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First published online 20 July 2004
doi: 10.1242/jcs.01247

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Analysis of Chlamydia caviae entry sites and involvement of Cdc42 and Rac activity

Agathe Subtil^*, Benjamin Wyplosz^*, María Eugenia Balañá^* and Alice Dautry-Varsat

Unité de Biologie des Interactions Cellulaires, Institut Pasteur, CNRS URA 2582, 25 rue du Docteur Roux, 75015 Paris, France

View larger version (94K): [in a new window]   Fig. 1. Actin rearrangements upon bacteria entry. HeLa cells were infected with FITC-coupled bacteria for the indicated times before fixation and actin labelling using Alexa546-coupled phalloidin. Notice that a bacterium is visible in each of the actin patches, which transiently appeared (5 minutes and 15 minutes, arrows). At later times, infection also induced a general actin remodelling (15 minutes and 45 minutes, asterisks).

View larger version (102K): [in a new window]   Fig. 2. Distribution of DRMs during bacterial entry. HeLa cells were infected with C. caviae GPIC for 0 minute (top) or 5 minutes (bottom) before fixation. Actin was visualized with Alexa546-coupled phalloidin (left) and GM1 gangliosides were stained using FITC-cholera toxin (middle). Superimposition of the two stainings is shown (right), clusters of GM1 are indicated (arrows).

View larger version (48K): [in a new window]   Fig. 3. Distribution of phosphorylated proteins during bacterial entry. Non-infected cells (top) and cells infected for 5 minutes with FITC-coupled C. caviae GPIC (bottom) were labelled with anti-phosphotyrosine antibody (middle) while actin was visualized using Alexa546-coupled phalloidin (left). Upon infection, phosphorylated proteins were concentrated at the sites of intense actin polymerization, where bacteria are found (arrows).

View larger version (38K): [in a new window]   Fig. 4. Effect of the inhibition of small GTPases of the Rho family on Chlamydia entry. (A) Illustration of the method used for the quantification of entry efficiency. Transiently transfected HeLa cells were infected with C. caviae for 4 hours as described in the Materials and Methods section. Extracellular bacteria were labelled in far-red (CyTM-5, left) in non-permeabilized cells. After permeabilization, total bacteria were labelled in green (middle) and transfected cells were labelled in red (they appear blue in the merged image). Images were acquired in the three channels and were merged (right). In this example, the cells had been transfected with the GTPase binding domain of WASP. Notice that, overexpression of this constuct (as well as of the GTPase binding domain of PAK) induced an overall decrease of bacterial attachment of about 50%, which was taken into account when measuring the entry efficiency. (B) Quantification of C. caviae entry efficiency. HeLa cells were transfected with the indicated plasmids and infected with C. caviae GPIC the following day. Extracellular and intracellular bacteria, as well as transfected cells, were labelled as described above. The number of surface-associated and intracellular bacteria were counted in the transfected and non-transfected population (n>25 cells) and the efficiency of entry (intracellular/total cell-associated) was calculated. For each experiment, the efficiency of entry into transfected cells is expressed relative to that into non-transfected cells. The data shown are the average of three experiments. Last column: cells were pretreated for 2 hours with 10–7 M EDIN prior to measuring bacteria entry. Efficiency of entry is expressed relative to that in non-treated cells.

View larger version (65K): [in a new window]   Fig. 5. Kinetics of activation of Cdc42 and Rac upon infection. HeLa cells in suspension were centrifuged for 20 seconds in DMEM alone (control lane) or with bacteria (other lanes), and incubated for the indicated times at 37°C. Cells were lysed and incubated with GST-CRIB. GST-CRIB-associated proteins were pulled-down using glutathione-Sepharose and analysed by western blotting using anti-Rac1 (A) and anti-Cdc42 (C) antibodies. Aliquots of total cell lysates were immunoblotted for total Rac1 (B) and total Cdc42 (D), showing that the total amount of each small GTPase was identical at all time points. Data are representative for four experiments.

View larger version (101K): [in a new window]   Fig. 6. Effect of the inhibition of Cdc42 and Rac on actin polymerization during bacterial entry. Cells, transfected for 18 hours with dominant negative Cdc42 (A-E) or Rac (F-J), were inoculated with culture medium alone (A,B,F,G) or with FITC-coupled bacteria (C,D,E,H,I,J). Five minutes p.i. the cells were fixed, and transfected cells were identified using anti-myc antibodies (A,C,F,H) while actin was observed using phalloidin (B,D,G,I). E and J show the superimposition of the 3 images collected from the infected samples with the transfected cells (blue), actin (red) and bacteria (green). Both Cdc42N17 and RacN17 were recruited to the sites of actin polymerization, with which bacteria were associated (arrows). Notice that in transfected cells actin patches were larger than in non-transfected cells (arrowheads).

View larger version (82K): [in a new window]   Fig. 7. Effect of PI 3-kinase inhibitors on bacterial entry and on actin polymerization. (A) Control HeLa cells or cells pretreated with 100 nM wortmannin or 50 µM LY294002 were infected with C. caviae GPIC. Four hours after infection the cells were fixed and extracellular and intracellular bacteria were labelled as described in the Materials and Methods section. The number of surface-associated and intracellular bacteria were counted in control and treated cells (n=number of cells) and the efficiency of entry (intracellular/total cell-associated) was calculated. Results are expressed as the efficiency of entry relative to that in control cells. This experiment is representative of two. (B) Control HeLa cells (top row) or cells pretreated for 30 minutes with 50 µM LY294002 (bottom row) were infected with FITC-coupled C. caviae GPIC, centrifuged and incubated for 5 minutes at 37°C before fixation and actin labelling using Alexa-546 phalloidin.

View larger version (20K): [in a new window]   Fig. 8. A scheme of some early events during C. caviae entry. Interaction of a bacterium (grey circle) with the host cell plasma membrane induces the clustering of cholesterol-rich membrane domains (orange line), a phenomenon that might participate in initiating intracellular signalling. Signalling for entry might also be mediated by putative effector molecule(s) secreted by a type III mechanism. The small GTPases Cdc42 and Rac, which control proper actin polymerization, are rapidly activated. Abundant protein phosphorylation is detected and phosphorylated proteins (orange shapes) accumulate at the entry sites where they take part in the signalling cascade. In particular, several phosphorylated proteins can interact with the PI 3-kinase (blue), whose activity is required for C. caviae entry. Activation of Cdc42 and Rac is transient and is followed by the depolymerization of actin filaments, associated with a decrease in the accumulation of phosphorylated proteins around internalized bacteria. Although bacteria-associated actin patches have disappeared 45 minutes p.i., general remodelling of the cytoskeleton can be observed for longer times after infection.