Chlamydia psittaci IncA is phosphorylated by the host cell and is exposed on the cytoplasmic face of the developing inclusion

Chlamydiae are obligate intracellular bacteria that replicate within a non-acidified vacuole called an inclusion. Chlamydia psittaci (strain GPIC) produces a 39 kDa protein (IncA) that is localized to the inclusion membrane. While IncA is present as a single 39 kDa species in purified reticulate bodies, two additional higher M _r forms are found in C. psittaci -infected cells. This finding suggested that IncA may be post-translationally modified in the host cell. Here we present evidence that IncA is a serine/threonine phosphoprotein that is phosphorylated by host cell enzymes. This conclusion is supported by the following experimental findings: (i) treatment of infected cells with inhibitors of host cell phosphatases or kinases altered the electrophoretic migration pattern of IncA; (ii) treatment with calf intestinal alkaline phosphatase eliminated the multiple-banding pattern of IncA, leaving only the protein band with the lowest relative molecular weight; and (iii) radioimmunoprecipitation of lysates of [³²P]-orthophosphate-labelled infected HeLa cells with anti-IncA antisera demonstrated that the two highest M _r IncA bands were phosphorylated. A vaccinia-virus recombinant expressing incA was used to determine if HeLa cells can phosphorylate IncA in the absence of a chlamydial background. IncA in lysates of these cells migrated identically to that seen in C. psittaci -infected cells, indicating the host cell was responsible for the phosphorylation of the protein. Microinjection of fluorescently labelled anti-IncA antibodies into C. psittaci -infected HeLa cells resulted in immunostaining of the outer face of the inclusion membrane. Collectively, these results demonstrate that IncA is phosphorylated by the host cell, and regions of IncA are exposed at the cytoplasmic face of the inclusion.     

The Chlamydia trachomatis IncA protein is required for homotypic vesicle fusion

Chlamydiae replicate within an intracellular vacuole, termed an inclusion, that is non-fusogenic with vesicles of the endosomal or lysosomal compartments. Instead, the inclusion appears to intersect an exocytic pathway from which chlamydiae intercept sphingomyelin en route from the Golgi apparatus to the plasma membrane. Chlamydial protein synthesis is required to establish this interaction. In an effort to identify those chlamydial proteins controlling vesicle fusion, we have prepared polyclonal antibodies against several Chlamydia trachomatis inclusion membrane proteins. Microinjection of polyclonal antibodies against three C. trachomatis inclusion membrane proteins, IncA, F and G, into the cytosol of cells infected with C. trachomatis demonstrates reactivity with antigens on the cytoplasmic face of the inclusion membrane, without apparent inhibition of chlamydial multiplication. Microinjection of antibodies against the C. trachomatis IncA protein, however, results in the development of an aberrant multilobed inclusion structure remarkably similar to that of C. psittaci GPIC. These results suggest that the C. trachomatis IncA protein is involved in homotypic vesicle fusion and/or septation of the inclusion membrane that is believed to accompany bacterial cell division in C. psittaci . This proposal is corroborated by the expression of C. trachomatis and C. psittaci IncA in a yeast two-hybrid system to demonstrate C. trachomatis , but not C. psittaci , IncA interactions. Despite the inhibition of homotypic fusion of C. trachomatis inclusions, fusion of sphingomyelin-containing vesicles with the inclusion was not suppressed.     

Conservation of the biochemical properties of IncA from Chlamydia trachomatis and Chlamydia caviae: oligomerization of IncA mediates interaction between facing membranes

The developmental cycle of Chlamydiaceae occurs in a membrane compartment called an inclusion. IncA is a member of a family of proteins synthesized and secreted onto the inclusion membrane by bacteria. IncA proteins from different species of Chlamydiaceae show little sequence similarity. We report that the biochemical properties of Chlamydia trachomatis and Chlamydia caviae are conserved. Both proteins self-associate to form multimers. When artificially expressed by the host cell, they localize to the endoplasmic reticulum. Strikingly, heterologous expression of IncA in the endoplasmic reticulum completely inhibits concomitant inclusion development. Using truncated forms of IncA from C. caviae, we show that expression of the C-terminal cytoplasmic domain of the protein at the surface of the endoplasmic reticulum is sufficient to disrupt the bacterial developmental cycle. On the other hand, development of a C. trachomatis strain that does not express IncA is not inhibited by artificial IncA expression, showing that the disruptive effect observed with the wild-type strain requires direct interactions between IncA molecules at the inclusion and on the endoplasmic reticulum. Finally, we modeled IncA tetramers in parallel four helix bundles based on the structure of the SNARE complex, a conserved structure involved in membrane fusion in eukaryotic cells. Both C. trachomatis and C. caviae IncA tetramers were highly stable in this model. In conclusion, we show that the property of IncA proteins to assemble into multimeric structures is conserved between chlamydial species, and we propose that these proteins may have co-evolved with the SNARE machinery for a role in membrane fusion.     

SNARE protein mimicry by an intracellular bacterium

Many intracellular pathogens rely on host cell membrane compartments for their survival. The strategies they have developed to subvert intracellular trafficking are often unknown, and SNARE proteins, which are essential for membrane fusion, are possible targets. The obligate intracellular bacteria Chlamydia replicate within an intracellular vacuole, termed an inclusion. A large family of bacterial proteins is inserted in the inclusion membrane, and the role of these inclusion proteins is mostly unknown. Here we identify SNARE-like motifs in the inclusion protein IncA, which are conserved among most Chlamydia species. We show that IncA can bind directly to several host SNARE proteins. A subset of SNAREs is specifically recruited to the immediate vicinity of the inclusion membrane, and their accumulation is reduced around inclusions that lack IncA, demonstrating that IncA plays a predominant role in SNARE recruitment. However, interaction with the SNARE machinery is probably not restricted to IncA as at least another inclusion protein shows similarities with SNARE motifs and can interact with SNAREs. We modelled IncA's association with host SNAREs. The analysis of intermolecular contacts showed that the IncA SNARE-like motif can make specific interactions with host SNARE motifs similar to those found in a bona fide SNARE complex. Moreover, point mutations in the central layer of IncA SNARE-like motifs resulted in the loss of binding to host SNAREs. Altogether, our data demonstrate for the first time mimicry of the SNARE motif by a bacterium.     

An α-Helical Core Encodes the Dual Functions of the Chlamydial Protein IncA

Chlamydia is an intracellular bacterium that establishes residence within parasitophorous compartments (inclusions) inside host cells. Chlamydial inclusions are uncoupled from the endolysosomal pathway and undergo fusion with cellular organelles and with each other. To do so, Chlamydia expresses proteins on the surface of the inclusion using a Type III secretion system. These proteins, termed Incs, are located at the interface between host and pathogen and carry out the functions necessary for Chlamydia survival. Among these Incs, IncA plays a critical role in both protecting the inclusion from lysosomal fusion and inducing the homotypic fusion of inclusions. Within IncA are two regions homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) domains referred to as SNARE-like domain 1 (SLD1) and SNARE-like domain 2 (SLD2). Using a multidisciplinary approach, we have discovered the functional core of IncA that retains the ability to both inhibit SNARE-mediated fusion and promote the homotypic fusion of Chlamydia inclusions. Circular dichroism and analytical ultracentrifugation experiments show that this core region is composed almost entirely of α-helices and assembles into stable homodimers in solution. Altogether, we propose that both IncA functions are encoded in a structured core domain that encompasses SLD1 and part of SLD2.     

Impact of urban environment and host phenotype on the epidemiology of Chlamydiaceae in feral pigeons (Columba livia)

Chlamydiaceae are obligate intracellular Gram-negative bacteria found all over the world and known to cause various forms of disease in animals and humans. Urban pigeons are known to be an important reservoir of Chlamydia psittaci, the agent of human psittacosis. In this study, we examined the influence of pigeon houses used to regulate pigeon populations and of melanin-based coloration on several epidemiological parameters of Chlamydiaceae in 708 urban pigeons in Paris. We also identified species and genotypes of Chlamydiaceae present in Parisian populations. First, our results revealed that pigeons roosting and breeding in pigeon houses were equally infected by Chlamydiaceae as those that did not. Second, we found that dark melanic pigeons excreted more Chlamydiaceae than pale melanic ones. Finally, species and strain diversities were very low: all samples were of C. psittaci genotype B. Nevertheless, two atypical Chlamydiaceae were identified based on 16S rRNA and ompA sequences. Our study thus highlights the importance of considering environmental and host phenotype when investigating the epidemiology of infectious diseases.     

Interaction between components of the type III secretion system of Chlamydiaceae

Members of the family Chlamydiaceae possess at least 13 genes, distributed throughout the chromosome, that are homologous with genes of known type III secretion systems (TTS). The aim of this study was to use putative TTS proteins of Chlamydophila pneumoniae, whose equivalents in other bacterial TTS function as chaperones, to identify interactions between chlamydial proteins. Using the BacterioMatch Two-Hybrid Vector system (Stratagene, La Jolla, Calif.), lcrH-2 and sycE, positions 1021 and 0325, respectively, from C. pneumoniae CM-1 were used as "bait" to identify target genes (positions 0324, 0705, 0708, 0808 to 0810, 1016 to 1020, and 1022) in close proximity on the chromosome. Interaction between the products of the lcrH-2 (1021) and lcrE (copN) (0324) genes was detected and confirmed by pull-down experiments and enzyme immunoassays using recombinant LcrH-2 and LcrE. As further confirmation of this interaction, the homologous genes from Chlamydia trachomatis, serovar E, and Chlamydophila psittaci, Texas turkey, were also cloned in the two-hybrid system to determine if LcrH-2 and LcrE would interact with their orthologs in other species. Consistent with their genetic relatedness, LcrH-2 from C. pneumoniae interacted with LcrE produced from the three species of Chlamydiaceae; LcrH-2 from C. psittaci reacted with LcrE from C. pneumoniae but not from C. trachomatis; and C. trachomatis LcrH-2 did not react with LcrE from the other two species. Deletions from the N and C termini of LcrE from C. pneumoniae identified the 50 C-terminal amino acids as essential for the interaction with LcrH-2. Thus, it appears that in the Chlamydiaceae TTS, LcrH-2 interacts with LcrE, and therefore it may serve as a chaperone for this protein.     

Overexpression of c-Myc alters G(1)/S arrest following ionizing radiation

Study of the mechanism(s) of genomic instability induced by the c-myc proto-oncogene has the potential to shed new light on its well-known oncogenic activity. However, an underlying mechanism(s) for this phenotype is largely unknown. In the present study, we investigated the effects of c-Myc overexpression on the DNA damage-induced G(1)/S checkpoint, in order to obtain mechanistic insights into how deregulated c-Myc destabilizes the cellular genome. The DNA damage-induced checkpoints are among the primary safeguard mechanisms for genomic stability, and alterations of cell cycle checkpoints are known to be crucial for certain types of genomic instability, such as gene amplification. The effects of c-Myc overexpression were studied in human mammary epithelial cells (HMEC) as one approach to understanding the c-Myc-induced genomic instability in the context of mammary tumorigenesis. Initially, flow-cytometric analyses were used with two c-Myc-overexpressing, nontransformed immortal lines (184A1N4 and MCF10A) to determine whether c-Myc overexpression leads to alteration of cell cycle arrest following ionizing radiation (IR). Inappropriate entry into S phase was then confirmed with a bromodeoxyuridine incorporation assay measuring de novo DNA synthesis following IR. Direct involvement of c-Myc overexpression in alteration of the G(1)/S checkpoint was then confirmed by utilizing the MycER construct, a regulatable c-Myc. A transient excess of c-Myc activity, provided by the activated MycER, was similarly able to induce the inappropriate de novo DNA synthesis following IR. Significantly, the transient expression of full-length c-Myc in normal mortal HMECs also facilitated entry into S phase and the inappropriate de novo DNA synthesis following IR. Furthermore, irradiated, c-Myc-infected, normal HMECs developed a sub-G(1) population and a >4N population of cells. The c-Myc-induced alteration of the G(1)/S checkpoint was also compared to the effects of expression of MycS (N-terminally truncated c-Myc) and p53DD (a dominant negative p53) in the HMECs. We observed inappropriate hyperphosphorylation of retinoblastoma protein and then the reappearance of cyclin A, following IR, selectively in full-length c-Myc- and p53DD-overexpressing MCF10A cells. Based on these results, we propose that c-Myc attenuates a safeguard mechanism for genomic stability; this property may contribute to c-Myc-induced genomic instability and to the potent oncogenic activity of c-Myc.     

Ataxia-Telangiectasia Group D Complementing Gene (ATDC) Promotes Lung Cancer Cell Proliferation by Activating NF-κB Pathway

Previous studies suggested Ataxia-telangiectasia group D complementing gene (ATDC) as an oncogene in many types of cancer. However, its expression and biological functions in non-small cell lung cancer (NSCLC) remain unclear. Herein, we investigated its expression pattern in 109 cases of human NSCLC samples by immunohistochemistry and found that ATDC was overexpressed in 62 of 109 NSCLC samples (56.88%). ATDC overexpression correlated with histological type (p<0.0001), tumor status (p = 0.0227) and histological differentiation (p = 0.0002). Next, we overexpressed ATDC in normal human bronchial epithelial cell line HBE and depleted its expression in NSCLC cell lines A549 and H1299. MTT and colony formation assay showed that ATDC overexpression promoted cell proliferation while its depletion inhibited cell growth. Furthermore, cell cycle analysis showed that ATDC overexpression decreased the percentage of cells in G1 phase and increased the percentage of cells in S phase, while ATDC siRNA treatment increased the G1 phase percentage and decreased the S phase percentage. Further study revealed that ATDC overexpression could up-regulate cyclin D1 and c-Myc expression in HBE cells while its depletion down-regulated cyclin D1 and c-Myc expression in A549 and H1299 cells. In addition, ATDC overexpression was also associated with an increased proliferation index, cyclin D1 and c-Myc expression in human NSCLC samples. Further experiments demonstrated that ATDC up-regulated cyclin D1 and c-Myc expression independent of wnt/β-catenin or p53 signaling pathway. Interestingly, ATDC overexpression increased NF-κB reporter luciferase activity and p-IκB protein level. Correspondingly, NF-κB inhibitor blocked the effect of ATDC on up-regulation of cyclin D1 and c-Myc. In conclusion, we demonstrated that ATDC could promote lung cancer proliferation through NF-κB induced up-regulation of cyclin D1 and c-Myc.     

Mammalian 14-3-3beta associates with the Chlamydia trachomatis inclusion membrane via its interaction with IncG

Chlamydiae replicate intracellularly within a vacuole that is modified early in infection to become fusogenic with a subset of exocytic vesicles. We have recently identified four chlamydial inclusion membrane proteins, IncD-G, whose expression is detected within the first 2 h after internalization. To gain a better understanding of how these Inc proteins function, a yeast two-hybrid screen was employed to identify interacting host proteins. One protein, 14-3-3beta, was identified that interacted specifically with IncG. The interaction between 14-3-3beta and IncG was confirmed in infected HeLa cells by indirect immunofluorescence microscopy and interaction with a GFP-14-3-3beta fusion protein. 14-3-3 proteins are phosphoserine-binding proteins. Immunoprecipitation studies with [32P]-orthophosphate-labelled cells demonstrated that IncG is phosphorylated in both chlamydia-infected HeLa cells and in yeast cells expressing IncG. Site-directed mutagenesis of predicted 14-3-3 phosphorylation sites demonstrated that IncG binds to 14-3-3beta via a conserved 14-3-3-binding motif (RS164RS166F). Finally, indirect immunofluorescence demonstrated that 14-3-3beta interacts with Chlamydia trachomatis inclusions but not C. psittaci or C. pneumoniae inclusions. 14-3-3beta is the first eukaryotic protein found to interact with the chlamydial inclusion; however, its unique role in C. trachomatis pathogenesis remains to be determined.     

Trafficking of chlamydial antigens to the endoplasmic reticulum of infected epithelial cells

Confinement of the obligate intracellular bacterium Chlamydia trachomatis to a membrane-bound vacuole, termed an inclusion, within infected epithelial cells neither prevents secretion of chlamydial antigens into the host cytosol nor protects chlamydiae from innate immune detection. However, the details leading to chlamydial antigen presentation are not clear. By immunoelectron microscopy of infected endometrial epithelial cells and in isolated cell secretory compartments, chlamydial major outer membrane protein (MOMP), lipopolysaccharide (LPS) and the inclusion membrane protein A (IncA) were localized to the endoplasmic reticulum (ER) and co-localized with multiple ER markers, but not with markers of the endosomes, lysosomes, Golgi nor mitochondria. Chlamydial LPS was also co-localized with CD1d in the ER. Since the chlamydial antigens, contained in everted inclusion membrane vesicles, were found within the host cell ER, these data raise additional implications for antigen processing by infected uterine epithelial cells for classical and non-classical T cell antigen presentation.     

c-Myc is a novel target of cell cycle arrest by honokiol in prostate cancer cells

Honokiol (HNK), a highly promising phytochemical derived from Magnolia officinalis plant, exhibits in vitro and in vivo anticancer activity against prostate cancer but the underlying mechanism is not fully clear. This study was undertaken to delineate the role of c-Myc in anticancer effects of HNK. Exposure of prostate cancer cells to plasma achievable doses of HNK resulted in a marked decrease in levels of total and/or phosphorylated c-Myc protein as well as its mRNA expression. We also observed suppression of c-Myc protein in PC-3 xenografts upon oral HNK administration. Stable overexpression of c-Myc in PC-3 and 22Rv1 cells conferred significant protection against HNK-mediated growth inhibition and G₀-G₁ phase cell cycle arrest. HNK treatment decreased expression of c-Myc downstream targets including Cyclin D1 and Enhancer of Zeste Homolog 2 (EZH2), and these effects were partially restored upon c-Myc overexpression. In addition, PC-3 and DU145 cells with stable knockdown of EZH2 were relatively more sensitive to growth inhibition by HNK compared with control cells. Finally, androgen receptor overexpression abrogated HNK-mediated downregulation of c-Myc and its targets particularly EZH2. The present study indicates that c-Myc, which is often overexpressed in early and late stages of human prostate cancer, is a novel target of prostate cancer growth inhibition by HNK.     

Tracking and elucidating alphavirus-host protein interactions

Viral infections cause profound alterations in host cells. Here, we explore the interactions between proteins of the Alphavirus Sindbis and host factors during the course of mammalian cell infection. Using a mutant virus expressing the viral nsP3 protein tagged with green fluorescent protein (GFP) we directly observed nsP3 localization and isolated nsP3-interacting proteins at various times after infection. These results revealed that host factor recruitment to nsP3-containing complexes was time dependent, with a specific early and persistent recruitment of G3BP and a later recruitment of 14-3-3 proteins. Expression of GFP-tagged G3BP allowed reciprocal isolation of nsP3 in Sindbis infected cells, as well as the identification of novel G3BP-interacting proteins in both uninfected and infected cells. Note-worthy interactions include nuclear pore complex components whose interactions with G3BP were reduced upon Sindbis infection. This suggests that G3BP is a nuclear transport factor, as hypothesized previously, and that viral infection may alter RNA transport. Immunoelectron microscopy showed that a portion of Sindbis nsP3 is localized at the nuclear envelope, suggesting a possible site of G3BP recruitment to nsP3-containing complexes. Our results demonstrate the utility of using a standard GFP tag to both track viral protein localization and elucidate specific viral-host interactions over time in infected mammalian cells.     

Rictor regulates FBXW7-dependent c-Myc and cyclin E degradation in colorectal cancer cells

Rictor (Rapamycin-insensitive companion of mTOR) forms a complex with mTOR and phosphorylates and activates Akt. Activation of Akt induces expression of c-Myc and cyclin E, which are overexpressed in colorectal cancer and play an important role in colorectal cancer cell proliferation. Here, we show that rictor associates with FBXW7 to form an E3 complex participating in the regulation of c-Myc and cyclin E degradation. The Rictor-FBXW7 complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Moreover, knocking down of rictor in serum-deprived colorectal cancer cells results in the decreased ubiquitination and increased protein levels of c-Myc and cyclin E while overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Genetic knockout of FBXW7 blunts the effects of rictor, suggesting that rictor regulation of c-Myc and cyclin E requires FBXW7. Our findings identify rictor as an important component of FBXW7 E3 ligase complex participating in the regulation of c-Myc and cyclin E protein ubiquitination and degradation. Importantly, our results suggest that elevated growth factor signaling may contribute to decrease rictor/FBXW7-mediated ubiquitination of c-Myc and cyclin E, thus leading to accumulation of cyclin E and c-Myc in colorectal cancer cells.     

The effect of growth state on the activity of the protein kinase isoenzymes. An increase in type II cyclic AMP-dependent protein kinase is correlated with growth arrest in G1 phase

Native polyacrylamide gels have been used to resolve protein kinase isoenzymes from cultured cells and the protein kinases have been identified by carrying out phosphorylation reactions in the gel. Following electrophoresis the gels were incubated with histone and [gamma-32P]ATP. The gels were then thoroughly washed and dried down, and the protein kinases were located by autoradiography. Protein kinase activity as measured in the gel system was a linear function of cytosol protein concentration up to about 100 microgram per channel and incorporation of 32P into histone was time dependent. Three bands of protein kinase activity were resolved in cytosol samples from baby hamster kidney (BHK) fibroblasts. The band with the lowest relative mobility utilized histone IIA or casein equally well as substrate protein whereas bands 2 and 3 demonstrated a clear preference for histone. Bands 2 and 3 displayed a relative mobility in electrophoresis that was identical to that observed for cyclic AMP-dependent protein kinases I and II from rat liver. Treatment of cytosol samples with cyclic AMP prior to electrophoresis resulted in the disappearance of cyclic AMP-dependent protein kinases from the gel profile. This method was employed to identify bands 2 and 3 as cyclic AMP-dependent protein kinases. The protein kinases in growth-arrested cells were compared with proliferating cells. We have observed a 3.5-fold increase in the activity of Type II protein kinase as the cells arrest growth in G1 phase of the cell cycle. This increase in Type II is correlated with the increase in cells blocked in G1 and a decrease in Type II activity appears to be an early event in permitting cells to leave G1 and resume growth.