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 mTOR inhibitor 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 https://www.selleckchem.com/products/BIBW2992.html 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 LXH254 cost 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 Methamphetamine 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).