Identification of Chlamydia trachomatis CT621, a protein delivered through the type III secretion system to the host cell cytoplasm and nucleus

Abstract

Chlamydiae are obligate intracellular bacteria, developing inside host cells within chlamydial inclusions. From these inclusions, the chlamydiae secrete proteins into the host cell cytoplasm. A pathway through which secreted proteins can be delivered is the type III secretion system (T3SS). The T3SS is common to several gram-negative bacteria and the secreted proteins serve a variety of functions often related to the modulation of host signalling. To identify new potentially secreted proteins, the cytoplasm was extracted from Chlamydia trachomatis L2-infected HeLa cells, and two-dimensional polyacrylamide gel electrophoresis profiles of [(35)S]-labelled chlamydial proteins from this extract were compared with profiles of chlamydial proteins from the lysate of infected cells. In this way, CT621 was identified. CT621 is a member of a family of proteins containing a domain of unknown function DUF582 that is only found within the genus Chlamydia. Immunofluorescence microscopy and immunoblotting demonstrated that CT621 is secreted late in the chlamydial developmental cycle and that it is the first chlamydial protein found to be localized within both the host cell cytoplasm and the nucleus. To determine whether CT621 is secreted through the T3SS, an inhibitor of this apparatus was added to the infection medium, resulting in retention of the protein inside the chlamydiae. Hence, the so far uncharacterized CT621 is a new type III secretion effector protein.

Fig. 1

Autoradiographs of 2D-PAGE-separated Chlamydia trachomatis L2 proteins labelled from 36 to 38 hpi with [³⁵S]-methionine/cysteine in the presence of cycloheximide. (a) Cytoplasmic extract of infected HeLa cells. Hollow arrows indicate CPAF C- and N-terminal parts. The solid arrow indicates the 26-kDa CT621 cleavage product. Regions surrounding these protein spots are boxed. (b) Whole lysate of infected HeLa cells. Boxed regions correspond to the regions mentioned in (a). (c) Enlargement of the boxed regions in (a), picturing the protein spots representing the CT621 cleavage product and both parts of CPAF. (d) Enlargement of the boxed regions in (b).

Fig. 2

Complete amino acid sequence of the Chlamydia trachomatis L2 protein CT621. The DUF582 domain is marked with a dashed line; the leucine zipper is double-underlined, and peptides identified with LC-MS/MS are shown in bold.

Fig. 3

Phylogenetic tree of CT621 protein homologues. Based on a multiple alignment performed in clustalx, a phylogenetic tree was constructed with the minimum evolution method in mega 3.1. The five Chlamydia trachomatis serovar L2 homologues are shown in bold. The abbreviations used are listed: Chlamydia trachomatis serovars: L2 (CtrL2), D (CtrD) and A (CtrA); Chlamydia pneumoniae isolates: CWL029 (CpnCWL), AR39 (CpnAR), J138 (CpnJ), TW-183 (CpnTW), Chlamydia muridarum (Cmu), Chlamydia caviae (Cca), Chlamydia felis (Cfe) and Chlamydia abortus (Cab). Where identical sequences are seen, only one isolate is listed.

Fig. 4

Subcellular localization of CT621 (a, c) and CPAF (b, d) in Chlamydia trachomatis L2-infected HeLa cells fixed with paraformaldehyde/glutaraldehyde at 46 hpi. (a, b) CT621 and CPAF are visualized in double stainings using antibodies directed against each of these proteins and against MOMP. The presence of CT621 and CPAF was revealed using FITC (green)-conjugated secondary antibodies and MOMP using rhodamine (red)-conjugated secondary antibodies. DNA was stained with DAPI (blue). (c, d) FITC channel shown separately. (e, f) Corresponding Nomarsky images. Scale bar=10 μm.

Fig. 5

Immunofluorescence microscopy of Chlamydia trachomatis L2-infected cells fixed with paraformaldehyde/glutaraldehyde at different points in time after infection. (a–e) Double stainings with primary antibodies against CT621 and MOMP, visualized using FITC (green)-conjugated and rhodamine (red)-conjugated secondary antibodies, respectively. (f–j) FITC channel shown separately. (k–o) Corresponding Nomarsky images. White arrows indicate an uninfected cell in which neither CT621 nor MOMP can be detected (c, h and m). CT621 secretion is initiated between 32 and 34 hpi. Scale bar=10 μm.

Fig. 6

Immunoblotting with CT621-specific antibodies. The inserted scale is created using stained Mark 12 molecular size markers. Polyvinylidene fluoride membranes containing EB: purified Chlamydia trachomatis EB; IC, lysate of infected cells; UC, lysate of uninfected cells; I, cytoplasmic extract; II, membrane/organelle/EB fraction; III, nuclear fraction; and IV, cytoskeletal fraction were reacted with anti-CT621 polyclonal antibodies.

Fig. 7

Immunofluorescence microscopy showing the effects of addition of 10 μM of the T3S inhibitor, C1, to Chlamydia trachomatis L2-infected cells at 30, 32 or 34 hpi. At 36 hpi, all the cells were fixed with paraformaldehyde/glutaraldehyde. (a–c) Double stainings with primary antibodies against CT621 and MOMP, visualized using FITC (green)-conjugated and rhodamine (red)-conjugated secondary antibodies, respectively. (d–f) FITC channel shown separately. (g–i) Corresponding Nomarsky images. Scale bar=10 μm.