Quantification of <i>C. trachomatis</i> adhesion by flow cytometry.

<p>Cells were incubated for 4°C with the indicated MOI of L2^incDGFP purified EBs, before fixation and analysis in the green channel by flow cytometry. (A) Histograms of fluorescence in the green (FL-1) channel. In each panel, the fluorescence of non-infected cells is reported for comparison (dashed line). The horizontal bar delimits the fluorescence above background level and the percentage of cells (out of total cells) reaching these fluorescence values is indicated above. NI  =  non-infected. (B) The mean fluorescence of non-infected cells was subtracted from the mean fluorescence of infected cells and the resulting fluorescence value was plotted against the MOI. The inset shows an enlargement of the values obtained at low MOI. This experiment is representative of two.</p

GFP-expressing <i>C. trachomatis</i> serovar L2 strains are suitable to track RBs and EBs.

<p>(A) Genomic DNA accumulation over time for the parental strain and the two GFP-expressing strains, L2^nmGFP and L2^incDGFP, with GFP expression controlled by a neisserial (nm) promoter or the promoter for the chlamydial <i>incD</i> gene, respectively. Bacterial DNA was quantified by Q-PCR on the <i>ompA</i> gene. An experiment representative of three is shown. In this experiment, the MOI for the parental strain was about 2 to 3 times less than that for the two other strains, accounting for the lower bacterial DNA amount at all time points. Nevertheless, the growth curves are similar for the three strains (B) Coverslips were coated with L2 (top) or L2^incDGFP (bottom) EBs. After fixation, the bacterial envelope was stained with an anti-MOMP/LPS antibody followed with Cy5 coupled secondary antibodies, and bacterial DNA was labeled with Hoechst. The scale bar represents 5 µm.</p

Quantitative measurement of the <i>C. trachomatis</i> growth cycle using flow cytometry.

<p>(A) Kinetics of chlamydial growth. Cells were infected at a MOI = 0.5 and fixed at the indicated times. Samples were analyzed by flow cytometry as described in the Methods section, and histograms of fluorescence in the green channel (FL-1) are shown. For each time point, 10,000 cells were analyzed. The horizontal bar delimits the fluorescence above background level( = infected cells). The percentage of cells included in this gate, and their mean fluorescence, are indicated. NI  =  non-infected. (B) Comparison of the determination of infection rates by flow cytometry and microsocopy. Cells were infected at MOI = 0.2 in a 36 mm dish with one coverslip in the dish. Twenty-four hours later, cells on the coverslips were fixed and processed for microscopy toma nually count the percentage of infected cells (top left panel, cellular DNA appears in blue and inclusions are green, bar = 20 µm). The rest of the dish was used for determining the infection rate by flow cytometry (top right panel). The average of five measurements with each method is shown. A t-test showed no statistical difference between the two methods. (C) Determination of tetracycline IC-50 by flow cytometry. Cells were infected (MOI = 0.3) for one hour prior to tetracycline addition and incubated for 24 hrs before analysis by flow cytometry as described above. The histogram shows the mean fluorescence in the infected population relative to the value measured in infected cells treated with ethanol only, the error bars show the standard deviation in the triplicate of this experiment. The experiment has been reproduced three times. (D) Analysis of infection rates in a non-homogenous population. Cells were transfected for 24 hrs with the indicated construct prior to infection with L2^incDGFP. At 24 hrs post-infection, cells were detached and fixed, and the His tag was stained in red as described in the Methods section. Transfected cells were positive in the red channel (FL-4), infected cells in the green channel (FL-1). A dot-plot analysis of the two parameters is shown for cells transfected with IncA-His (left) or Δ75IncA-His (right). For each condition, the percentage of infected cells in the transfected and non-transfected populations is reported in the histogram. The experiment shown is representative of three independent assays with error bars showing the standard deviation. In panels B, C and D results of t-tests are reported.</p

Quantification of <i>C. trachomatis</i> internalization by automated microscopy.

<p>Cells were pre-incubated at 37 °C for 30 min in culture medium alone (untreated) or supplemented with 1 µg/ml cytochalasin D or solvent (DMSO). Cells were then transfered to 4 °C and incubated with bacteria (MOI = 10) for 30 min. At time zero, the plates were transferred to 37 °C and incubated for the indicated times. The cells were fixed, extracellular bacteria were labeled with anti-MOMP antibodies followed with Cy5-coupled secondary antibodies, and DNA was labeled with Hoechst. (A) Representative fields of the untreated control in the blue (first column), green (second column) and far-red (third column) channels with the merged pictures shown on the right. Prior to transfer to 37 °C (top) all bacteria (green) are extracellular (red), while after 10 min at 37 °C half of the bacteria are internalized (arrowheads). The scale bar represents 5 µm. (B) Kinetics of bacterial entry. Images were processed with the ICY software as described in the methods section. Each time point represents averages on more than 80 bacteria from 10 different fields. One experiment representative of three is shown.</p

Table_2_The Loss of Expression of a Single Type 3 Effector (CT622) Strongly Reduces Chlamydia trachomatis Infectivity and Growth.PDF

<p>Invasion of epithelial cells by the obligate intracellular bacterium Chlamydia trachomatis results in its enclosure inside a membrane-bound compartment termed an inclusion. The bacterium quickly begins manipulating interactions between host intracellular trafficking and the inclusion interface, diverging from the endocytic pathway and escaping lysosomal fusion. We have identified a previously uncharacterized protein, CT622, unique to the Chlamydiaceae, in the absence of which most bacteria failed to establish a successful infection. CT622 is abundant in the infectious form of the bacteria, in which it associates with CT635, a putative novel chaperone protein. We show that CT622 is translocated into the host cytoplasm via type three secretion throughout the developmental cycle of the bacteria. Two separate domains of roughly equal size have been identified within CT622 and a 1.9 Å crystal structure of the C-terminal domain has been determined. Genetic disruption of ct622 expression resulted in a strong bacterial growth defect, which was due to deficiencies in proliferation and in the generation of infectious bacteria. Our results converge to identify CT622 as a secreted protein that plays multiple and crucial roles in the initiation and support of the C. trachomatis growth cycle. They reveal that genetic disruption of a single effector can deeply affect bacterial fitness.</p

Table_4_The Loss of Expression of a Single Type 3 Effector (CT622) Strongly Reduces Chlamydia trachomatis Infectivity and Growth.PDF

<p>Invasion of epithelial cells by the obligate intracellular bacterium Chlamydia trachomatis results in its enclosure inside a membrane-bound compartment termed an inclusion. The bacterium quickly begins manipulating interactions between host intracellular trafficking and the inclusion interface, diverging from the endocytic pathway and escaping lysosomal fusion. We have identified a previously uncharacterized protein, CT622, unique to the Chlamydiaceae, in the absence of which most bacteria failed to establish a successful infection. CT622 is abundant in the infectious form of the bacteria, in which it associates with CT635, a putative novel chaperone protein. We show that CT622 is translocated into the host cytoplasm via type three secretion throughout the developmental cycle of the bacteria. Two separate domains of roughly equal size have been identified within CT622 and a 1.9 Å crystal structure of the C-terminal domain has been determined. Genetic disruption of ct622 expression resulted in a strong bacterial growth defect, which was due to deficiencies in proliferation and in the generation of infectious bacteria. Our results converge to identify CT622 as a secreted protein that plays multiple and crucial roles in the initiation and support of the C. trachomatis growth cycle. They reveal that genetic disruption of a single effector can deeply affect bacterial fitness.</p

Image_2_The Loss of Expression of a Single Type 3 Effector (CT622) Strongly Reduces Chlamydia trachomatis Infectivity and Growth.TIF

<p>Invasion of epithelial cells by the obligate intracellular bacterium Chlamydia trachomatis results in its enclosure inside a membrane-bound compartment termed an inclusion. The bacterium quickly begins manipulating interactions between host intracellular trafficking and the inclusion interface, diverging from the endocytic pathway and escaping lysosomal fusion. We have identified a previously uncharacterized protein, CT622, unique to the Chlamydiaceae, in the absence of which most bacteria failed to establish a successful infection. CT622 is abundant in the infectious form of the bacteria, in which it associates with CT635, a putative novel chaperone protein. We show that CT622 is translocated into the host cytoplasm via type three secretion throughout the developmental cycle of the bacteria. Two separate domains of roughly equal size have been identified within CT622 and a 1.9 Å crystal structure of the C-terminal domain has been determined. Genetic disruption of ct622 expression resulted in a strong bacterial growth defect, which was due to deficiencies in proliferation and in the generation of infectious bacteria. Our results converge to identify CT622 as a secreted protein that plays multiple and crucial roles in the initiation and support of the C. trachomatis growth cycle. They reveal that genetic disruption of a single effector can deeply affect bacterial fitness.</p

Table_3_The Loss of Expression of a Single Type 3 Effector (CT622) Strongly Reduces Chlamydia trachomatis Infectivity and Growth.PDF

<p>Invasion of epithelial cells by the obligate intracellular bacterium Chlamydia trachomatis results in its enclosure inside a membrane-bound compartment termed an inclusion. The bacterium quickly begins manipulating interactions between host intracellular trafficking and the inclusion interface, diverging from the endocytic pathway and escaping lysosomal fusion. We have identified a previously uncharacterized protein, CT622, unique to the Chlamydiaceae, in the absence of which most bacteria failed to establish a successful infection. CT622 is abundant in the infectious form of the bacteria, in which it associates with CT635, a putative novel chaperone protein. We show that CT622 is translocated into the host cytoplasm via type three secretion throughout the developmental cycle of the bacteria. Two separate domains of roughly equal size have been identified within CT622 and a 1.9 Å crystal structure of the C-terminal domain has been determined. Genetic disruption of ct622 expression resulted in a strong bacterial growth defect, which was due to deficiencies in proliferation and in the generation of infectious bacteria. Our results converge to identify CT622 as a secreted protein that plays multiple and crucial roles in the initiation and support of the C. trachomatis growth cycle. They reveal that genetic disruption of a single effector can deeply affect bacterial fitness.</p

Table_1_The Loss of Expression of a Single Type 3 Effector (CT622) Strongly Reduces Chlamydia trachomatis Infectivity and Growth.PDF

<p>Invasion of epithelial cells by the obligate intracellular bacterium Chlamydia trachomatis results in its enclosure inside a membrane-bound compartment termed an inclusion. The bacterium quickly begins manipulating interactions between host intracellular trafficking and the inclusion interface, diverging from the endocytic pathway and escaping lysosomal fusion. We have identified a previously uncharacterized protein, CT622, unique to the Chlamydiaceae, in the absence of which most bacteria failed to establish a successful infection. CT622 is abundant in the infectious form of the bacteria, in which it associates with CT635, a putative novel chaperone protein. We show that CT622 is translocated into the host cytoplasm via type three secretion throughout the developmental cycle of the bacteria. Two separate domains of roughly equal size have been identified within CT622 and a 1.9 Å crystal structure of the C-terminal domain has been determined. Genetic disruption of ct622 expression resulted in a strong bacterial growth defect, which was due to deficiencies in proliferation and in the generation of infectious bacteria. Our results converge to identify CT622 as a secreted protein that plays multiple and crucial roles in the initiation and support of the C. trachomatis growth cycle. They reveal that genetic disruption of a single effector can deeply affect bacterial fitness.</p

Image_1_The Loss of Expression of a Single Type 3 Effector (CT622) Strongly Reduces Chlamydia trachomatis Infectivity and Growth.TIF

<p>Invasion of epithelial cells by the obligate intracellular bacterium Chlamydia trachomatis results in its enclosure inside a membrane-bound compartment termed an inclusion. The bacterium quickly begins manipulating interactions between host intracellular trafficking and the inclusion interface, diverging from the endocytic pathway and escaping lysosomal fusion. We have identified a previously uncharacterized protein, CT622, unique to the Chlamydiaceae, in the absence of which most bacteria failed to establish a successful infection. CT622 is abundant in the infectious form of the bacteria, in which it associates with CT635, a putative novel chaperone protein. We show that CT622 is translocated into the host cytoplasm via type three secretion throughout the developmental cycle of the bacteria. Two separate domains of roughly equal size have been identified within CT622 and a 1.9 Å crystal structure of the C-terminal domain has been determined. Genetic disruption of ct622 expression resulted in a strong bacterial growth defect, which was due to deficiencies in proliferation and in the generation of infectious bacteria. Our results converge to identify CT622 as a secreted protein that plays multiple and crucial roles in the initiation and support of the C. trachomatis growth cycle. They reveal that genetic disruption of a single effector can deeply affect bacterial fitness.</p