A chlamydial type III translocated protein is tyrosine-phosphorylated at the site of entry and associated with recruitment of actin

Abstract

The obligate intracellular bacterium Chlamydia trachomatis rapidly induces its own entry into host cells. Initial attachment is mediated by electrostatic interactions to heparan sulfate moieties on the host cell, followed by irreversible binding to an unknown secondary receptor. This secondary binding leads to the recruitment of actin to the site of attachment, formation of an actin-rich, pedestal-like structure, and finally internalization of the bacteria. How chlamydiae induce this process is unknown. We have identified a high-molecular-mass tyrosine-phosphorylated protein that is rapidly phosphorylated on attachment to the host cell. Immunoelectron microscopy studies revealed that this tyrosine-phosphorylated protein is localized to the cytoplasmic face of the plasma membrane at the site of attachment of surface-associated chlamydiae. The phosphoprotein was isolated by immunoprecipitation with the antiphosphotyrosine antibody 4G10 and identified as the chlamydial protein CT456, a hypothetical protein with unknown function. The chlamydial protein (Tarp) appears to be translocated into the host cell by type III secretion because it is exported in a Yersinia heterologous expression assay. Phosphotyrosine signaling across the plasma membrane preceded the recruitment of actin to the site of chlamydial attachment and may represent the initial signal transduced from pathogen to the host cell. These results suggest that C. trachomatis internalization is mediated by a chlamydial type III-secreted effector protein.

Fig. 1.

Detection of a unique tyrosine-phosphorylated protein in C. trachomatis-infected cells. (A) Mock-infected (Unt) and C. trachomatis serovar L2- or D-infected HeLa cells were incubated at 37°C for 30 min. Total cell lysates were separated by SDS/PAGE, and immunoblots were probed with mAb 4G10. The asterisk indicates the unique phosphoproteins. Molecular mass in kDa is indicated on the left. (B) HeLa cells were infected with L2 or mock-infected (Unt) for the indicated times (in hours, except where ′= minute) and subjected to SDS/PAGE and immunoblotting. The immunoblot was split, and upper blot was probed with 4G10 and lower blot was probed with anti-MOMP mAb I-45. (C) HeLa cells were infected with L2 with the indicated MOI for 30 min and immunoblotted against 4G10. (D) HeLa cells were infected (L2) or left uninfected (Unt) for 30 min, and total cell lysates were subjected to 2D gel electrophoresis and probed with 4G10. The arrow identifies the unique phosphoprotein. Molecular mass markers (kDa) are indicated.

Fig. 2.

Immunoelectron microscopy reveals polar localization of 4G10 with EBs. HeLa cells were infected with L2 EBs (MOI ≈ 300) for 5 or 30 min. Coverslips were stained with 4G10 and visualized with an immunoperoxidase-conjugated secondary antibody. Arrowheads indicate areas of positive 4G10 reactivity. (Bar = 500 nm.)

Fig. 3.

Identification of the unique phosphoprotein as hypothetical protein CT456. (A) The complete protein sequence for L2 CT456. Peptide fragments that match the serovar D CT456 protein sequence are underlined and sequenced peptides are boxed. Brackets indicate the repeat region. (B) HeLa cells were mock-(lanes 1 and 2) or L2-infected (MOI ≈ 1,000; lanes 3 and 4) for 30 min, and cell lysates were immunoprecipitated with 4G10 (lanes 2 and 4) or the irrelevant isotype control mAb 13-3 (lanes 1 and 3) and separated by SDS/PAGE. Immunoblots were probed with 4G10, rabbit preimmune sera, or a rabbit polyclonal antiserum prepared against CT456. Molecular mass markers (kDa) are indicated. (C) An immunoblot of L2 EB total lysates was probed with anti-CT456 pAb (lane 2) or preimmune sera (lane 1).

Fig. 4.

CT456 is secreted by a type III system. WT Y. pseudotuberculosis YPIII pIB102 (WT) or a type III secretion null strain YPIII pIB68 (yscS) expressing CT456-FT were grown in the presence (lanes 1, 2, 5, and 6) or absence (lanes 3, 4, 7, and 8) of 2.5 mM Ca²⁺. Cultures were induced at 37°C for 4 h and fractionated into cell-free supernatants (CS); whole cells (WC) were then subjected to SDS/PAGE. Immunoblots were probed with α-Flag M2 to detect CT456-FT, α-YopD, or α-LcrH.

Fig. 5.

CT456 is not exposed on the EB surface but is translocated and phosphorylated before actin recruitment. (A) HeLa cells were infected with L2 at 4°C, the medium was replaced, and the cultures were then held at 4°C or shifted to 37°C for 30 min. Cells were fixed and stained by indirect immunofluorescence with anti-CT456 and anti-MOMP mAb L2-I-10 or 4G10. (Bar = 1 μm.) (B) Cos7 cells were electroporated with actin-EGFP, microinjected with Alexa Fluor 660-labeled 4G10, and infected with CMTMR-labeled L2 EBs. Images of a GFP-expressing, microinjected, and infected cell were acquired every 2 s by spinning-disk confocal microscopy. The timeline begins with the first visualization of EB fluorescence in contact with the cell. The EB is indicated by the arrow. A representative experiment is shown. (Bar = 1 μm.)

Fig. 6.

Cells expressing full-length CT456-EGFP colocalize with phalloidin and 4G10. HeLa cells expressing CT456-EGFP or CT229-EGFP were fixed, and the EGFP signal was viewed in conjunction with staining of actin with phalloidin or indirect immunofluorescence of 4G10. (Bar = 10 μm.)