Cytokinesis is blocked in mammalian cells transfected with Chlamydia trachomatis gene CT223
© Alzhanov et al; licensee BioMed Central Ltd. 2009
Received: 28 August 2008
Accepted: 05 January 2009
Published: 05 January 2009
The chlamydiae alter many aspects of host cell biology, including the division process, but the molecular biology of these alterations remains poorly characterized. Chlamydial inclusion membrane proteins (Incs) are likely candidates for direct interactions with host cell cytosolic proteins, as they are secreted to the inclusion membrane and exposed to the cytosol. The inc gene CT223 is one of a sequential set of orfs that encode or are predicted to encode Inc proteins. CT223p is localized to the inclusion membrane in all tested C. trachomatis serovars.
A plasmid transfection approach was used to examine the function of the product of CT223 and other Inc proteins within uninfected mammalian cells. Fluorescence microscopy was used to demonstrate that CT223, and, to a lesser extent, adjacent inc genes, are capable of blocking host cell cytokinesis and facilitating centromere supranumeracy defects seen by others in chlamydiae-infected cells. Both phenotypes were associated with transfection of plasmids encoding the carboxy-terminal tail of CT223p, a region of the protein that is likely exposed to the cytosol in infected cells.
These studies suggest that certain Inc proteins block cytokinesis in C. trachomatis-infected cells. These results are consistent with the work of others showing chlamydial inhibition of host cell cytokinesis.
Chlamydiae are obligate intracellular bacteria that replicate in a cytoplasmic vacuole (the inclusion) within host cells [1, 2]. All Chlamydia spp. are significant pathogens, and infections occur in a wide variety of animal species. Chlamydia trachomatis infections lead to serious mucosal diseases of humans including blinding trachoma  and diseases of the genital tract . The study of chlamydial host-pathogen relationships is complicated by the lack of a genetic system to manipulate the chlamydial genome, and thus, alternate approaches must be used to understand chlamydial virulence properties. One approach that has been particularly useful in these studies is the use of surrogate genetic systems including yeast, mammalian cells, and other bacterial species [5–10].
Inhibition of the host cell cycle by chlamydiae was demonstrated by early researchers [11, 12] and was expanded upon recently by Greene and Zhong . Other recent investigations have demonstrated that chlamydial infection alters the cell cycle in a variety of ways, leading to centrosomal defects  and slowing of host cell division . The molecular mechanisms leading to these changes are poorly understood. Recent studies have suggested a possible role of chlamydiae in cancers of different infected tissues [16–18] and, thus, the role of chlamydiae in alterations of cell cycle biology are of significance.
The different chlamydial species each produce a set of proteins, termed Incs, that are localized to the chlamydial inclusion membrane and exposed to the cytosol of the host cell . Each sequenced chlamydial genome encodes over 40 candidate Incs, and there are both conserved and species-specific Incs among the different chlamydiae. The demonstrated function of a limited number of Inc proteins is known [9, 20–23], but most are poorly characterized.
Chlamydia trachomatis encodes a species-specific set of Incs within orfs CT223-CT229. CT224 and CT225 have no clear homologs in any other chlamydiae, while CT223, and CT226–CT229 have homologs only in C. muridarum, a closely related chlamydial species . The localization to the inclusion membrane of the products of CT223, CT225, CT226, and CT229 was confirmed via fluorescence microscopy . Transcription of CT228 and CT229 is initiated very early following infection of cells  and, therefore, the encoded proteins are hypothesized to be essential to early inclusion development. Recent work by Rzomp et al. demonstrated that CT229p associated with Rab4 in a two-hybrid assay and in mammalian cells , but the function of any of the proteins encoded by the other orfs in this group is not known. To address possible functions of candidate C. trachomatis Incs, we used a plasmid transfection system to introduce genes encoding different Incs into mammalian cells, and then characterized any resulting phenotypes with fluorescence microscopy. These investigations demonstrated that transfection with plasmids expressing CT223, and to a lesser extent, CT224 and CT225, led to a block in host cell cytokinesis. Cells transfected with plasmids encoding CT223p led to an inhibition of cytokinesis that was similar to that seen in C. trachomatis- infected cells. The block was shown to be associated with the carboxy-terminal end of CT223p, the region of the protein hypothesized to be exposed to the host cell cytosol at the surface of the inclusion. Alleles of CT223 from different strains yielded similar inhibition of cytokinesis, consistent with the inhibitory effect on cytokinesis by all tested C. trachomatis serovars .
Chlamydial strains, DNA preparation, and host cell lines
Elementary bodies (EB) of Chlamydia trachomatis strains D/UW3, J/UW36, J9235, J(s)1980, J(s)6686 and LGV-434, C. caviae strain GPIC, and C. muridarum strain Nigg were used in infections and/or for preparation of genomic DNA samples that were used as PCR templates. Genomic DNA was prepared by boiling EB suspensions in a water bath for 10 minutes followed by removal of bacterial debris via centrifugation. HeLa or McCoy cells (ATCC) were cultured in Minimal Essential Medium supplemented with 10% fetal bovine serum and 10 μg/ml gentamicin and grown at 37°C in 5% CO2.
Construction of plasmids and oligonucleotides used in transfection experiments
Oligonucleotides used for amplification of inc genes by PCR.
DA71 Eco RI
CT223 C. trachomatis
DA97 Kpn I
CT223 C. trachomatis
DA116 Eco RV
CT223 C. trachomatis
DA119 Eco RV
CT223/179 C. trachomatis
DA121 Eco RV
CT223/91 C. trachomatis
DA122 Eco RI
CT223/56 C. trachomatis
DA76 Bam HI
CT229 C. trachomatis
DA99 Kpn I
CT229 C. trachomatis
DA98 Bam HI
CT228 C. trachomatis
DA74 Pst I
CT228 C. trachomatis
DA 131 Eco RI
CT227 C. trachomatis
DA 132 Eco RV
CT227 C. trachomatis
DA 129 EcoR I
CT226 C. trachomatis
DA130 Eco RV
CT226 C. trachomatis
DA127 Eco RI
CT225 C. trachomatis
DA128 Eco RV
CT225 C. trachomatis
DA125 Eco RI
CT224 C. trachomatis
DA126 Xho l
CT224 C. trachomatis
DA34 Eco RI
incA C. trachomatis
DA21 Eco RV
incA C. trachomatis
DA22 Eco RI
incC C. trachomatis
DA23 Eco RV
incC C. trachomatis
JL003 Eco RI
incA C. caviae
JL010 Eco RV
incA C. caviae
JL007 Eco RI
incB C. caviae
JL006 Eco RV
incB C. caviae
JL014 Eco RI
incC C. caviae
JL013 Eco RV
incC C. caviae
DA114 Eco RI
DA115 Eco RV
Antibodies, transfection experiments and immunofluorescence microscopy
Monoclonal antibody recognizing chlamydial lipopolysaccharide was a gift from Harlan Caldwell of the Rocky Mountain laboratories, Hamilton, MT. Monoclonal antibody A57B9 (anti-HSP60) recognizes a genus common epitope on chlamydial HSP60 protein . Monoclonal antibodies used in the analysis of CT223p localization in C. trachomatis-infected HeLa or McCoy cells were produced and used as previously described . Rabbit polyclonal anti-CT223p antisera was generated against the peptide sequence NH3-NGINDLSPAPEAKKTGSGL and were produced commercially (Proteintech, Chicago, IL). For these experiments, cells were infected with chlamydiae and incubated for time periods indicated in the figure legends. Cells were then fixed with 100% methanol and used for immunofluorescence.
Transfection of plasmids into HeLa or McCoy cells grown on sterile glass coverslips was conducted using Lipofectamine 2000 (Gibco) according to the manufacturer's instructions. Transfected cells were incubated for 36 hours and then fixed with methanol. The efficiency of transfection was determined by labeling with monoclonal anti-6-His antibody (Clontech) and secondary FITC or TRITC fluorescent antibodies (Southern Biotechnology Associates) to detect the product of the transgene. Monoclonal anti-γ-tubulin antibodies (Sigma) were used to detect centrosomes. Cells expressing gfp were analyzed without labeling. Coverslips were examined under 1000× magnification using a Leica fluorescence microscope and images were collected using the SPOT digital camera system (Diagnostic Instruments Inc., Sterling Heights, MI). The rates of cells with a polynuclear phenotype were determined by counting transfected cells with two or more nuclei among the total population of transfected cells.
The number of transfected cells having a polynuclear phenotype was evaluated in at least three independent experiments for each plasmid construct tested. A total of at least 500 individual transfected cells were counted for each tested plasmid construct. Standard deviations were calculated for each individual plasmid construct examined and the significance of differences between means was evaluated using both the Student's t-test and the Kruskall-Wallis test, as calculated using the Instat software program (GraphPad Software, San Diego, CA).
Examination of the association between infection and a reduced rate of cytokinesis
Distribution of CT223p at the inclusion membrane varies in different C. trachomatis strains
We have shown that CT223p in certain strains – including J(s)1980 and J(s)6686 – is not recognized in fluorescent microscopic analysis using two different anti-CT223p monoclonal antibodies [25, 29] (Fig. 2F, H). However, peptide-specific polyclonal antibodies demonstrate that the protein is produced in all tested strains (Fig. 2E, G).
Delivery of full length and carboxy-terminal C. trachomatis CT223p to the host cell cytosol alters host cell phenotype
The multinuclear phenotype was manifested by the carboxy-terminal 179 amino acids of CT223p (Fig. 4). A reduced but still significant level of multinuclear cells were identified in cells transfected with a plasmid encoding only the carboxy-terminal 56 amino acids of CT223p, but, transfection of a plasmid encoding an internal fragment of CT223p (CT223/CT91p) did not lead to a significant level of multinuclear cells. These data suggest that the domain of the protein responsible for blocking cytokinesis was present in the carboxy-terminal 56 amino acids.
Cytosolic expression of other incs
The orf encoding CT223p is within a likely operon encoding known and candidate inc genes CT223–CT227, and is adjacent to a very early operon containing two inc genes (CT228 and CT229). We tested each orf in these operons for an association with a polynuclear phenotype. Each orf was expressed in transfected cells and there was no apparent difference in expression level, based on fluorescence microscopy of transfected cells (not shown). Orfs CT224 and CT225 also were associated with a reduced but still statistically significant percentage of polynuclear cells in a transfected population (Fig. 4). None of the other tested orfs were associated with an increased number of polynuclear cells.
The same approach was used for testing the effects on cell cytokinesis of other Inc proteins. HeLa or McCoy cells transfected with plasmids encoding each protein from C. trachomatis incA and incC, and C. caviae incA, incB and incC were compared with cells expressing full length and truncated CT223. None of these plasmids led to an increase in polynuclear cells relative to controls (Fig. 4).
The CT223 coding sequence from different C. trachomatis strains encode proteins with up to 5% difference in amino acid sequence (22). We therefore tested plasmids encoding CT223p from strains with known amino acid sequence differences for their ability to block cytokinesis. Transfection of plasmids encoding each CT223p sequence was associated with an increase in multinucleate cells (Fig. 5). In contrast, transfection of a plasmid expressing C. muridarum TC0495, which is a syntenous, apparent CT223 homolog (less than 30% predicted amino acid sequence identity), did not lead to an increase in the number of multinucleate cells relative to controls (Fig. 5).
Cells producing CT223p and CT223/179p have increased numbers of centrosomes
CT223p is a chlamydial Inc protein that varies antigenically but is produced by all tested C. trachomatis isolates. The protein was detected in our analysis at 8 h p.i. (not shown) and was abundant on the inclusion membrane at all subsequent time points. This is consistent with the transcriptional profiling of Belland et al. , who demonstrate that the transcript for CT223 is first detected 8 h p.i. and remains actively transcribed for the rest of the developmental cycle. The gene is clustered with a set of orfs (CT223–CT229) encoding known or candidate inclusion membrane proteins that are only found in the C. trachomatis and C. muridarum genomes . CT223p is localized as patches or short ribbon-like distribution in all strains examined prior to 30 h p.i. At later time points the protein is differently distributed in different strains, shown in this work in a comparison between a serovar J strain and a serovar L2 strain. Tested isolates of serovar D appear similarly to the serovar L2 strain (not shown).
The ability of C. trachomatis to affect the mammalian cell cycle has been documented by several authors. Greene and Zhong  established that infection by C. trachomatis affects host cell cytokinesis in a multiplicity of infection-dependent manner, results that were confirmed in our work (Fig. 1). Grieshaber et al.  demonstrated that chlamydial inclusions associate with the centrosome leading to increased numbers of centrosomes and chromosome segregation defects in infected cells. Molecular interactions between chlamydiae and host molecules important in cell division were explored by Balsara et al.  who showed that chlamydial infection leads to alterations in the abundance of cyclin-dependent kinases and to the cleavage of cyclin B1. However, any chlamydial proteins that might participate in the alteration of the host cell cycle have not been identified.
While it is possible that the observed multinuclear phenotype is a function of cellular fusion, as opposed to inhibition of cytokinesis, Greene and Zhong  discuss several lines of evidence that point to the latter possibility. This includes the lack of observed fusion intermediates, the presence of mitotic forms, and normal DNA synthesis in chlamydiae-infected host cells. These observations support the likelihood that cells are being blocked in a terminal state of division, as opposed to being stimulated to fusion with neighboring cells, following chlamydial infection.
CT223p was first examined as a candidate Inc protein because of the presence of an amino-terminal bi-lobed hydrophobic domain that is proposed to be a membrane anchor for Incs . Like many Incs, CT223p also contains a long carboxy-terminal tail that is largely hydrophilic. It is likely that this carboxy-terminal region of the protein is responsible for direct interactions between Incs and proteins in the host cell cytosol, a property shown to be true for tested Inc proteins [7, 21, 22]. Transfection of cells with plasmids encoding only the carboxy-terminal 179 amino acids or (to a lesser extent) the 56 carboxy-terminal amino acids of CT223p led to increased accumulation of host cell nuclei within cells. We have sequence data for CT223p from several C. trachomatis isolates and, while there is sequence variation among strains, the carboxy-terminal third of the protein is highly conserved [; data not shown].
Two other Inc proteins, CT224p and CT225p, also affected host cell cytokinesis, although the effect was less than that observed with CT223p. These proteins are encoded sequentially in the C. trachomatis genome and are unique to this species. However, the predicted protein sequences of these three proteins share very limited primary amino acid identity. In contrast, the protein product of C. muridarum orf TC0495, an apparent homolog of CT223 that is encoded in a syntenous operon  did not block cytokinesis in our assays. The degree of identity between CT223p and TC0495p is not high, and it is likely that key structures between the two proteins are not conserved in a way that allows the C. muridarum protein to affect cytokinesis in this assay. The degree of identity among CT223p, CT224p and CT225p is even lower, and, therefore, it is even less intuitive that these proteins would share a common phenotype when produced within mammalian cells. Therefore, the molecular mechanisms associated with the inhibition of cytokinesis observed in these studies remain unclear.
There are many possible steps in the complicated process of cell division that might be affected by the Incs that affect cytokinesis. The cell cycle is under control of a family of protein kinases known as Cyclin-dependent kinases (Cdks), which are under control of various regulatory proteins such as CAK and CKIs [31, 32]. Some of these proteins are differently processed or differently abundant in chlamydiae-infected vs. uninfected cultured cells . We hypothesize that CT223p and other Inc proteins directly or indirectly disrupt Cdk, cyclin, or possibly other protein functions and, thus, affect cell cycle control. We are currently using surrogate systems to identify possible host cell cycle-specific proteins that interact directly with CT223p at the inclusion membrane surface.
Plasmid-based expression of the chlamydial inclusion membrane protein CT223p caused a reduction in mammalian cell cytokinesis in vitro. Other Inc proteins had a lesser effect on cytokinesis in this assay. These results support the conclusion that Ct223 expression by C. trachomatis and localization of the protein to the inclusion membrane is associated with the observed inhibition of host cell cytokinesis in C. trachomatis-infected host cells.
This work was supported by P.H.S. grants AI42869 and AI48769, and through the Oregon State University Department of Microbiology Tartar Scholarship Fund. We thank Dr. Aishu Ramakrishnan and all members of the Rockey laboratory for technical assistance and support. Dr. Hencelyn Chu is acknowledged for coordinating the production and testing of the polyclonal anti-CT223p antisera.
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