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Infection and Immunity, January 2007, p. 497-503, Vol. 75, No. 1
0019-9567/07/$08.00+0     doi:10.1128/IAI.00935-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Hypothetical Protein Cpn0308 Is Localized in the Chlamydia pneumoniae Inclusion Membrane{triangledown}

Jianhua Luo,1 Tianjun Jia,1,2 Rhonda Flores,1 Ding Chen,1 and Guangming Zhong1*

Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78229,1 Department of Immunology, Hebei Medical University, Shijiazhuang, Hebei, China 0500172

Received 12 June 2006/ Returned for modification 16 August 2006/ Accepted 20 October 2006


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ABSTRACT
 
The hypothetical protein encoded by Chlamydia pneumoniae open reading frame cpn0308 was detected in inclusion membranes of C. pneumoniae-infected cells using antibodies raised with Cpn0308 fusion proteins. The anti-Cpn0308 antibodies did not cross-react with IncA, a known C. pneumoniae inclusion membrane protein, although the anti-Cpn0308 antibody staining overlapped with the anti-IncA antibody labeling. The labeling of the inclusion membrane by the anti-Cpn0308 antibody was specifically blocked by the Cpn0308 but not IncA fusion proteins. The Cpn0308 antigen was detectable 24 h after infection and remained in the inclusion membrane throughout the infection course.


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TEXT
 
Although both Chlamydia trachomatis and C. pneumoniae are human pathogens and share a common obligate intravacuolar biphasic life cycle (9, 10, 18), they differ in tissue tropism. C. trachomatis primarily infects ocular and urogenital tissues, leading to preventable blindness in some developing nations and becoming a major cause of sexually transmitted bacterial diseases in the developed world. The species C. pneumoniae infects the human respiratory tract. Although the respiratory infection with C. pneumoniae is often asymptomatic in immune-competent individuals (8, 18), it is associated with pathologies in other systems, including the cardiovascular system (3, 4, 14, 16, 17, 19, 23). The chlamydial ability to replicate inside a cytoplasmic vacuole (designated inclusion) of host cells likely contributes to the chlamydial pathogenicity (2, 18). In order to establish and maintain a successful intravacuolar growth, Chlamydia has to exchange both materials and signals with host cells via the inclusion membrane. For example, Chlamydia has possessed the capacity of both importing nutrients and metabolic intermediates from host cells (5, 11, 12, 22, 26) and secreting chlamydial factors into host cells (6, 25, 29, 30). However, the pathways that Chlamydia organisms use to interact with host cells are largely unknown. Since the first identification of Chlamydia-encoded proteins in the inclusion membrane (designated Inc proteins [20]), it has been hypothesized that the Inc proteins may either participate in the transmission of signals and materials or serve as effector molecules during chlamydial interactions with host cells (13, 21). Identification of Inc proteins may facilitate our understanding of how Chlamydia interacts with host cells. Therefore, searching for novel inclusion membrane proteins has become a hot topic under intensive investigation. We have recently employed an anti-fusion protein antibody approach for identifying new inclusion membrane proteins in C. pneumoniae-infected cells and found that the hypothetical protein Cpn0308 is localized in the C. pneumoniae inclusion membrane.

We expressed the hypothetical proteins encoded by open reading frames (ORFs) from the C. pneumoniae AR39 genome as fusion proteins with GST (glutathione-S-transferase) as the N-terminal fusion tag (24). Mice were immunized with the purified chlamydial fusion proteins to raise both polyclonal antisera (pAb) and monoclonal antibodies (MAb) (31). The mouse antibodies were used to localize the endogenous proteins in C. pneumoniae-infected cells via an indirect immunofluorescence assay (30). The antibodies raised against the Cpn0308 fusion protein labeled the C. pneumoniae inclusion membrane (Fig. 1). Both the anti-Cpn0308 pAb and MAbs consistently detected a dominant inclusion membrane signal similar to the signal revealed by the anti-IncA, but not the anti-CPAF, anti-MOMP, or anti-HSP60 antibodies. We further verified the inclusion membrane localization of Cpn0308 using confocal microscopy. The anti-Cpn0308 labeling did not colocalize with CPAF, MOMP, or HSP60 but clearly overlapped with the anti-IncA labeling, even at different focal points along the Z axis. IncA, encoded by the C. pneumoniae ORF cpn0186, is a known inclusion membrane protein in C. pneumoniae-infected cells (1, 15). The above observations have demonstrated that Cpn0308, like IncA, is also an inclusion membrane protein. We further used several approaches to confirm the antibody binding specificities. The anti-Cpn0308 MAbs reacted with the GST-Cpn0308 but not the GST-IncA or GST-CPAFcp fusion proteins, although all fusion proteins were detectable by their corresponding homologous antibodies (data not shown). The antibodies raised with the chlamydial GST fusion proteins were further reacted with the red fluorescence protein (RFP)-C. pneumoniae fusion proteins expressed in transfected cells. The anti-Cpn0308 antibodies detected the RFP-Cpn0308 but not the RFP-IncA fusion proteins (Fig. 2A). More importantly, the detection of the endogenous antigens in the C. pneumoniae-infected cells by the anti-Cpn0308 and anti-IncA antibodies was blocked by the corresponding homologous but not the heterologous GST fusion proteins in an immunofluorescence assay (Fig. 2B). Finally, a Western blot assay was carried out to further evaluate the anti-Cpn0308reactivity with the C. pneumoniae-infected cell lysates (Fig. 2C). The anti-Cpn0308 antibody only recognized a protein band corresponding to the endogenous Cpn0308 from the lysates of C. pneumoniae-infected HeLa cells but not HeLa cells alone or C. trachomatis-infected cells. Together, the above experiments have demonstrated that the anti-Cpn0308 antibody specifically detected the Cpn0308 antigen in the inclusion membrane of the C. pneumoniae-infected cells. When the expression of Cpn0308 protein was monitored along the infection time, Cpn0308 was first detected 24 h after infection and remained in the inclusion membrane throughout the infection course (Fig. 3).


Figure 1
Figure 1
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  		FIG.1. Detection of Cpn0308 in C. pneumoniae inclusion membrane. HeLa cells were infected with C. pneumoniae AR39 organisms at a multiplicity of infection of 0.5 in the presence of 2 mg/ml of cycloheximide for 72 h. The infected cultures grown on coverslips were processed for various immunostainings. (A) Cpn0308 was probed with a mouse antiserum (panel a) and monoclonal antibodies (MAb) 2D7 (b), 3A6 (c), 3H5 (d), and 5E10 (e), all of which were raised with the GST-Cpn0308 fusion protein and visualized with a Cy3-conjugated goat anti-mouse immunoglobulin G (IgG) (red). A rabbit anti-AR39 antiserum (R12AR39) together with a Cy2-conjugated goat anti-rabbit IgG (green) was used to visualize the C. pneumoniae organisms, and Hoechst was used to visualize DNA. (B) The AR39 organism-infected cell samples were costained with the anti-Cpn0308 MAb 2D7 (green) and DNA Hoechst dye (blue) in combination with antibodies recognizing other C. pneumoniae reference proteins, including CPAFcp, IncA, MOMP, and HSP60 (all in red). Images of the immunostainings were obtained using an AX70 fluorescence microscope equipped with a charge-coupled device camera as described previously (30). (C) The samples were costained as described for panel B, except that the DNA dye was replaced with the rabbit anti-AR39 antiserum R12AR39 plus a goat anti-rabbit IgG conjugated with Cy5 (blue). The images were acquired sequentially one color at a time and overlaid in tri-color using a confocal microscope (Olympus; provided by the UTHSCSA imaging core). (D) The colocalization of Cpn0308 and IncA was further analyzed at three different focal points along the Z axis using confocal microscopy. Note that the anti-Cpn0308 antibodies detected strong inclusion membrane signals similar to and overlapping that obtained with the anti-IncA but not the other antibodies. DIC, differential interference contrast.


Figure 2
Figure 2
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  		FIG.2. The anti-Cpn0308 antibody detection of C. pneumoniae inclusion membrane is specific. (A) HeLa cells transfected with the recombinant plasmids pDsRed-C1 monomer/Cpn0308 or Cpn0186 (IncA; expressed as RFP fusion proteins; red) for 24 h were processed for immunostaining with various antibodies listed along the left side of the figure (green) plus Hoechst (blue). It is clear that the antibodies only labeled the corresponding homologous gene-transfected cells without cross-reacting with the unrelated gene-transfected cells. (B) Anti-Cpn0308 MAb 2D7 and anti-IncA MAb 2B12.1 were preabsorbed with or without the GST fusion proteins listed on top of the figure, followed by immunostaining as described in the legend to Fig. 1B. Note that antibody staining was only blocked by preabsorption with the corresponding homologous GST fusion proteins. (C) HeLa cells with or without either C. trachomatis (L2) or C. pneumoniae (AR39) infection were harvested using a sodium dodecyl sulfate (SDS) buffer and loaded onto a SDS polyacrylamide gel along with the GST-Cpn0308 fusion protein sample, as indicated in the figure. The gel-resolved proteins bands were blotted onto nitrocellulose membrane for Western analysis using anti-mammalian HSP70, anti-chlamydial HSP60, and anti-Cpn0308 as the primary antibodies as indicated on top of the figure, and the primary antibody reactivities were detected with a goat anti-mouse IgG conjugated with horseradish peroxidase and visualized with standard enhanced chemiluminescence. MW, molecular size; IFA, indirect immunofluorescence assay.


Figure 3
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  		FIG. 3. Time course detection of Cpn0308. HeLa cells were infected with AR39 organisms for various periods of time as indicated on top of the figure, and the culture samples were subjected to immunostaining with the MAb 2D7 for Cpn0308 (red) and the rabbit antibody R12AR39 for AR39 organisms (green) and Hoechst dye for DNA (blue). The images were acquired using a conventional fluorescence microscope as described in the legend to Fig. 1B. Note that Cpn0308 protein was detected as early as 24 h after infection and remained in the inclusion membrane during the entire infection course.

In the current study, we have confirmed the localization of IncA in the C. pneumoniae inclusion membranes and, more importantly, identified the hypothetical protein Cpn0308 as a novel inclusion protein using both polyclonal antisera and monoclonal antibodies. The antibodies raised against IncA and Cpn0308 both labeled the C. pneumoniae inclusion membranes. However, no cross-reactivity was found between these two, which is consistent with the finding that there is no significant amino acid sequence homology between IncA and Cpn0308 (http://www.ncbi.nlm.nih.gov/BLAST/BLAST.cgi). Cpn0308 is a 133-amino-acid protein encoded by a hypothetical ORF flanked by two known genes, glgP (alpha glycan phosphorylase) and dnaA (DnaA replication protein; http://www.stdgen.lanl.gov/), in the C. pneumoniae genome. Cpn0308 seems to be unique for the C. pneumoniae species, since no homologues are found in any other chlamydia or non-chlamydia species by BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/BLAST.cgi). It is not clear why cpn0308 was not listed as a C. pneumoniae-specific hypothetical ORF (http://www.stdgen.lanl.gov/). Despite the lack of primary sequence homology, computer-based prediction programs did predict Cpn0308 as an Inc protein (28), and Cpn0308 was secretable in a heterologous type III system (27). We have now provided the first experimental evidence directly demonstrating the localization of Cpn0308 in the C. pneumoniae inclusion membrane. Since not all predicted inclusion membrane proteins are localized in the inclusion membrane (1) and not all experimentally demonstrated inclusion membrane proteins were predicted by computer-based methods (1, 7, 21, 28), it is both necessary and significant to experimentally search for new inclusion membrane proteins in Chlamydia-infected cells. Once sufficient inclusion membrane proteins are identified, we may be able to more precisely unravel the structural features of the inclusion membrane proteins and further understand the functions of these proteins.


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ACKNOWLEDGMENTS
 
This work was supported in part by grants (to G. Zhong) from the National Institutes of Health.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229. Phone: (210) 567-1169. Fax: (210) 567-0293. E-mail: Zhongg{at}UTHSCSA.EDU. Back

{triangledown} Published ahead of print on 13 November 2006. Back

Editor: J. B. Bliska


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Infection and Immunity, January 2007, p. 497-503, Vol. 75, No. 1
0019-9567/07/$08.00+0     doi:10.1128/IAI.00935-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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