Chlamydia pneumoniae inclusion membrane protein Cpn0585 interacts with multiple Rab GTPases

Chlamydiae are intracellular bacteria that develop within a membrane-bound vacuole called an inclusion. To ensure that the inclusion is a safe niche for chlamydial replication, chlamydiae exploit a number of host cell processes, including membrane-trafficking pathways. Recently, several Rab GTPases were found to associate with the inclusions of various chlamydial species. Here we report that Cpn0585, a Chlamydia pneumoniae inclusion membrane protein (Inc), interacts with multiple Rab GTPases. The results from yeast two-hybrid experiments revealed that an amino-terminally truncated form of Cpn0585 (Cpn0585_102-651) interacts with Rab1, Rab10, and Rab11 but not with Rab4 or Rab6. Cpn0585-Rab GTPase interactions are direct and GTP dependent as shown in glutathione S-transferase pull-down assays using native and recombinant Cpn0585. In C. pneumoniae-infected HEp-2 cells transfected with enhanced green fluorescent protein (EGFP)-tagged Rab GTPases, the colocalization with Cpn0585 at the inclusion membrane was partial for EGFP-Rab1 and EGFP-Rab10, but extensive for wild-type EGFP-Rab11A and the constitutively active GTPase-deficient EGFP-Rab11AQ70L. Moreover, Cpn0585 colocalized with EGFP-Rab11AQ70L as early as 2 h postinfection. Upon delivery into live C. pneumoniae-infected cells, Cpn0585_628-651-specific antibodies bound to the inclusion membrane, demonstrating that the Rab GTPase-interacting domain of Cpn0585 faces the host cell cytosol. Finally, ectopic expression of Cpn0585_102-651 partially inhibited the development of C. pneumoniae inclusions in EGFP. but not in EGFP-Rab11AQ70L-expressing HEp-2 cells. Collectively, these data suggest that Cpn0585 is involved in the recruitment of Rab GTPases to the inclusion membrane and that interfering with this function may adversely impact the fitness of the C. pneumoniae inclusion for chlamydial replication.     

Localization of the hypothetical protein Cpn0585 in the inclusion membrane of Chlamydia pneumoniae-infected cells

Cpn0585, encoded by a hypothetical open reading frame in Chlamydia pneumoniae genome, was detected in the inclusion membrane during C. pneumoniae infection using both polyclonal and monoclonal antibodies raised with Cpn0585 fusion protein. The anti-Cpn0585 antibodies specifically recognized the endogenous Cpn0585 without cross-reacting with IncA (a known inclusion membrane protein of C. pneumoniae) or other control antigens. A homologue of Cpn0585 in the C. caviae species (encoded by the ORF CCA00156) was also localized in the inclusion membrane of the C. caviae-infected cells. The Cpn0585 protein became detectable 24h while CCA00156 as early as 8h after infection. Once expressed, both proteins remained in the inclusion membrane throughout the rest of infection course.     

The hypothetical protein CT813 is localized in the Chlamydia trachomatis inclusion membrane and is immunogenic in women urogenitally infected with C. trachomatis

Using antibodies raised with chlamydial fusion proteins, we have localized a protein encoded by hypothetical open reading frame CT813 in the inclusion membrane of Chlamydia trachomatis. The detection of the C. trachomatis inclusion membrane by an anti-CT813 antibody was blocked by the CT813 protein but not unrelated fusion proteins. The CT813 protein was detected as early as 12 h after chlamydial infection and was present in the inclusion membrane during the entire growth cycle. All tested serovars from C. trachomatis but not other chlamydial species expressed the CT813 protein. Exogenously expressed CT813 protein in HeLa cells displayed a cytoskeleton-like structure similar to but not overlapping with host cell intermediate filaments, suggesting that the CT813 protein is able to either polymerize or associate with host cell cytoskeletal structures. Finally, women with C. trachomatis urogenital infection developed high titers of antibodies to the CT813 protein, demonstrating that the CT813 protein is not only expressed but also immunogenic during chlamydial infection in humans. In all, the CT813 protein is an inclusion membrane protein unique to C. trachomatis species and has the potential to interact with host cells and induce host immune responses during natural infection. Thus, the CT813 protein may represent an important candidate for understanding C. trachomatis pathogenesis and developing intervention and prevention strategies for controlling C. trachomatis infection.     

Isolates of Chlamydia trachomatis that occupy nonfusogenic inclusions lack IncA, a protein localized to the inclusion membrane

The chlamydiae are obligate intracellular pathogens that occupy a nonacidified vacuole, termed an inclusion, throughout their developmenal cycle. When an epithelial cell is infected with multiple Chlamydia trachomatis elementary bodies, they are internalized by endocytosis into individual phagosomal vacuoles that eventually fuse to form a single inclusion. In the course of large-scale serotyping studies in which fluorescent antibody staining of infected cells was used, a minority of strains that had an alternate inclusion morphology were identified. These variants formed multiple nonfusogenic inclusions in infected cells, with the number of independent inclusions per cell varying directly with the multiplicity of infection. Overall the nonfusogenic phenotype was found in 1.5% (176 of 11,440) of independent isolates. Nonfusing variants were seen in C. trachomatis serovars B, D, D-, E, F, G, H, Ia, J, and K. The nonfusing phenotype persisted through repeated serial passage, and the phenotype was consistent in four mammalian host cell lines. Fluorescence microscopy and immunoblotting with antisera directed at proteins in the C. trachomatis inclusion membrane revealed that one such protein, IncA, was not detected in the inclusion membrane in each tested nonfusogenic strain. The distributions of other chlamydial proteins, including one additional Inc protein, were similar in wild-type and variant strains. The incA coding and upstream regions were amplified and sequenced from the prototype serovar D and two nonfusing serovar D((s)) strains. Three nucleotide changes were discovered in the D((s)) incA gene, leading to two amino acid changes within the predicted D((s)) IncA sequence. These studies demonstrate a subgroup of variant C. trachomatis isolates that form nonfusing inclusions; the variant phenotype is associated with the absence of detectable IncA and with an altered incA sequence that modifies the characteristic hydrophobic domain of the IncA protein.     

肺炎嗜衣原体Cpn0810重组蛋白诱导THP-1细胞产生促炎因子和凋亡的研究

目的:在E.coliBL21中表达肺炎嗜衣原体Cpn0810,并研究该蛋白能否诱导人单核细胞( THP-1)产生TNF-α和IL-6等促炎因子和细胞凋亡.方法:PCR扩增Cpn0810蛋白编码基因,构建pGEX6p-2/Cpn0810重组质粒,在E.coliBL21中诱导表达,经ToxinEraser纯化柱纯化后,用不同浓度的GST-Cpn0810作用THP-1细胞,ELISA法检测TNF-α和IL-6的水平,Hoechst33258荧光染色、AnnexinV-F1TC-PI染色法检测细胞凋亡情况.结果:构建了重组质粒pGEX6p-2/Cpn0810,并在E.coliBL21菌中高效表达.该蛋白能诱导THP-1细胞以时间、剂量依赖方式表达TNF-α和IL-6;当10mg/L GST-Cpn08 10处理THP-1细胞24h后,形态学上,细胞表现为皱缩、核碎裂、细胞起泡及凋亡小体等凋亡特征.结论:表达并纯化的Cpn0810蛋白能诱导THP-1细胞分泌TNF-α和IL-6等促炎因子和诱导其发生凋亡.     

Sequence analysis of the major outer membrane protein gene of Chlamydia pneumoniae.

Compared with the major outer membrane proteins (MOMPs) of the other chlamydial species, the Chlamydia pneumoniae MOMP appears to be less antigenically complex, and as determined by immunoblot analysis, it does not appear to be the immunodominant antigen recognized during infection. Nucleotide sequence analysis of the C. pneumoniae MOMP gene (ompA) revealed that it consisted of a 1,167-base open reading frame with an inferred 39,344-dalton mature protein of 366 amino acids plus a 23-amino-acid leader sequence. A ribosomal-binding site was located in the 5' upstream region, and two stop codons followed by an 11-base dyad forming a stable stem-loop structure were identified. This sequence shares 68 and 71% DNA sequence homology to the Chlamydia trachomatis serovar L2 and Chlamydia psittaci ovine abortion agent MOMP genes, respectively. Interspecies alignment identified regions, corresponding to the variable domains, which share little sequence similarity with the other chlamydial MOMPs. All seven cysteines conserved in the C. trachomatis and C. psittaci MOMPs, which are involved in the formation of disulfide cross-linkages, are found in the C. pneumoniae MOMP.     

The GTPase Rab4 interacts with Chlamydia trachomatis inclusion membrane protein CT229

Chlamydiae, which are obligate intracellular bacteria, replicate in a nonlysosomal vacuole, termed an inclusion. Although neither the host nor the chlamydial proteins that mediate the intracellular trafficking of the inclusion have been clearly identified, several enhanced green fluorescent protein (GFP)-tagged Rab GTPases, including Rab4A, are recruited to chlamydial inclusions. GFP-Rab4A associates with inclusions in a species-independent fashion by 2 h postinfection by mechanisms that have not yet been elucidated. To test whether chlamydial inclusion membrane proteins (Incs) recruit Rab4 to the inclusion, we screened a collection of chlamydial Incs for their ability to interact with Rab4A by using a yeast two-hybrid assay. From our analysis, we identified a specific interaction between Rab4A and Chlamydia trachomatis Inc CT229, which is expressed during the initial stages of infection. CT229 interacts with only wild-type Rab4A and the constitutively active GTPase-deficient Rab4AQ67L but not with the dominant-negative GDP-restricted Rab4AS22N mutant. To confirm the interaction between CT229 and Rab4A, we demonstrated that DsRed-CT229 colocalized with GFP-Rab4A in HeLa cells and more importantly wild-type and constitutively active GFP-Rab4A colocalized with CT229 at the inclusion membrane in C. trachomatis serovar L2-infected HeLa cells. Taken together, these data suggest that CT229 interacts with and recruits Rab4A to the inclusion membrane and therefore may play a role in regulating the intracellular trafficking or fusogenicity of the chlamydial inclusion.     

Localization of the hypothetical protein Cpn0797 in the cytoplasm of Chlamydia pneumoniae-infected host cells

Using antibodies raised with chlamydial fusion proteins, we have localized a protein encoded by the hypothetical open reading frame Cpn0797 in the cytoplasm of Chlamydia pneumoniae-infected host cells. The anti-Cpn0797 antibodies specifically recognized Cpn0797 protein without cross-reacting with either CPAFcp or Cpn0796, the only two proteins known to be secreted into the host cell cytosol by C. pneumoniae organisms. Thus, Cpn0797 represents the third C. pneumoniae protein secreted into the host cell cytosol experimentally identified so far.     

Flotillin-1 (Reggie-2) contributes to Chlamydia pneumoniae growth and is associated with bacterial inclusion

Chlamydiae are obligate intracellular pathogens replicating only inside the eukaryotic host. Here, we studied the effect of human flotillin-1 protein on Chlamydia pneumoniae growth in human line (HL) and A549 epithelial cell lines. RNA interference was applied to disrupt flotillin-1-mediated endocytosis. Host-associated bacteria were detected by quantitative PCR, and C. pneumoniae growth was evaluated by inclusion counts. C. pneumoniae attachment to host cells was unaffected, but bacterial intracellular growth was attenuated in the flotillin-1-silenced cells. By using confocal microscopy, we detected flotillin-1 colocalized with the inclusion membrane protein A (IncA) in the C. pneumoniae inclusion membranes. In addition, flotillin-1 was associated with IncA in detergent-resistant membrane microdomains (DRMs) in biochemical fractioning. These results suggest that flotillin-1 localizes to the C. pneumoniae inclusion membrane and plays an important role for intracellular growth of C. pneumoniae.     

Evidence that the major outer membrane protein of Chlamydia trachomatis is glycosylated.

The major outer membrane protein (MOMP) of Chlamydia trachomatis was determined to be a glycoprotein on the basis of susceptibility to glycosidase digestion and the presence of carbohydrate by staining and radiolabeling. The MOMP of the serovar L2 organisms was isolated by electroelution from the protein band excised from the gel after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The incubation of MOMP with N-glycosidase F, an endoglycosidase that cleaves the N-glycan, and periodate resulted in two new molecular weight species. While MOMP treated with N-glycosidase F showed a lower-molecular-weight mobility, the periodate-treated MOMP increased in molecular weight. Both treatments abolished the ability of the MOMP to bind to HeLa cell components. In the immunoblot, the reactivity to the monoclonal antibody specific against the C. trachomatis species was preserved. The endoglycosidase specific to O-linked glycan, endo-alpha-N-acetylgalactosaminidase, had no visible effect on the isolated MOMP. Carbohydrate was detected in the MOMP by p-phenylenediamine staining of the protein band in the gel following SDS-PAGE. Autoradiograms of proteins of chlamydial organisms metabolically labeled with [3H]galactose or [3H]glucosamine and separated by SDS-PAGE revealed the MOMP band. The isolated MOMP was shown to bind specifically to concanavalin A, wheat germ agglutinin, and Dolichos biflorus agglutinin in the lectin binding assay. No binding was observed with Ulex europaeus agglutinin I, soybean agglutinin, or Ricinus communis agglutinin.     

Similarity of Chlamydia pneumoniae strains in the variable domain IV region of the major outer membrane protein gene.

DNA was amplified by polymerase chain reaction from the gene encoding the major outer membrane protein (MOMP) of Chlamydia pneumoniae in order to examine the relatedness of strains isolated from diverse geographical regions. Primers for this reaction were chosen to span a 207-bp region comparable to that of the fourth variable segment of the MOMP gene of Chlamydia trachomatis. Among C. trachomatis, sequence heterogeneity is characteristic within variable sequence domain IV (VDIV) and correlates with serovar type. In contrast, sequence analysis of polymerase chain reaction products from 13 C. pneumoniae isolates indicated that all tested strains were identical in this segment of the MOMP gene. The predicted amino acid sequences from the C. pneumoniae VDIV gene products shared only 13.3 to 30% homology with published VDIV regions from serovars of C. trachomatis. Homology of these VDIV amino acid sequences with sequences from strains of C. psittaci ranged from 45.7 to 60%. The sequence conservation of the VDIV region of the MOMP gene indicates that C. pneumoniae strains may be more genetically homogeneous than C. trachomatis or Chlamydia psittaci strains. Future investigations of antigenic diversity among C. pneumoniae strains should be aimed at the evaluation of variation in other regions of the C. pneumoniae genome.     

Characterization of fifty putative inclusion membrane proteins encoded in the Chlamydia trachomatis genome

Although the Chlamydia trachomatis genome is predicted to encode 50 inclusion membrane proteins, only 18 have been experimentally localized in the inclusion membrane of C. trachomatis-infected cells. Using fusion proteins and anti-fusion protein antibodies, we have systematically evaluated all 50 putative inclusion membrane proteins for their localization in the infected cells, distribution patterns, and effects on subsequent chlamydial infection when expressed ectopically, as well as their immunogenicity during chlamydial infection in humans. Twenty-two of the 50 proteins were localized in the inclusion membrane, and 7 were detected inside the inclusions, while the location of the remaining 21 was not defined. Four (CT225, CT228, CT358, and CT440) of the 22 inclusion membrane-localized proteins were visualized in the inclusion membrane of Chlamydia-infected cells for the first time in the current study. The seven intra-inclusion-localized proteins were confirmed to be chlamydial organism proteins in a Western blot assay. Further characterization of the 50 proteins revealed that neither colocalization with host cell endoplasmic reticulum nor inhibition of subsequent chlamydial infection by ectopically expressed proteins correlated with the inclusion membrane localization. Interestingly, antibodies from women with C. trachomatis urogenital infection preferentially recognized proteins localized in the inclusion membrane, and the immunodominant regions were further mapped to the region predicted to be on the cytoplasmic side of the inclusion membrane. These observations suggest that most of the inclusion membrane-localized proteins are both expressed and immunogenic during C. trachomatis infection in humans and that the cytoplasmic exposure may enhance the immunogenicity.     

The immunodominant 90-kilodalton protein is localized on the terminal tip structure of Mycoplasma pneumoniae.

Immunoblot analysis of convalescent-phase sera of experimentally infected chimpanzees or monoclonal antibodies (MAbs) specific to the 90- and 40-kDa proteins of Mycoplasma pneumoniae indicated that both proteins were present in cytadsorbing, pathogenic strains PI-1428, M129, and FH but absent in noncytadsorbing, nonpathogenic strain M129-B176. Adsorption of convalescent-phase chimpanzee sera with virulent strain PI-1428 removed reactivity, whereas adsorption with avirulent strain M129-B176 did not remove reactivity to these two proteins. By using proteolysis and specific MAbs, we demonstrated that the 90- and 40-kDa proteins were surface exposed. Immunoelectron microscopy employing specific MAbs showed that the 90-kDa protein is localized on the terminal tip attachment apparatus. However, the MAb specific for the 40-kDa protein failed to indicate a similar localization. Nevertheless, these data, taken together, indicate that the immunodominant 90- and 40-kDa proteins are surface exposed, are localized on the terminal tip apparatus, and might be involved in the attachment mechanism.     

Hypothetical membrane mechanisms in essential tremor

Background  

Essential tremor (ET) is the most common movement disorder and its pathophysiology is unknown. We hypothesize that increased membrane excitability in motor circuits has a key role in the pathogenesis of ET. Specifically, we propose that neural circuits controlling ballistic movements are inherently unstable due to their underlying reciprocal innervation. Such instability is enhanced by increased neural membrane excitability and the circuit begins to oscillate. These oscillations manifest as tremor.     

Antibodies to 60-kilodalton heat shock protein and outer membrane protein 2 of Chlamydia pneumoniae in patients with coronary heart disease.

Evidence linking Chlamydia pneumoniae infection to atherosclerosis and to atherothrombotic events has recently emerged. A primary candidate implicated in these pathogenetic events is the 60-kDa chlamydial heat shock protein (HSP60). Another putative candidate to activate a potential proinflammatory mechanism is the chlamydial outer membrane protein 2 (OMP2). We have generated both HSP60 and OMP2 recombinant antigens in a nondenatured form and shown that (i) the two antigens were highly immunogenic in mice and (ii) murine antisera thus generated recognized the native C. pneumoniae proteins. We measured by enzyme linked immunosorbent assay (ELISA) and immunoblot assay antibody titers to the recombinant antigens in samples from 219 patients with coronary heart disease (CHD), 179 patients with unstable angina (UA), 40 patients with acute myocardial infarction (AMI), and 100 age-, sex-, and risk factor-matched healthy controls. We also examined whether anti-HSP60 and/or anti-OMP2 antibodies correlated with anti-C. pneumoniae antibodies assessed by a commercial microimmunofluorescence (MIF) assay. Immunoglobulin G (IgG), but neither IgA nor IgM, antibodies against the two recombinant proteins were detected by ELISA. In particular, anti-HSP60 antibodies were detected in >99% of CHD patients versus 0% of the controls, whereas the proportions of anti-OMP2 positive subjects were >70 and 27%, respectively. Nonetheless, among CHD patients, similar frequencies of positive subjects and titers of anti-HSP60 or anti-OMP2 antibodies were present in UA and AMI subjects. The anti-OMP2, but not the anti-HSP60, antibodies showed high specificity. Consistently, high serological correlation was observed between IgG MIF titers and IgG ELISA reactivity to OMP2 but not to HSP60. Overall, the results of this study demonstrate a strong correlation between CHD and anti-HSP60 IgG levels, as measured by our in-house ELISA. They also suggest that recombinant OMP2 ELISA, because of its high specificity and strong correlation with MIF assay, could be a candidate diagnostic marker for C. pneumoniae infection, which would be of potential usefulness for its specificity and nonsubjective nature.     

Sequence analysis of the gene encoding the Chlamydia pneumoniae DnaK protein homolog.

The antigen-coding region of a 4.2-kb PstI fragment of Chlamydia pneumoniae (pLC3), which encodes a 75-kDa immunoreactive protein recognized during human C. pneumoniae infection, was localized to a 2.0-kb EcoRI fragment. This subclone expressed an immunoreactive fusion protein of ca. 82 kDa. Nucleotide sequence analysis of the C. pneumoniae gene revealed that it consisted of a 1,980-base open reading frame with an inferred 71,550-Da protein of 660 amino acids. Putative Escherichia coli-like promoters and a ribosomal binding site were located in the 5' upstream region, and an 11-base dyad forming a stable stem-loop structure following two in-frame stop codons was identified. The C. pneumoniae 75-kDa protein is a member of the hsp70 family of heat shock proteins and has 87% amino acid similarity with the Chlamydia trachomatis protein.     

Inhibition of phagolysosome fusion is localized to Chlamydia psittaci-laden vacuoles.

Intracellular survival of Chlamydia psittaci is in part dependent on the ability of the organism to thwart phagolysosome formation. Circumvention of phagolysosome fusion could be either localized to chlamydia-laden vacuoles or generalized to all phagosomes in the host cell. To determine which of these modes is in operation the ability of chlamydia elementary and reticulate bodies to protect Saccharomyces cerevisiae from degradation in macrophage phagolysosomes was examined via acridine orange and Giemsa staining. No statistically significant difference was evident between the amount of fusion observed in coinfected macrophages and those infected with yeast cells alone. This was ot dependent on some unique interaction between the chlamydia and the yeast cells since viable count studies to determine the protection of a second organism, Escherichia coli, also failed to show significantly different amounts of inactivation of the bacteria by macrophages in the presence of C. psittaci. Therefore, the inhibition of phagolysosome fusion is localized to chlamydia-laden phagosomes.