i. Cells were then labeled with either polyclonal anti-CT223p antisera (E, G) or monoclonal anti-CT223p antibody (F, H), both of which are labeled red. Note that CT223p is labeled by the polyclonal antisera in each strain, while the monoclonal anti-CT223p does not label the protein in strain J(s)1980. We have shown that CT223p

in certain strains – including J(s)1980 and J(s)6686 – is not recognized in fluorescent microscopic analysis GSK2118436 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 Plasmids encoding CT223p from several C. trachomatis strains were transfected into both McCoy or HeLa cells and the effect on cellular cytokinesis was observed using fluorescent microscopy. Transfection with each of these plasmids led to a high proportion of multinucleate cells 30 hours post transfection (Fig. 3A). A similar phenotype was observed when cells were transfected with plasmids encoding the carboxy-terminal tail of CT223p (Fig. 3B). The average number of polynuclear cells following expression of a CT223 transgene was approximately 20%, regardless of the isolate from which the gene was amplified (Figs. 4 and 5).

In contrast, cells transfected with a plasmid encoding GFP, or cells transfected with ACP-196 an empty vector (mock transfected) as control, all had levels of polynuclear cells of approximately 2–4%. Figure 3 Cytosolic production of CT223p and CT223/179p from C. trachomatis serovar D/UW3 leads to a

multinuclear phenotype within mammalian cells. The vector pcDNA4/HisMaxC was used in each construct. Full length CT223p (panel A) and CT223/179p (panel B) were produced within cells following transfection of pcDNA4-based plasmids. Each was detected with anti-6 × His monoclonal antibodies (red). Microtubules were detected by labeling with specific anti-tubulin antibodies (green). The nuclei are labeled with DAPI (blue). Panel A; McCoy cell transfected with pcDNA4/HisMaxC encoding CT223p. Three nuclei are localized inside of a single cell expressing CT223. Panel B; McCoy cells transfected with pcDNA4/HisMaxC encoding Decitabine nmr carboxy-terminal CT223/179p. The scale bar in B indicates 10 microns for each panel. Figure 4 Quantification of multinuclear cells following expression of different inc genes in McCoy cells. This graph represents percentage of polynuclear cells among McCoy cells following transfection of pcDNA4/HisMaxC-based plasmids encoding different Inc proteins. Unless indicated, the sequences were derived from the published C. trachomatis D/UW3 genome sequence. Statistical significance is indicated with the asterisk above the individual treatment groups, as compared to pCDNA-transfected cells (Student’s t-test, p < 0.01).