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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 (Cy^TM-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.

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