Measurement of effector protein injection by type III and type IV secretion systems by using a 13-residue phosphorylatable glycogen synthase kinase tag

Abstract

Numerous bacterial pathogens use type III secretion systems (T3SSs) or T4SSs to inject or translocate virulence proteins into eukaryotic cells. Several different reporter systems have been developed to measure the translocation of these proteins. In this study, a peptide tag-based reporter system was developed and used to monitor the injection of T3S and T4S substrates. The glycogen synthase kinase (GSK) tag is a 13-residue phosphorylatable peptide tag derived from the human GSK-3beta kinase. Translocation of a GSK-tagged protein into a eukaryotic cell results in host cell protein kinase-dependent phosphorylation of the tag, which can be detected with phosphospecific GSK-3beta antibodies. A series of expression plasmids encoding Yop-GSK fusion proteins were constructed to evaluate the ability of the GSK tag to measure the injection of Yops by the Yersinia pestis T3SS. GSK-tagged YopE, YopH, LcrQ, YopK, YopN, and YopJ were efficiently phosphorylated when translocated into HeLa cells. Similarly, the injection of GSK-CagA by the Helicobacter pylori T4SS into different cell types was measured via phosphorylation of the GSK tag. The GSK tag provides a simple method to monitor the translocation of T3S and T4S substrates.

FIG. 1.

Nucleotide and amino acid sequences of the AKT, GSK, MEK, STAT, and ELK tags. DNA sequences encoding the AKT tag (aa 466 to 479 of AKT), the GSK tag (aa 1 to 13 of GSK-3β), the MEK tag (aa 214 to 226 of MEK1/2), the STAT tag (aa 697 to 711 of STAT3), and the ELK tag (SV40 large tumor antigen NLS fused to aa 375 to 392 of ELK-1), flanked by PstI and XhoI restriction sites, were inserted into PstI- and XhoI-digested plasmid pYopE, generating plasmids pYopE₁₂₉-AKT, pYopE₁₂₉-GSK, pYopE₁₂₉-MEK, pYopE₁₂₉-STAT, and pYopE₁₂₉-ELK. Phosphorylation of circled residues, corresponding to AKT Ser473, GSK-3β Ser9, MEK1/2 Ser217/Ser221, STAT3 Tyr705, and ELK-1 Ser383, is required for detection with the corresponding phosphospecific antibody preparation (Cell Signaling Technology).

FIG. 2.

Secretion and translocation of YopE₁₂₉-AKT, YopE₁₂₉-GSK, YopE₁₂₉-MEK, YopE₁₂₉-STAT, and YopE₁₂₉-ELK. (A) Expression and secretion of AKT-, GSK-, MEK-, STAT-, and ELK-tagged YopE₁₂₉. Y. pestis KIM8-3002.P39 (ΔyopE) carrying plasmid pYopE₁₂₉-AKT, pYopE₁₂₉-GSK, pYopE₁₂₉-MEK, pYopE₁₂₉-STAT, or pYopE₁₂₉-ELK was grown in the presence or absence of 2.5 mM CaCl₂ in TMH medium for 5 h at 37°C. Expression and secretion of tagged YopE₁₂₉ proteins were determined by SDS-PAGE and immunoblot analysis of cell pellet (pellet) and culture supernatant (sup.) fractions with antiserum specific for YopE. (B) Injection and phosphorylation of YopE₁₂₉-GSK and YopE₁₂₉-ELK in infected HeLa cells. Y. pestis KIM8-3002.P39 (ΔyopE) carrying plasmid pYopE, pYopE₁₂₉-AKT, pYopE₁₂₉-GSK, pYopE₁₂₉-MEK, pYopE₁₂₉-STAT, or pYopE₁₂₉-ELK was grown in HIB for 2 h at 30°C and for 1 h at 37°C. HeLa cell monolayers (six-well dishes) were infected at an MOI of 30 for 3 h at 37°C. Infected monolayers were solubilized with SDS-PAGE sample buffer and analyzed by SDS-PAGE and immunoblotting with antibodies that recognize each tag sequence regardless of tag phosphorylation (α-ELK, α-AKT, α-GSK, α-MEK, or α-STAT) or that recognize the tag only if phosphorylated (α-P-ELK, α-P-AKT, α-P-GSK, α-P-MEK, or α-P-STAT).

FIG. 3.

Injection and phosphorylation of YopE₁₂₉-GSK and YopK-GSK require the expression of a functional YopB/YopD translocon. HeLa cell monolayers were infected at an MOI of 30 with Y. pestis KIM5-3001.P39 (ΔyopE) and KIM5-3001.P64 (ΔyopE ΔyopB) carrying plasmid pYopE₁₂₉-GSK (A) or plasmid pYopK-GSK (B). Infected monolayers were subjected to SDS-PAGE and immunoblot analysis with anti-GSK antibodies (α-GSK) and phosphospecific anti-GSK antibodies (α-P-GSK). Levels of HeLa GSK-3β (used as a loading control) are shown. No translocation and phosphorylation of YopE₁₂₉-GSK or YopK-GSK was detected in lysates from HeLa cell monolayers infected with the yopB deletion strain. Complementation (/C) of the yopB deletion strains with plasmid pYopB2 restored normal levels of YopE₁₂₉-GSK or YopK-GSK translocation and phosphorylation.

FIG. 4.

Construction of plasmid vectors that express GSK-tagged or ELK-tagged YopH, YopD, SycE, LcrQ, YopT, YopK, YopN, YopJ, or LcrV. DNA fragments encoding full-length YopH, YopD, SycE, LcrQ, YopT, YopK, YopN, YopJ, or LcrV fused to sequences encoding the GSK tag or the ELK tag were generated by the PCR-ligation-PCR technique, digested with EcoRI (or MfeI) and HindIII (or XbaI), and inserted into EcoRI- and HindIII (or XbaI)-digested pBAD30 (see Materials and Methods).

FIG. 5.

Injection and phosphorylation of YopH-GSK, LcrQ-GSK, YopK-GSK, YopN-GSK, and YopJ-GSK in HeLa cells. HeLa cell monolayers were infected at an MOI of 30 with Y. pestis KIM5-3001.P39 (ΔyopE) carrying derivatives of pBAD30 that express GSK-tagged (left panel) or ELK-tagged (right panel) proteins (arrowheads). Infected monolayers were analyzed by SDS-PAGE and immunoblotting with anti-GSK antibodies (α-GSK), anti-ELK antibodies (α-ELK), phosphospecific anti-GSK antibodies (α-P-GSK), or phosphospecific anti-ELK antibodies (α-P-ELK). The molecular masses (in kilodaltons) of protein standards are indicated on the left of each blot.

FIG. 6.

Injection and phosphorylation of YopH-GSK, LcrQ-GSK, YopK-GSK, YopN-GSK, and YopJ-GSK require the expression of a functional YopB/YopD translocon. HeLa cell monolayers were infected at an MOI of 30 with the Y. pestis yop polymutant deletion strain (KIM5-3001.P48) and an isogenic yopB deletion strain (KIM5-3001.P49) carrying plasmid pYopH-GSK, pSycE-GSK, pLcrQ-GSK, pYopK-GSK, pYopN-GSK, or pYopJ-GSK. Infected monolayers were subjected to SDS-PAGE and immunoblot analysis with anti-GSK antibodies (α-GSK) and phosphospecific anti-GSK antibodies (α-P-GSK). The YopH-GSK, LcrQ-GSK, YopK-GSK, YopN-GSK, and YopJ-GSK proteins expressed by Y. pestis KIM5-3001.P49 (arrowheads in top panel) were injected into the HeLa cells, and the GSK tag was phosphorylated (arrowheads in bottom panel). No translocation and phosphorylation of GSK-tagged proteins was observed in lysates from HeLa cell monolayers infected with the yopB deletion strain (− yopB). The molecular masses (in kilodaltons) of protein standards are indicated on the left of each blot.

FIG. 7.

Analysis of YopN-GSK function and translocation. (A) Regulation of Yop secretion by YopN-GSK. Expression and secretion of YopN-GSK and YopM by Y. pestis KIM5-3001 (parent), KIM5-3001.6 (ΔyopN), and KIM5-3001.6 carrying plasmid pYopN-GSK grown in the presence (+) or absence (−) of 2.5 mM CaCl₂ for 5 h in TMH medium. Volumes of culture supernatant (sup.) proteins and cell pellet fractions (pellet) corresponding to equal numbers of bacteria were subjected to SDS-PAGE and immunoblot analysis with antisera specific for YopN (α-YopN), the GSK tag (α-GSK), and YopM (α-YopM). (B) Roles of SycN, YscB, TyeA, and LcrG in the translocation of YopN-GSK. HeLa cell monolayers were infected at an MOI of 30 with Y. pestis KIM5-3001.6 (ΔyopN), KIM5-3001.P72 (ΔyopN ΔyopB), KIM5-3001.P55 (ΔyopN ΔtyeA), KIM5-3001.P68 (ΔyopN ΔsycN), KIM5-3001.P70 (ΔyopN ΔyscB), and KIM5-3001.P69 (ΔyopN ΔlcrG) carrying pYopN-GSK. Infected monolayers were subjected to SDS-PAGE and immunoblot analysis with anti-GSK antibodies (α-GSK) and phosphospecific anti-GSK antibodies (α-P-GSK). Levels of HeLa cell GSK-3β (used as a loading control) are shown.

FIG. 8.

H. pylori T4SS-mediated injection of GSK-CagA into St3051 gastric epithelial cells and J774 macrophage cells. St3051 and J774 cells were infected with H. pylori P12ΔcagA expressing GSK-CagA or GSK-CagA_1-1194 (GSK-CagAΔ20C) at an MOI of 100 for 4 h at 37°C. Infected monolayers were examined by SDS-PAGE and immunoblot analysis with anti-GSK antibodies (α-GSK) and phosphospecific anti-GSK antibodies (α-P-GSK). Levels of HeLa cell GSK-3β (used as a loading control) are shown. Injected GSK-CagA was processed partially in St3051 cells, and completely in J774 cells, from ca. 135 kDa to an N-terminal ca.100-kDa fragment and a C-terminal ca. 35-kDa fragment (not shown).