SGM Journals
PATHOGENS AND PATHOGENICITY

Host α-adducin is redistributed and localized to the inclusion membrane in Chlamydia- and Chlamydophila-infected cells

Hencelyn G. Chu, Sara K. Weeks, Diana M. Gilligan, Daniel D. Rockey

  • 1Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331-4804, USA
  • 2Department of Microbiology, Oregon State University, Corvallis, OR 97331-3804, USA
  • 3Puget Sound Blood Center, University of Washington, Seattle, WA 98104, USA
  • Correspondence
    Daniel D. Rockey
    rockeyd{at}orst.edu

    • Microbiology 2008; 154(12):3848–3855 · https://doi.org/10.1099/mic.0.2008/020941-0

    View at publisher PubMed

    Abstract

    A large-scale analysis of proteins involved in host-cell signalling pathways was performed using chlamydia-infected murine cells in order to identify host proteins that are differentially activated or localized following infection. Two proteins whose distribution was altered in Chlamydia trachomatis-infected cells relative to mock-infected cells were the actin-binding protein adducin and the regulatory kinase Raf-1. Immunoblot analysis with antibodies to both phosphorylated and non-phosphorylated forms of these proteins demonstrated that the abundance of each protein was markedly reduced in the cytosolic fraction of C. trachomatis- and Chlamydophila caviae-infected cells, but the total cellular protein abundance remained unaffected by infection. Fluorescence microscopy of chlamydia-infected cells using anti-α-adducin antibodies demonstrated labelling at or near the chlamydial inclusion membrane. Treatment of infected cells with nocodazole or cytochalasin D did not affect α-adducin that was localized to the margins of the inclusion. The demonstration of α-adducin and Raf-1 redistribution within cells infected by different chlamydiae provides novel opportunities for analysis of host–pathogen interactions in this system.

    • ↵†Present address: Department of Pathology and Laboratory Medicine, University of California, Irvine, CA 92697-4800, USA.

    • A supplementary table comparing the abundance of phosphorylated proteins in infected vs mock-infected samples or lysates is available with the online version of this paper.

    Edited by: T. P. Hatch

    INTRODUCTION

    The obligately intracellular chlamydiae grow and develop within a unique and dynamic intravacuolar environment (the inclusion; Valdivia, 2008), and chlamydial development requires modulation of different aspects of host-cell biology to the benefit of the pathogen. While the chlamydiae are known to affect many host cellular processes, including apoptosis (Miyairi & Byrne, 2006), vesicular morphogenesis (Beatty, 2006; Carabeo et al., 2003; Grieshaber et al., 2003; Rzomp et al., 2003; Scidmore & Hackstadt, 2001; Scidmore et al., 1996a, b, 2003), cytokinesis (Greene & Zhong, 2003), lipid metabolism (Hackstadt et al., 1995), and host-cell immune responses (Lad et al., 2005), many aspects of intracellular host–pathogen interactions remain unclear. Chlamydial proteins that are involved in such alterations include proteins secreted into the host cytosol and those localized to the inclusion membrane (Rockey & Rosquist, 1994; Zhong et al., 2001).

    In order to explore possible unique host-cell responses that are altered during infection, we screened chlamydia-infected and mock-infected cells with an array of antibodies specific to phosphorylated forms of host-cell proteins involved in major eukaryotic signalling pathways. These experiments demonstrated that the host proteins α-adducin and Raf-1 are redistributed during chlamydial growth within infected cells. Immunofluorescence microscopy demonstrated that α-adducin is localized to the margins of chlamydial inclusions, in a microtubule- and microfilament-independent process.

    METHODS

    Cell culture and antibodies.

    McCoy and HeLa cells were grown in Minimum Essential Medium (Gibco) supplemented with 10 % fetal bovine serum and 0.5 μg gentamicin ml−1 (MEM-10). Protease and phosphatase inhibitors were purchased from Roche. Anti-phospho-α-adducinSer726, phospho-Raf-1Ser259, c-Raf1 and phospho-PKBSer473 (protein kinase B) were obtained from Upstate Biotechnology. Antisera to recombinant human erythrocyte α-adducin were used to examine the abundance of total α-adducin (Gilligan et al., 2002; Joshi et al., 1991). Monoclonal antibody directed at chlamydial Hsp60 was used to label and visualize chlamydial developmental forms (Yuan et al., 1992). Antibodies directed at host 14-3-3β were obtained from Dr Marci Scidmore (Scidmore & Hackstadt, 2001). Secondary antibodies conjugated to fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) were purchased from Southern Biotechnologies. Secondary antibodies conjugated to IRdye 800 or IRdye 700 were purchased from Rockland Immunochemicals. The DNA-specific fluorescent label 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Vector Laboratories) was used at 2 μg ml−1 in mounting medium to label host and chlamydial DNA in immunofluorescence experiments. Actin filaments were labelled by FITC-conjugated phalloidin (100 ng ml−1; Sigma) and microtubules were labelled by monoclonal antibodies to α-tubulin (Sigma).

    Chlamydia strains and cell culture.

    Chlamydia trachomatis strains J/UW-36, J(s)/893, D/UW-3, LGV 434, Chlamydophila caviae GPIC and Chlamydophila pneumoniae TWAR were used to separately infect host cells at a m.o.i. of 3 (for the phosphoprotein assays) or 0.5 (for fluorescence microscopy). Chlamydiae were diluted in SPG (0.25 M sucrose, 10 mM sodium phosphate, 5 mM l-glutamic acid) prior to inoculation onto cells. Mock-infected cells were incubated with SPG only. Infected and mock-infected cells were centrifuged at 2000 g for 1 h, followed by replacement of inocula with MEM-10 plus cycloheximide (1.0 μg ml−1). Cells were incubated at 37 °C in 5 % CO2. Unless indicated, cells were incubated 24 h prior to lysis or fixation.

    Kinetworks phosphorylation site screen (KPSS) kinome analysis.

    Protein samples prepared from McCoy cells separately infected with two C. trachomatis strains [J/UW-36 and J(s)/893, both of serovar J] and mock-infected cell lysates were submitted for the KPSS-1.3 phosphoprotein panel analysis by Kinexus Bioinformatics. Samples were prepared according to instructions from the company. Briefly, infected or mock-infected cells were homogenized in lysis buffer [20 mM Tris/HCl (pH 7.0), 2 mM EGTA, 5 mM EDTA, 30 mM sodium fluoride, 40 mM β-glycerophosphate (pH 7.2), 2 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride and 1 % Triton X-100] at 4 °C, and ultracentrifuged at 90 000 g. Total protein concentrations in crude cell lysates were determined using the Bradford assay (Bio-Rad). Approximately 300 μg of each sample were resolved by SDS-PAGE and transferred onto a nitrocellulose membrane. Cocktails of antibody were applied to the membrane through a 20-lane Immunetics multiblotter. The immunoblots were then developed using a chemiluminescent reagent (ECL Plus, GE Healthcare) and signals were detected with a Fluor-S-MultiImager (Bio-Rad). Raw immunoblot data (not shown) were quantified with Quantity One Software (Bio-Rad). Quantitative comparisons among different test groups for each phosphoprotein were conducted by averaging the replicates for each tested strain, and dividing that value by the value for the control strain. Thus, values >1 indicate increased abundance of phosphorylated protein relative to the control, and values <1 indicate decreased abundance of phosphorylated protein relative to the control. For a result to be considered significant, a change of phosphoprotein quantity in a sample relative to mock-infected cells must be greater than 25 %, as it is suggested by the company that any change less than 25 % may be attributable to experimental variation.

    Immunoblot detection of individual host proteins.

    Infected and mock-infected cells were incubated at 37 °C in 5 % CO2. Protein samples used were prepared either by extraction of total cell proteins or by fractionation with Triton X-100 (Gilligan et al., 2002). For the Triton fractionation, infected and mock-infected cells were homogenized in lysis buffer, sonicated, centrifuged (16 000 g) and the Triton-soluble (TS) supernatant and Triton-insoluble pellet fractions were collected. Protein content in samples was determined by Coomassie blue staining of control gels and/or through the Bradford assay. In some cases, the relative abundance of phospho-PKBSer473 was used as a control for total protein in mock-infected and C. trachomatis-infected cell protein lysates. Samples were normalized to equalize the total protein per well, subjected to 12 % SDS-PAGE and transferred to nitrocellulose. The immunoblots were then blocked with Odyssey (Licor) blocking buffer and probed with antibodies to phospho-α-adducinSer726, phospho-Raf-1Ser259, recombinant full-length α-adducin, c-Raf1, phospho-PKBSer473 or C. trachomatis Hsp60. The blots were then probed with goat anti-rabbit or goat anti-mouse antibodies conjugated to IRdye 700 or IRdye 800, respectively. After a final wash, the Odyssey Infrared Imager (160 μm resolution, 0 mm offset) was used to scan the membranes. Images were processed and assembled in Adobe Photoshop (Adobe Systems).

    Immunofluorescence.

    Infected cell monolayers were fixed for 1 h in 4 % paraformaldehyde at designated times post-infection (p.i.). Fixed cells were then probed with primary antibodies to chlamydial Hsp60 and either antisera against phospho-α-adducinSer726 or antisera against recombinant α-adducin that were diluted in 2 % BSA in PBS (FA block). Anti-α-adducin antibodies were incubated on cells overnight at 4 °C but all other antibodies were incubated on blots for 1 h at room temperature. Following washing of monolayers with PBS, the cells were incubated for 1 h at room temperature with the appropriate secondary antibodies (Southern Biotechnology Associates). All coverslips were examined under oil immersion on a Leica DMLB microscope. Images were captured with a Spot digital camera and software (Diagnostics Instruments), and assembled using Adobe Photoshop.

    Nocodazole or cytochalasin D treatment of cells.

    Infected and mock-infected cells cultured for 24 h p.i. were treated with 10 μM nocodazole, 10 μM cytochalasin D, or 0.1 % DMSO for 2 h at 37 °C. Cells were then fixed with 4 % paraformaldehyde as described above, probed with monoclonal antibodies to α-tubulin or FITC-conjugated phalloidin and viewed by fluorescence microscopy.

    RESULTS

    Kinome analysis of C. trachomatis-infected cells

    A multi-phosphoprotein analysis was used to evaluate common eukaryotic phosphoproteins that are differentially phosphorylated in C. trachomatis-infected vs mock-infected cells. Each of these samples was screened for the phosphorylation status of 31 host phosphoproteins (Table 1⇓). Several phosphoproteins displayed changes in abundance in the extracts from infected versus uninfected cells. These included α-adducin, γ-adducin, cAMP response element binding protein (CREB), extracellular regulated kinase 1 and 2 (ERK 1/2), c-Jun oncoprotein, N-methyl-d-aspartate glutamate receptor subunit (NMDA) NR1, p38 α-MAP kinase, MAP kinase 3/6, Raf-1, ribosomal S6 kinase 1, and signal transducer and activator of transcription (STAT) 1 and 3. Because the interactions of some of these proteins with the chlamydial infectious process have been discussed by others (Lad et al., 2005; Su et al., 2004), we focused primarily on two relatively uncharacterized proteins, α-adducin and Raf-1.

    Table 1.

    Phosphoprotein signal strength in infected cells relative to mock-infected cells, for both non-fusogenic [J(s)/893] and wild-type (J/UW-36) C. trachomatis strains

    The two strains used in these studies were a wild-type isolate (J/UW-36) and J(s)/893, a serotype-matched, IncA-negative, non-fusogenic isolate (Suchland et al., 2000), which have differences in several aspects of host–microbe interactions relative to wild-type strains (Geisler et al., 2001; Rockey et al., 2002; Xia et al., 2005). The observed reductions in phosphorylated α-adducin and Raf-1 were common to lysates of both wild-type and IncA-negative strains (Table 1⇑). The phosphorylation assay also identified candidate proteins that may be differentially phosphorylated in cells infected with the wild-type versus non-fusogenic IncA-negative strain (i.e. CDK1, CREB, MEK1/2, MEK6; Table 1⇑; see also Supplementary Table S1, available with the online version of this paper). These differences were not pursued because of variation within individual samples in the phosphorylation assay.

    Immunoblotting with specific anti-adducin and anti-Raf-1 antibodies

    Immunoblotting was used to confirm that both phospho-α-adducin (Fig. 1⇓A) and phospho-Raf-1 (Fig. 1⇓C) were significantly reduced in the Triton-soluble (TS) fractions of infected cells relative to mock-infected cells. Immunoblots probed with antisera to recombinant α-adducin (Fig. 1⇓B) and Raf-1 (Fig. 1⇓D) demonstrated that the reduction in band intensity for each phosphorylated protein resulted from a reduction in the abundance of each protein in the TS fractions, and not from differential phosphorylation. The abundance of total adducin and Raf-1 within the infected cells, as measured by immunoblotting of total protein lysates from these cells, was unaffected by the chlamydial infection (Figs 1⇓ and 2⇓). While we were able to demonstrate α-adducin and Raf-1 in the Triton-insoluble fraction of infected cells (not shown), the recovery of these proteins from the Triton-insoluble fractions of the cells or the pellets was inconsistent and not quantitative. Collectively, these immunoblotting results demonstrated that α-adducin and Raf-1 were redistributed within C. trachomatis-infected cells.

    Figure image not available in archive
    Fig. 1.

    The phosphoproteins α-adducin and Raf-1 are depleted in the TS fraction of infected cells. For blots in rows A–E, lane representations are as follows: 1, whole-cell protein lysate of mock-infected McCoy cells; 2, whole-cell protein lysate of C. trachomatis J/UW-36-infected McCoy cells; 3, TS fraction of mock-infected McCoy cells; 4, TS fraction of C. trachomatis J/UW-36-infected McCoy cells. Row A was probed with anti-phospho-α-adducin, row B with anti-recombinant-α-adducin, row C with anti-phospho-Raf-1, row D with anti-c-Raf-1, and row E with anti-phospho-PKB/Akt.

    Figure image not available in archive
    Fig. 2.

    α-Adducin abundance is reduced in the TS fractions of cells infected with other chlamydial strains. Blots were probed with anti-phospho-α-adducin. Lanes: 1, total protein lysate of mock-infected McCoy cells; 2, total protein lysates of infected cells; 3, TS fraction of mock-infected McCoy cells; 4, TS fraction of infected cells. The strains tested were C. trachomatis D/UW-3 (D), C. trachomatis LGV-434 (L2) and Cp. caviae GPIC (GPIC).

    Protein lysates obtained from McCoy cells infected with different strains of C. trachomatis and with Cp. caviae GPIC were also probed with antibodies to phospho-α-adducin (Fig. 2⇑) and total α-adducin (Fig. 1⇑B). These immunoblots demonstrated that the altered abundance of α-adducin was common to infection by both Chlamydia and Chlamydophila strains. Additionally, blots of lysates of chlamydiae grown in cells of human origin (HeLa) demonstrated that the differences between infected and uninfected cells were not unique to the murine McCoy cell line (not shown).

    The different isoforms of adducin function as heterodimers or heteromultimers and the α and γ isoforms are present within epithelial cells (Gilligan et al., 1999; Matsuoka et al., 1996). While this work focused on the α isoform of the protein, the original kinome screen also demonstrated that phosphorylated γ-adducin was reduced in the TS fraction of infected cells (Table 1⇑).

    Temporal analysis of α-adducin distribution

    Two experiments were conducted to evaluate whether chlamydial development was required for the reduction in α-adducin in the TS fraction of infected cells. We first analysed the abundance of phospho-α-adducin in the TS fractions of infected and mock-infected McCoy cells as a function of incubation time. All samples were equalized for total protein prior to electrophoresis and immunoblotting. The depletion of phospho-α-adducin in the TS fractions of infected cells was observed first at 12 h p.i. and was increasingly apparent at 24 h p.i. (Fig. 3A⇓). No change in phospho-α-adducin abundance was observed in TS fractions of parallel mock-infected cells throughout the time-course assay.

    Figure image not available in archive
    Fig. 3.

    The subcellular localization of α-adducin is altered at the mid-time points post-C. trachomatis J/UW-36 infection (A) and occurs as a result of infection, not attachment (B). In (A), the TS fractions of mock-infected McCoy cells are labelled ‘M’, and the TS fractions of infected cells are labelled ‘I’. For each time point represented, both M and I samples were probed with anti-phospho-α-adducin. For (B), lane designations for both the tetracycline-treated (+ Tet) and untreated (No Tet) samples are as follows: 1, mock-infected McCoy whole-cell protein lysate; 2, C. trachomatis J/UW-36 infected whole-cell protein lysate; 3, TS fractions of mock-infected cells; 4, TS fractions of C. trachomatis J/UW-36-infected cells. Both immunoblots in (B) are probed with anti-recombinant-α-adducin (mol. mass 100 kDa).

    The role of chlamydial development in the observed changes in α-adducin localization was also examined by culturing chlamydia-infected cells in medium containing tetracycline, which effectively blocks chlamydial development and growth. Total α-adducin (Fig. 3B⇑) or phospho-α-adducin (not shown) in the TS fractions was not reduced in the tetracycline-treated infected cells. However, in parallel monolayers of infected cells not treated with tetracycline, α-adducin was depleted as expected. Collectively, these data demonstrate that chlamydial growth was required for the observed alteration in α-adducin localization within infected cells.

    It was possible that α-adducin was digested by chlamydial or host proteinases during culture or during the extraction procedure. To test the latter possibility, TS fractions from infected and uninfected cells were mixed and incubated on ice for time periods between 0 and 1 h post-extraction. Immunoblots were performed on these mixed lysates and there was no difference in α-adducin levels between these mixtures and control, uninfected lysates. While the possibility remains that α-adducin is degraded in the soluble fraction during chlamydial growth, immunoblots of dilutions of total protein lysates from cycloheximide-treated, chlamydia-infected cells did not identify differences in total α-adducin abundance within cells (not shown). The possibility that a fraction of α-adducin is degraded within cells following chlamydial infection remains to be formally examined.

    Microscopic analysis of phospho-α-adducin in chlamydia-infected cells

    The cellular localization of phospho-α-adducin was explored using immunofluorescence microscopy of C. trachomatis and Cp. pneumoniae-infected McCoy cells fixed 24 h p.i. In uninfected cells, phospho-α-adducin was observed dispersed throughout the cytoplasm with no specific pattern of accumulation (Fig. 4A⇓). In contrast, phospho-α-adducin was observed in close proximity to the chlamydial inclusion membrane of cells infected with either species (Fig. 4A, B⇓). Labelling with anti-phospho-α-adducin antibodies co-localized with antibodies to chlamydial inclusion membrane protein CT223p (Fig. 5D, E⇓) and with host 14-3-3β (Fig. 5A, B, C⇓), a protein known to localize to the inclusion membrane within infected cells (Scidmore & Hackstadt, 2001). These data demonstrate that α-adducin localizes to the margin of the chlamydial inclusion within cells infected by different chlamydial species.

    Figure image not available in archive
    Fig. 4.

    Phosphorylated α-adducin (red) accumulates around the chlamydial inclusion in McCoy cells infected with C. trachomatis J/UW-36 (A) and Cp. pneumoniae TWAR (B) fixed at 28 h p.i. Scale bar, 10 μm (both images). (C–F) The association of phospho-α-adducin with chlamydial inclusions is independent of intact actin or microtubules. C. trachomatis J/UW-36-infected cells at 24 h p.i. were treated with DMSO as a solvent control (C and E), 30 μM nocodazole (D) or 10 μM cytochalasin D (F), for 3 h prior to fixation and staining with FITC-conjugated α-tubulin (green) (C and D) and phospho-α-adducin (red), or FITC-phalloidin (green; E and F) and phospho-α-adducin. DNA is labelled with DAPI (blue) in each image. The asterisks (*) in (C–F) indicate chlamydial inclusions and the arrowhead in panels (C and F) shows an uninfected cell. Scale bar, 10 μm (all four images C–F).

    Figure image not available in archive
    Fig. 5.

    Double labelling of infected HeLa cells to demonstrate α-adducin co-localization with chlamydial and host proteins at the inclusion membrane. (A–C) Adducin co-localizes with 14-3-3β at the surface of inclusions formed by C. trachomatis L2-434. These panels are split images of the same cells labelled by antibodies against 14-3-3β (C), DAPI-labelled host and bacterial DNA (B), and phospho-α-adducin (A). The asterisks (*) in (B) indicate chlamydial inclusions. (D, E) α-Adducin (D) co-localizes with the inclusion membrane protein CT223p (E) in cells infected by C. trachomatis J/UW-36. Scale bars in (C) and (E) indicate 10 μm for each linked set of images.

    The role of microtubules and microfilaments in the distribution of α-adducin to the inclusion membrane was examined using nocodazole or cytochalasin D treatment of infected cells prior to fixation, and examination using immunofluorescence. Evidence for disruption of these structures was sought by using anti-α-tubulin or labelled phalloidin, respectively. In infected cells treated with either inhibitor, phospho-α-adducin accumulation around the chlamydial inclusions appeared undisturbed (Fig. 4D, F⇑).

    DISCUSSION

    The adducins are actin-capping proteins that are involved in a variety of cellular signalling pathways ranging from cytoskeletal arrangements to apoptosis (Gilligan et al., 1999, 2002; Imamdi et al., 2004; Larsson, 2006; Matsuoka et al., 1996). Central to many aspects of adducin biology is its association with the dynactin complex. Adducin co-immunoprecipitates with components of this complex, such as p150Glued, Arp1 and dynamitin (Holleran et al., 1998). Grieshaber et al. (2003) recently demonstrated that, while chlamydial inclusions traverse intracellularly via dynein-mediated microtubular interactions, chlamydial migration is unaffected by disruption of the dynactin complex. The localization of α-adducin in close proximity to the chlamydial inclusion membrane may provide clues to novel interactions between components of the dynactin complex and the developing chlamydial inclusion.

    Possible functions of adducin in the chlamydial infectious process remain to be characterized. Phosphorylation by protein kinase C at Ser726 releases α-adducin from actin–spectrin complexes and the phosphorylated protein localizes to the host cytoplasm (Imamdi et al., 2004; Larsson, 2006). Phosphorylation by Rho-kinase at Thr445, as observed in platelet activation, causes enhanced association of α-adducin with actin–spectrin complexes (Tamaru et al., 2005). Future experiments will examine the phosphorylation status of the Rho kinase-targeted residue on α-adducin in chlamydia-infected cells, and the possibility that other host and chlamydial proteins co-localize with α-adducin following infection.

    Certain host-cell Rab proteins were reported to accumulate around the inclusion independently of intact microtubules (Rzomp et al., 2003). Our data showed that phospho-α-adducin similarly localizes to the chlamydial inclusion independently of intact microtubules. Disruption of actin filaments also did not affect α-adducin localization to the inclusion membrane. While it cannot be ruled out that the microtubules and/or actin filaments are involved in the initial events of α-adducin localization to the inclusion, our data indicate that these structures are not required to maintain α-adducin localization around the inclusion. These results are intriguing, as adducins facilitate interactions between actin and spectrin during extensions of microfilaments, and it is therefore surprising that disruption of microfilaments does not disrupt α-adducin localization to the inclusion membrane.

    Raf-1 was shown to be activated during chlamydial infection (Su et al., 2004) and, in other systems, activation of Raf-1 leads to accumulation of the protein on the cell membrane. Our work is consistent with these studies, as chlamydial infection led to removal of total Raf-1 from the TS fraction of the infected cell.

    Chlamydiae manipulate cytoskeletal structures to facilitate entry (Jewett et al., 2006), redirect vesicular traffic (Scidmore et al., 1996a), and maintain chlamydia-filled inclusions in close proximity to the nucleus and other organelles (Grieshaber et al., 2003). The chlamydia-induced alteration of α-adducin and Raf-1 distribution described in this report may influence the interaction of these proteins with downstream substrates, perhaps contributing to chlamydial development and growth. Additional research is required to determine the specific signalling pathways leading to alterations in these phosphoproteins in infected cells, the molecular steps that lead to redistribution of the proteins, and the roles these changes play during chlamydial development.

    Acknowledgments

    Dr Scott Grieshaber of the University of Florida and Dr Hong Zhang of Kinexus Corp. provided valuable editorial assistance. We thank Dr Luiz Bermudez for providing cytochalasin D, Dr Dahong Zhang for providing nocodazole, and Dr Kathy Magnusson for use of the Odyssey Licor Scanner. Robert Suchland of the University of Washington Chlamydia Laboratory is acknowledged for technical assistance with Cp. pneumoniae culture. This research is supported in part by grants from the National Institutes of Health (AI48769, AI031448) and a National Institutes of Allergy and Infectious Diseases Research Supplement for Underrepresented Minorities (RSUM) pre-doctoral fellowship.

    References

    1. Beatty, W. L. (2006). Trafficking from CD63-positive late endocytic multivesicular bodies is essential for intracellular development of Chlamydia trachomatis. J Cell Sci 119, 350–359.
    2. Carabeo, R. A., Mead, D. J. & Hackstadt, T. (2003). Golgi-dependent transport of cholesterol to the Chlamydia trachomatis inclusion. Proc Natl Acad Sci U S A 100, 6771–6776.
    3. Geisler, W. M., Suchland, R. J., Rockey, D. D. & Stamm, W. E. (2001). Epidemiology and clinical manifestations of unique Chlamydia trachomatis isolates that occupy nonfusogenic inclusions. J Infect Dis 184, 879–884.
    4. Gilligan, D. M., Lozovatsky, L., Gwynn, B., Brugnara, C., Mohandas, N. & Peters, L. L. (1999). Targeted disruption of the beta adducin gene (Add2) causes red blood cell spherocytosis in mice. Proc Natl Acad Sci U S A 96, 10717–10722.
    5. Gilligan, D. M., Sarid, R. & Weese, J. (2002). Adducin in platelets: activation-induced phosphorylation by PKC and proteolysis by calpain. Blood 99, 2418–2426.
    6. Greene, W. & Zhong, G. (2003). Inhibition of host cell cytokinesis by Chlamydia trachomatis infection. J Infect 47, 45–51.
    7. Grieshaber, S. S., Grieshaber, N. A. & Hackstadt, T. (2003). Chlamydia trachomatis uses host cell dynein to traffic to the microtubule-organizing centre in a p50 dynamitin-independent process. J Cell Sci 116, 3793–3802.
    8. Hackstadt, T., Scidmore, M. A. & Rockey, D. D. (1995). Lipid metabolism in Chlamydia trachomatis-infected cells: directed trafficking of Golgi-derived sphingolipids to the chlamydial inclusion. Proc Natl Acad Sci U S A 92, 4877–4881.
    9. Holleran, E. A., Karki, S. & Holzbaur, E. L. (1998). The role of the dynactin complex in intracellular motility. Int Rev Cytol 182, 69–109.
    10. Imamdi, R., de Graauw, M. & van de Water, B. (2004). Protein kinase C mediates cisplatin induced loss of adherens junctions followed by apoptosis of renal proximal tubular epithelial cells. J Pharmacol Exp Ther 311, 892–903.
    11. Jewett, T. J., Fischer, E. R., Mead, D. J. & Hackstadt, T. (2006). Chlamydial TARP is a bacterial nucleator of actin. Proc Natl Acad Sci U S A 103, 15599–15604.
    12. Joshi, R., Gilligan, D. M., Otto, E., McLaughlin, T. & Bennett, V. (1991). Primary structure and domain organization of human alpha and beta adducin. J Cell Biol 115, 665–675.
    13. Lad, S. P., Fukuda, E. Y., Li, J., de la Maza, L. M. & Li, E. (2005). Up-regulation of the JAK/STAT1 signal pathway during Chlamydia trachomatis infection. J Immunol 174, 7186–7193.
    14. Larsson, C. (2006). Protein kinase C and the regulation of the actin cytoskeleton. Cell Signal 18, 276–284.
    15. Matsuoka, Y., Hughes, C. A. & Bennett, V. (1996). Adducin regulation. Definition of the calmodulin-binding domain and sites of phosphorylation by protein kinases A and C. J Biol Chem 271, 25157–25166.
    16. Miyairi, I. & Byrne, G. I. (2006). Chlamydia and programmed cell death. Curr Opin Microbiol 9, 102–108.
    17. Rockey, D. D. & Rosquist, J. L. (1994). Protein antigens of Chlamydia psittaci present in infected cells but not detected in the infectious elementary body. Infect Immun 62, 106–112.
    18. Rockey, D. D., Viratyosin, W., Bannantine, J. P., Suchland, R. J. & Stamm, W. E. (2002). Diversity within inc genes of clinical Chlamydia trachomatis variant isolates that occupy non-fusogenic inclusions. Microbiology 148, 2497–2505.
    19. Rzomp, K. A., Scholtes, L. D., Briggs, B. J., Whittaker, G. R. & Scidmore, M. A. (2003). Rab GTPases are recruited to chlamydial inclusions in both a species-dependent and species-independent manner. Infect Immun 71, 5855–5870.
    20. Scidmore, M. A. & Hackstadt, T. (2001). Mammalian 14-3-3β associates with the Chlamydia trachomatis inclusion membrane via its interaction with IncG. Mol Microbiol 39, 1638–1650.
    21. Scidmore, M. A., Fischer, E. R. & Hackstadt, T. (1996a). Sphingolipids and glycoproteins are differentially trafficked to the Chlamydia trachomatis inclusion. J Cell Biol 134, 363–374.
    22. Scidmore, M. A., Rockey, D. D., Fischer, E. R., Heinzen, R. A. & Hackstadt, T. (1996b). Vesicular interactions of the Chlamydia trachomatis inclusion are determined by chlamydial early protein synthesis rather than route of entry. Infect Immun 64, 5366–5372.
    23. Scidmore, M. A., Fischer, E. R. & Hackstadt, T. (2003). Restricted fusion of Chlamydia trachomatis vesicles with endocytic compartments during the initial stages of infection. Infect Immun 71, 973–984.
    24. Su, H., McClarty, G., Dong, F., Hatch, G. M., Pan, Z. K. & Zhong, G. (2004). Activation of Raf/MEK/ERK/cPLA2 signaling pathway is essential for chlamydial acquisition of host glycerophospholipids. J Biol Chem 279, 9409–9416.
    25. Suchland, R. J., Rockey, D. D., Bannantine, J. P. & Stamm, W. E. (2000). Isolates of Chlamydia trachomatis that occupy nonfusogenic inclusions lack IncA, a protein localized to the inclusion membrane. Infect Immun 68, 360–367.
    26. Tamaru, S., Fukuta, T., Kaibuchi, K., Matsuoka, Y., Shiku, H. & Nishikawa, M. (2005). Rho-kinase induces association of adducin with the cytoskeleton in platelet activation. Biochem Biophys Res Commun 332, 347–351.
    27. Valdivia, R. H. (2008). Chlamydia effector proteins and new insights into chlamydial cellular microbiology. Curr Opin Microbiol 11, 53–59.
    28. Xia, M., Suchland, R. J., Bumgarner, R. E., Peng, T., Rockey, D. D. & Stamm, W. E. (2005). Chlamydia trachomatis variant with nonfusing inclusions: growth dynamic and host-cell transcriptional response. J Infect Dis 192, 1229–1236.
    29. Yuan, Y., Lyng, K., Zhang, Y. X., Rockey, D. D. & Morrison, R. P. (1992). Monoclonal antibodies define genus-specific, species-specific, and cross-reactive epitopes of the chlamydial 60-kilodalton heat shock protein (hsp60): specific immunodetection and purification of chlamydial hsp60. Infect Immun 60, 2288–2296.
    30. Zhong, G., Fan, P., Ji, H., Dong, F. & Huang, Y. (2001). Identification of a chlamydial protease-like activity factor responsible for the degradation of host transcription factors. J Exp Med 193, 935–942.