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

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

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

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

MAP1B is highly expressed in kidney podocytes.

<p>Immunofluorescence staining of kidney sections derived from adult human and rat kidney. WT1 served as a marker for podocyte nuclei. Nephrin and synaptopodin served as foot process markers. DAPI was used for staining nuclei. MAP1B is expressed highly specific in podocytes (scale for overview: 50 μm; higher magnification: 25 μm) (A,C). Expression of MAP1B during glomerulogenesis in newborn rat kidney. Glomerular differentiation advances from cortex to medulla (upper right: immature; lower left: mature) (scale for overview: 50 μm; higher magnification: 25 μm) (B). Transmission electron microscopy of kidney sections. Immunolabeling of MAP1B confirms an enrichment in areas with a dense MT cytoskeleton in primary processes and the cell body (primary processes (PP), foot processes (arrowheads), Nucleus (Nu)), glomerular basement membrane (GBM); scale: 200 nm) (D).</p

Constitutive MAP1B Knock out (KO) mice do not exhibit a glomerular phenotype.

<p>Immunofluorescence microscopy of kidney sections from constitutive MAP1B KO mice versus control littermates. Autofluorescence of PFA treated tissue is used to display glomerular anatomy. Total depletion of MAP1B HC in podocytes of KO animals as well as the specificity of our antibody for MAP1B expression is confirmed (scale: 50 μm) (A). We did not observe alterations of glomerular morphology using light microscopy of Periodic-acid Schiff (PAS) stained kidney sections of MAP1B KO animals (scale: 50 μm) (B). MAP1B podocyte foot process morphology examination using transmission electron microscopy of MAP1B KO podocytes (Podocyte (Pd), capillary (C), Bowman’s urinary space (B), podocyte foot processes (arrow heads), scale: 10 μm) (C). Proteinuria in the physiological range in both KO and control littermate by measuring urinary albumin/creatinin ratios (n = 7 for KO and control animals, p = 0,205) (D). 9 week old MAP1B KO animals showed a significantly reduced body weight compared to control littermates (n = 7 for KO and control animals, p = 0,010) (E).</p

Podocyte-Specific Deletion of Murine CXADR Does Not Impair Podocyte Development, Function or Stress Response

<div><p>The coxsackie- and adenovirus receptor (CXADR) is a member of the immunoglobulin protein superfamily, present in various epithelial cells including glomerular epithelial cells. Beside its known function as a virus receptor, it also constitutes an integral part of cell-junctions. Previous studies in the zebrafish pronephros postulated a potential role of CXADR for the terminal differentiation of glomerular podocytes and correct patterning of the elaborated foot process architecture. However, due to early embryonic lethality of constitutive <i>Cxadr</i> knockout mice, mammalian data on kidney epithelial cells have been lacking. Interestingly, <i>Cxadr</i> is robustly expressed during podocyte development and in adulthood in response to glomerular injury. We therefore used a conditional transgenic approach to elucidate the function of <i>Cxadr</i> for podocyte development and stress response. Surprisingly, we could not discern a developmental phenotype in podocyte specific <i>Cxadr</i> knock-out mice. In addition, despite a significant up regulation of CXADR during toxic, genetic and immunologic podocyte injury, we could not detect any impact of <i>Cxadr</i> on these injury models. Thus these data indicate that in contrast to lower vertebrate models, mammalian podocytes have acquired molecular programs to compensate for the loss of <i>Cxadr</i>.</p></div

Nephrotoxic serum (NTS) enhances podocyte specific CXADR expression, but lack of CXADR does not influence the course of NTS induced disease.

<p><b>(A-C)</b> On both d4 and d5 after NTS injection CXADR was increased in glomerular podocytes as shown in co-labeling experiments with NEPHRIN (control: white arrows—parietal epithelial cells, NTS: arrow heads—podocytes). As can be easily depicted from the NEPHRIN specific panel <b>(A”, B”, C”)</b> NEPHRIN abundance is greatly reduced during the course of the disease, underlining the severity of the chosen stress model. <b>(D)</b> Schematic of the injection scheme and follow-up using urine collections. <b>(E&F)</b> Western blot using decapsulated glomerular lysates was used to quantify CXADR expression which was increased two fold 5 days after NTS injection. <b>(G&H)</b> As shown with immunofluorescence stainings CXADR induction was absent in podocyte specific <i>Cxadr</i> knock-out animals. <b>(I)</b> Proteinuria developed similarly in wild-type and knock-out animals showing no functional differences.</p

Glomerular function, histology and ultrastructure are maintained in podocyte specific <i>Cxadr</i> knock-out animals.

<p><b>(A)</b> Functional assessment only showed a slightly elevated albuminuria in <i>Cxadr</i> deficient animals at P2 which was lost during further maturation of the kidney. <b>(B&C)</b> Histology of glomeruli was normal in podocyte deficient <i>Cxadr</i> animals as was <b>(D&E)</b> ultrastructural assessment by SEM <b>(D–D”</b>, <b>E–E”)</b> and TEM <b>(D”‘</b>, <b>E”‘)</b>.</p