Golgi Fragmentation and Sphingomyelin Transport to <i>Chlamydia trachomatis</i> during Penicillin-Induced Persistence Do Not Depend on the Cytosolic Presence of the Chlamydial Protease CPAF

<div><p><i>Chlamydia</i> grows inside a cytosolic vacuole (the inclusion) that is supplied with nutrients by the host through vesicular and non-vesicular transport. It is unclear in many respects how <i>Chlamydia</i> organizes this transport. One model posits that the <i>Chlamydia</i>-induced fragmentation of the Golgi-apparatus is required for normal transport processes to the inclusion and for chlamydial development, and the chlamydial protease CPAF has been controversially implicated in Golgi-fragmentation. We here use a model of penicillin-induced persistence of infection with <i>Chlamydia trachomatis</i> to test this link. Under penicillin-treatment the inclusion grew in size for the first 24 h but after that growth was severely reduced. Penicillin did not reduce the number of infected cells with fragmented Golgi-apparatus, and normal Golgi-fragmentation was found in a CPAF-deficient mutant. Surprisingly, sphingomyelin transport into the inclusion and into the bacteria, as measured by fluorescence accumulation upon addition of labelled ceramide, was not reduced during penicillin-treatment. Thus, both Golgi-fragmentation and transport of sphingomyelin to <i>C. trachomatis</i> still occurred in this model of persistence. The portion of cells in which CPAF was detected in the cytosol, either by immunofluorescence or by immune-electron microscopy, was drastically reduced in cells cultured in the presence of penicillin. These data argue against an essential role of cytosolic CPAF for Golgi-fragmentation or for sphingomyelin transport in chlamydial infection.</p></div

Golgi-fragmentation during acute and persistent infection.

<p>(<b>A</b>) YFP-Golgi-HeLa cells were infected with <i>C. trachomatis</i> L2 with or without addition of 100 U/ml PenG, RST17 (CPAF−) and the corresponding control strain RST5 (CPAF+) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103220#pone.0103220-Snavely1" target="_blank">[14]</a>, incubated for the indicated times and processed for immunofluorescence. Blue: Hoechst DNA-stain, yellow: GA, pink: <i>Chlamydia</i>. All images were taken at 40-fold magnification. Arrows point to uninfected cells displaying a normal, not fragmented GA. (<b>B</b>) Quantification of the portion of infected cells showing a fragmented GA. All infected cells as well as all infected cells with fragmented GA were counted and the ratio of fragmentation-positive cells was calculated (number of infected cells counted: 499, 629, 478, 517, 397, 475, 380, 522 from left to right). Shown are means of 3 independent experiments ± SEM.</p

Inclusion size during acute and persistent chlamydial infection and in the presence of peptide inhibitors.

<p>HeLa, oviduct epithelial cells and MEFs were infected with <i>C. trachomatis</i> L2 at an MOI of 1 with or without addition of 100 U/ml PenG. At 24 or 48 h post infection, cells were fixed, processed for immunofluorescence and inclusion size was measured. Shown are the means of 3 independent experiments ± SEM, 15 view-fields per sample were evaluated using the AxioVision Software (number of inclusions measured = 244, 219, 299, 295 for HeLa; 201, 209, 164, 160 for oviduct and 135, 212, 181, 153 for MEFs). <i>C. trachomatis</i> is also inhibited by lower concentrations of PenG <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103220#pone.0103220-Dumoux1" target="_blank">[42]</a> but this concentration has been used before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103220#pone.0103220-Skilton1" target="_blank">[21]</a>.</p

CPAF expression during acute and persistent infection.

<p>HeLa, oviduct epithelial cells and MEFs were infected with <i>C. trachomatis</i> L2 at an MOI of 1 with or with addition of 100 U/ml PenG. Shown are representative Western Blots of uninfected (Control), acutely infected (Infection) or persistently (PenG) infected cells. Whole cell lysates were prepared with either RIPA (<b>A</b>) or UREA (<b>B</b>) extraction buffer (see methods) at 24, 40 and 48 h p.i. and 10 µg protein was loaded onto each lane. CPAFc: active CPAF; <i>c</i>Hsp60: chlamydial Hsp60 protein; Actin: used as loading control.</p

CPAF localisation during acute and persistent infection.

<p>(<b>A</b>) HeLa cells were infected with <i>C. trachomatis</i> L2 at an MOI of 1, with or without addition of 100 U/ml PenG, incubated for 24 or 48 h and processed for immunofluorescence. Blue: Hoechst DNA-stain, red: CPAF, green: <i>Chlamydia</i>. All images were taken at 100-fold magnification. For quantification of cell numbers with Golgi-fragmentation and detectable cytosolic CPAF see Fig. 5C. (<b>B</b>) Immunogold labelling of CPAF in HeLa cells infected for 30 h with <i>C. trachomatis</i> L2 at an MOI of 1 without (upper row) or with addition of 100 U/ml PenG (lower row). Black arrows indicate CPAF inside the inclusions, white or grey arrowheads indicate free CPAF in the cytoplasm. For quantification of the staining see Fig. 5D. (<b>C</b>) Relative numbers of infected cells that show cytosolic CPAF as well as Golgi fragmentation in immunofluorescence. YFP-Golgi-HeLa cells were infected for 24 h without or with addition of 100 U/ml PenG, fixed and stained for CPAF and DNA as in (<b>A</b>). All infected cells and all infected cells with fragmented GA and cytosolic CPAF were counted, and the relative number of infected cells with cytosolic CPAF as the share of total cells with fragmented GA was calculated (number of infected cells counted: 434, 318). Shown are means of 4 independent experiments ± SEM. (<b>D</b>) Quantification of immunogold-labelled CPAF as shown in (<b>B</b>). Regions of inclusions or cytoplasm were outlined using ITEM software and the number of gold particles was counted within these regions. Uninfected but stained cells were used as negative control.</p

Precisely adjusted heterogeneous RovA expression is crucial for virulence.

<p>(<b><i>A</i></b>) Fluorescence microscopy of cryosections allows detection of bacteria by expression of the constitutive P_<i>tet</i>-<i>mCherry</i> reporter (mCherry) and revealed heterogeneous expression of the P_<i>rovA</i>-<i>egfp</i>_<i>LVA</i> reporter (eGFP_LVA) in the caecum 3 days post infection. (<b><i>B</i></b>) Quantification of eGFP_LVA-positive cells of the wild-type (YPIII) and the isogenic mutant (YP287) expressing the more stable RovA_{P98S/SG127/128IK/G116A} variant. The mean percentage of RovA-expressing bacteria was significantly higher in the mutant (***, <i>P</i> < 0.001; two-tailed Student’s <i>t</i>-test; <i>n</i> = 40 for each genotype). (<b><i>C</i></b>) Survival of mice infected with <i>Y</i>. <i>pseudotuberculosis</i> revealed reduced virulence of mutants lacking RovA or producing more stable RovA derivatives (**, <i>P</i> < 0.01, ***, <i>P</i> < 0.001; log-rank (Mantel-Cox) test; <i>n</i> = 17 for each genotype). (<b><i>D</i></b>) Infection of mice with 2 x 10⁸ bacteria either deficient in RovA or producing a more stable RovA variant led to reduced colonization of MLNs 3 days post infection (**, <i>P</i> < 0.01; ***, <i>P</i> < 0.001; two-tailed Mann-Whitney test; <i>n</i> = 10 for each genotype). (<b><i>E</i></b>) Model of bistable <i>rovA</i> expression during <i>Y</i>. <i>pseudotuberculosis</i> infection. Upon uptake from the environment, the bacteria express RovA and RovA-induced invasin, which mediates internalization into M-cells. After transcytosis into the subepithelial lymphatic tissues (Peyer’s Patches), most bacteria have switched off <i>rovA</i> expression, thereby limiting their recognition by innate immune cells, but a small RovA ON subpopulation is still found within tissue lesions. During on-going infections heterogeneity could be advantageous for persistence in the caecum as well as reinfection and spreading to other hosts when bacteria are expelled into the intestinal lumen after tissue damage.</p

Identification of a temperature-responsive bistable switch.

<p>(<b><i>A</i></b>) The temperature-responsive <i>Yersinia</i> virulence regulator RovA is autoregulated through positive and negative feedback loops. At 25°C RovA is active and binds cooperatively to a high-affinity site upstream of P2 and activates <i>rovA</i> and <i>invA</i> transcription. When the RovA amount has reached a certain threshold, RovA binds to a low affinity site downstream of P1 to prevent uncontrolled <i>rovA</i> induction. An upshift to 37°C induces a reversible conformational change in RovA that leads to a strong reduction of its DNA-binding capacity and renders this regulator susceptible to proteolysis by the Lon protease. <i>rovA</i> transcription is further regulated by the nutrient-responsive repressor RovM. (<b><i>B</i></b>) <i>Y</i>. <i>pseudotuberculosis</i> wild-type carrying a P_<i>rovA</i>-<i>egfp</i>_<i>LVA</i> fusion was grown at different temperatures and analysed by fluorescence microscopy, and (<b><i>C</i></b>), flow cytometry (one representative replicate; 10⁵ cells). (<b><i>D</i></b>) The percentage of P_<i>rovA</i>-<i>egfp</i>_<i>LVA</i>-expressing wild-type cells quantified by flow cytometry (mean ± SEM; <i>n</i> = 3 for each temperature; 10⁵ cells per replicate), eGFP_LVA-positive cells (ON) are shown in green. The response of P_<i>rovA</i>-<i>egfp</i>_<i>LVA</i> to temperature corresponds to the average RovA level as determined by western blot. Relative RovA amounts were quantified and normalised to the highest temperature for which a homogenous RovA ON population was observed (mean ± SEM; <i>n</i> = 3 for each temperature). (<b><i>E</i></b>), Live cell imaging of <i>Y</i>. <i>pseudotuberculosis</i> expressing P_<i>rovA</i>-<i>egfp</i>_<i>LVA</i> at 32°C. Time series of individual bacteria starting from the OFF state demonstrates switching to the ON and back to the OFF state; representative overlays of eGFP_LVA and bright field images at different time points are shown (also see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006091#ppat.1006091.s013" target="_blank"><b>S</b>1 Video</a>).</p

Thermal shift experiments reveal hysteresis of the temperature-responsive bistable switch.

<p><i>Y</i>. <i>pseudotuberculosis</i> expressing P_<i>rovA</i>-<i>egfp</i>_<i>LVA</i> was grown to a continuous culture in a chemostat at 25°C, shifted for 8 h to 37°C and back to 25°C for 18 h. Bacteria were analyzed by (<b><i>A</i></b>) flow cytometry (mean ± SEM; <i>n</i> = 3 for each temperature; 10⁵ cells per replicate), or (<b><i>B</i></b>) by western blot. Relative RovA amounts were quantified using ImageJ (mean ± SEM; <i>n</i> = 3 for each temperature; c: a protein band unspecifically recognized by the antiserum served as loading control).</p