Solid box: signal sequence. Hatched box: enzymatic domain. IaC, the carboxyterminal residues 291–363 of IpaC containing the effector domain were fused to Ia*, corresponding to residues 41–454 of Ia with the E380A substitution that inactivates its enzymatic function [32]. (B) HeLa cells were challenged with the Iota Ib component and Ia* (IbIa) or IaC (IbIaC and bottom panels) and processed for fluorescent staining of F-actin. Arrows point at IbIaC-induced actin ruffles. Bottom panels: two series of focal planes corresponding to IbaC-induced actin foci. The numbers indicate the distance in µm from the cell basal surface.
Figure 9.
IaC-induced actin structures are enriched in Src.
HeLa cells transfected with Src-GFP were challenged with the Iota Ib component and Ia* (IbIa) or Ic (IbIaC) and processed for immunofluorescence staining of F-actin and PY416 (A) or cortactin (B). Arrows point at IbIaC-induced actin ruffles that contain activated Src. Actin foci detected in IbIc-stimulated cells are enriched in Src-GFP and cortactin (arrows). Scale bar, 5 µm. (C) Cells were challenged with IbIaC, IbIa, or IbIaC in the presence of 10 µM PP2. Src-GFP transfectants showing ruffles were scored microscopically. The numbers are representative of at least 300 transfected cells in three independent experiments.