Tyrosine phosphorylation of the chlamydial effector protein Tarp is species specific and not required for recruitment of actin

Abstract

Chlamydiae are obligate intracellular pathogens that efficiently induce their endocytosis by susceptible eukaryotic host cells. Recently, a Chlamydia trachomatis type III secreted effector protein, Tarp, was found to be translocated and tyrosine phosphorylated at the site of entry and associated with the recruitment of actin that coincides with endocytosis. C. trachomatis Tarp possesses up to six direct repeats of approximately 50 amino acids each. The majority of the tyrosine residues are found within this repeat region. Here we have ectopically expressed distinct domains of Tarp in HeLa 229 cells and demonstrated that tyrosine phosphorylation occurs primarily within the repeat region, while recruitment of actin is mediated by the C-terminal domain of the protein. A comparison of other sequenced chlamydial genomes revealed that each contains an ortholog of Tarp, although Chlamydia muridarum, Chlamydophila caviae, and Chlamydophila pneumoniae Tarp lack the large repeat region. Immunofluorescence and immunoblotting using an antiphosphotyrosine antibody show no evidence of phosphotyrosine at the site of entry of C. muridarum, C. caviae, and C. pneumoniae, although each species similarly recruits actin. Ectopic expression of full-length C. trachomatis and C. caviae Tarp confirmed that both recruit actin but only C. trachomatis Tarp is tyrosine phosphorylated. The data indicate that the C-terminal domain of Tarp is essential for actin recruitment and that tyrosine phosphorylation may not be an absolute requirement for actin recruitment. The results further suggest the potential for additional, unknown signal transduction pathways associated specifically with C. trachomatis.

FIG. 1.

Ectopic expression of C. trachomatis GFP-Tarp domains indicates tyrosine phosphorylation of the repeat region of Tarp. (A) Schematic of the C. trachomatis L2 Tarp domains expressed as C-terminal GFP fusions. (B) Immunoblots of protein extracts from HeLa 229 cells transfected with the various enhanced GFP (EGFP) fusion constructs. F, full-length Tarp; N, N-terminal domain; R, repeat region; C, C-terminal domain; E, EGFP vector control; H, uninfected HeLa cells. Blots were probed with MAb 4G10 for tyrosine phosphorylation and anti-GFP to confirm expression and relative migration. Asterisks identify tyrosine phosphorylation of TARP and the repeat region. An apparent degradation product of the repeat region is also detected. Please note that the repeat region fusion migrates aberrantly on SDS-PAGE, as does native Tarp (11). The same Kpn-Bam insert was also cloned into pRsetB for expression in E. coli, and the product was confirmed as the Tarp repeat region by matrix-assisted laser desorption ionization-time of flight mass spectroscopy (data not shown). Background tyrosine-phosphorylated host protein bands seen in HeLa cells are apparent in all lanes probed with MAb 4G10.

FIG. 2.

Immunofluorescent staining of C. trachomatis Tarp-enhanced GFP (EGFP) constructs for tyrosine phosphorylation. The constructs described in the legend to Fig. 1 were expressed in HeLa 229 cells and stained for tyrosine phosphorylation using MAb 4G10 with an Alexa Fluor 594-conjugated anti-mouse IgG secondary antibody or stained for actin recruitment using Texas Red phalloidin. Full-length Tarp and the C-terminal domain form aggregates in the host cell. The N-terminal domain and repeat region show diffuse localization throughout the cytoplasm and nucleus. CT229, an inclusion membrane protein (1, 34) expressed as a negative control, also forms aggregates within the cytoplasm. Bar, 10 μm.

FIG. 3.

Alignment of Tarp orthologs from C. trachomatis serovars L2 and D, C. muridarum, C. caviae, and C. pneumoniae. Sequences of C. trachomatis serovar L2 (LGV-434) CT456 (11) and serovar D (UW3-Cx) CT456 (35), Chlamydia muridarum (MoPn) TC0741 (27), Chlamydophila caviae (GPIC) CCA00170 (28), and Chlamydophila pneumoniae (CWL029) (CPn) CPn0572 (22) were aligned using Clustal W, version 1.82, multiple-sequence alignment software (http://www.ebi.ac.uk/clustalw). Asterisks indicate identities.

FIG. 4.

Actin is recruited to the site of entry by C. trachomatis, C. muridarum, C. caviae, and C. pneumoniae EBs. HeLa 229 cells expressing GFP-actin were infected with CMTMR-labeled (red) C. trachomatis serovars L2 and D, C. muridarum (MoPn), C. caviae (GPIC), and C. pneumoniae (Cpn) EBs and examined by spinning disk confocal microscopy for recruitment of actin. All chlamydial species and serovars examined demonstrated robust but transient recruitment of actin to the site of entry. The figure is a composite of single frames taken from a time-lapse series for each strain or species. Bar, 5 μm.

FIG. 5.

Immunofluorescent staining for tyrosine phosphorylation in association with C. trachomatis L2 and D, C. muridarum (MoPn), C. caviae (GPIC), and C. pneumoniae (Cpn) EBs during internalization by HeLa 229 cells. Cultures at 1 h postinfection were costained by indirect immunofluorescence for EBs using a rabbit polyclonal antiserum and tyrosine phosphorylation with MAb 4G10. Tyrosine phosphorylation is not seen in association with C. muridarum, C. caviae, and C. pneumoniae EBs but is readily observed with C. trachomatis serovars L2 and D. Bar, 5 μm.

FIG. 6.

C. muridarum, C. caviae, and C. pneumoniae Tarp is not tyrosine phosphorylated. Total protein lysates were collected from HeLa cells that were either mock-infected (Unt) or infected (MOI, ∼100) with C. trachomatis L2 or D, C. muridarum (MoPn), C. caviae (GPIC), or C. pneumoniae (CPn) for 1 h at 4°C (time = 0) or at 30 min post-temperature shift to 37°C. Equal volumes of parallel mock-infected or infected cultures were loaded on each lane. Immunoblots were probed with 4G10 and visualized by chemiluminescence. Asterisks indicate the positions of tyrosine-phosphorylated Tarp; molecular masses in kilodaltons are indicated on the left.

FIG. 7.

C. trachomatis Tarp secreted from Yersinia pseudotuberculosis is translocated via type III secretion into HeLa cells and tyrosine phosphorylated. Immunoblots of HeLa cells infected with Yersinia pseudotuberculosis MEKA (translocation competent) and MEKBA (translocation incompetent) vector controls or expressing C. trachomatis L2 full-length Tarp or Tarp with the repeat domain deleted are shown. Whole-cell lysates were probed for tyrosine phosphorylation with MAb 4G10 (A) or for Tarp expression with polyclonal rabbit anti-Tarp antibody (B). Asterisks indicate the positions of full-length Tarp; the diamond shows the position of the truncated product.

FIG. 8.

Staining of C. caviae Tarp-enhanced GFP constructs for tyrosine phosphorylation and actin recruitment. Full-length C. caviae (GPIC) Tarp was expressed in HeLa 229 cells and stained for tyrosine phosphorylation using MAb 4G10 with an Alexa Fluor 594-conjugated anti-mouse IgG secondary antibody. Actin recruitment was detected using Texas Red phalloidin. Full-length C. trachomatis Tarp-enhanced GFP was expressed as a control. Bar, 10 μm.