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 [19]. 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 LY2603618 nmr 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 selleck CT226-CT229 have homologs only in C. muridarum, a closely related chlamydial species [24]. The localization to the inclusion membrane of the products of CT223, CT225, CT226, and CT229 was confirmed via fluorescence microscopy [25]. Transcription of CT228 and CT229 is initiated very early following infection of cells [26] 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 [20], but the

function Apoptosis Compound Library high throughput 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 Sucrase 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 [13]. Methods 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.