DXD motif-dependent and -independent effects of the chlamydia trachomatis cytotoxin CT166

Abstract

The Gram-negative, intracellular bacterium Chlamydia trachomatis causes acute and chronic urogenital tract infection, potentially leading to infertility and ectopic pregnancy. The only partially characterized cytotoxin CT166 of serovar D exhibits a DXD motif, which is important for the enzymatic activity of many bacterial and mammalian type A glycosyltransferases, leading to the hypothesis that CT166 possess glycosyltransferase activity. CT166-expressing HeLa cells exhibit actin reorganization, including cell rounding, which has been attributed to the inhibition of the Rho-GTPases Rac/Cdc42. Exploiting the glycosylation-sensitive Ras(27H5) antibody, we here show that CT166 induces an epitope change in Ras, resulting in inhibited ERK and PI3K signaling and delayed cell cycle progression. Consistent with the hypothesis that these effects strictly depend on the DXD motif, CT166 with the mutated DXD motif causes neither Ras-ERK inhibition nor delayed cell cycle progression. In contrast, CT166 with the mutated DXD motif is still capable of inhibiting cell migration, suggesting that CT166 with the mutated DXD motif cannot be regarded as inactive in any case. Taken together, CT166 affects various fundamental cellular processes, strongly suggesting its importance for the intracellular survival of chlamydia.

Figure 1

Changes in protein levels in HeLa cells infected with Chlamydia trachomatis serovar D strain UW3 (Ctr D/UW3) and L₂/434. TRex™-HeLa cells were infected with either Ctr L₂/434 or Ctr D/UW3 (MOI of five) or they were mock infected for 48 h. Cells were lysed, and the levels of the indicated proteins were analyzed by Western blot. Representative Western blots (A) and signal intensities of a minimum of four independent experiments are presented as relative (rel.) individual signal intensities and as means (B). For statistical analysis, one-way ANOVA and Tukey’s multiple comparison test were used (* indicates statistically-significant differences: ** p ≤ 0.01).

Figure 2

Expression of CT166-wt or CT166-mut reduces the level of several proteins. To augment the expression of the chlamydial proteins, HeLa-control, HeLa-CT166-mut and HeLa-CT166-wt cells were incubated for 24 h in the presence of tetracycline (1 µg/mL). Cells were lysed, and the levels of the indicated protein were analyzed by Western blot. Representative Western blots (A) and quantified signal intensities (including the means) (B) of a minimum of three independent experiments are presented. To calculate the relative (rel.) intensity of bands, the signal levels of equally-treated, unaltered TRex™-HeLa cells were defined as 1.0 for each antibody. For statistical analysis, one-way ANOVA and Tukey’s multiple comparison test were used (* indicates statistically-significant differences: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

Figure 3

CT166 expression delays G1-S transition of cell cycle. HeLa-control, HeLa-CT166-mut and HeLa-CT166-wt cells were incubated in the presence of tetracycline (1 µg/mL) for 24 h. Cells were fixed, and the DNA was stained with propidium iodide. The DNA content was analyzed by flow cytometry. Depicted are two exemplary histograms for HeLa-control and HeLa-CT166-wt cells representing DNA-dependent fluorescence intensity versus cell frequency within 20,000 events analyzed by FACS (A). The same gates for the determination of the percentages of cells being in the 2n, synthesis (S) or 4n stages of the cell cycle, which were first set for the HeLa-control, were also applied for the other two cell lines. The results, i.e., the percentage of cells being in the indicated different stages of the cell cycle of three independent FACS experiments, each analyzing the three different cell lines in parallel and, in addition, their means, are depicted (B). One-way ANOVA and Tukey’s multiple comparison test were used for statistical evaluation (* indicates statistically-significant differences: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; n.s. = not significant).

Figure 4

CT166-wt induces the formation of multinucleated cells. HeLa-control, HeLa-CT166-mut and HeLa-CT166-wt cells were treated with tetracycline (1 µg/mL for 24 h) for protein expression, and then, the microtubule network and cell nuclei were stained (scale bar represents 50 µm). Representative fluorescence images (A) and the percentage of multinuclear cells ± SD (B) of three independent experiments are demonstrated (using the identical magnification factor). For statistics, one-way ANOVA and Tukey’s multiple comparison test were applied (* indicates statistically-significant differences: ** p ≤ 0.01; n.s. = not significant).

Figure 5

Reduced levels of RhoA, Rac1 and Cdc42 in HeLa-CT166-wt cells. Levels of RhoA, Rac1 and Cdc42 were analyzed in tetracycline-induced (1 µg/mL for 24 h) HeLa-control, HeLa-CT166-mut and HeLa-CT166-wt cells by Western blot. Representative Western blots (A) and quantified signal intensities (including means) (B) of a minimum of five independent experiments are depicted. To calculate the relative (rel.) intensity of bands, the signal levels of equally-treated, unaltered TRex™-HeLa cells were defined as 1.0 for each antibody. For statistical analysis, one-way ANOVA and Tukey’s multiple comparison test were used (* indicates statistically-significant differences: ** p ≤ 0.01; *** p ≤ 0.001).

Figure 5

Reduced levels of RhoA, Rac1 and Cdc42 in HeLa-CT166-wt cells. Levels of RhoA, Rac1 and Cdc42 were analyzed in tetracycline-induced (1 µg/mL for 24 h) HeLa-control, HeLa-CT166-mut and HeLa-CT166-wt cells by Western blot. Representative Western blots (A) and quantified signal intensities (including means) (B) of a minimum of five independent experiments are depicted. To calculate the relative (rel.) intensity of bands, the signal levels of equally-treated, unaltered TRex™-HeLa cells were defined as 1.0 for each antibody. For statistical analysis, one-way ANOVA and Tukey’s multiple comparison test were used (* indicates statistically-significant differences: ** p ≤ 0.01; *** p ≤ 0.001).

Figure 6

CT166 reduces the migration of HeLa-cells independently of the DXD motif. Confluent, tetracycline-induced (1 µg/mL for 24 h) HeLa-control, HeLa-CT166-mut and HeLa-CT166-wt monolayers were scratched and analyzed by phase contrast microscopy (A) after 24h and 48 h during 2 mM thymidine treatment (scale bar represents 200 µm). The determined mean ± SD of the covered area (in phase contrast images) of three independent experiments is depicted (B). For statistical evaluation, two-way ANOVA and the Bonferroni post-test were performed (* indicates statistically-significant differences: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; n.s. = not significant).

Figure 6

CT166 reduces the migration of HeLa-cells independently of the DXD motif. Confluent, tetracycline-induced (1 µg/mL for 24 h) HeLa-control, HeLa-CT166-mut and HeLa-CT166-wt monolayers were scratched and analyzed by phase contrast microscopy (A) after 24h and 48 h during 2 mM thymidine treatment (scale bar represents 200 µm). The determined mean ± SD of the covered area (in phase contrast images) of three independent experiments is depicted (B). For statistical evaluation, two-way ANOVA and the Bonferroni post-test were performed (* indicates statistically-significant differences: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; n.s. = not significant).