The adaptor molecules LAT and SLP-76 are specifically targeted by Yersinia to inhibit T cell activation

Abstract

T cell responses are critical to the survival of Yersinia-infected animals. Yersinia have the ability to directly suppress T lymphocyte activation through the virulence factor YopH, a tyrosine phosphatase. Using single cell video microscopy and FACS analysis, here we show that even an average of one Yersinia per T cell is sufficient to inhibit or alter T cell responses. This efficient inhibition is traced to specific targeting by YopH of the adaptor proteins, linker for activation of T cells (LAT) and SH2-domain-containing leukocyte protein of 76 kD (SLP-76), which are crucial for T cell antigen receptor (TCR) signaling. A catalytically inactive YopH translocated via the type III secretory pathway from the bacteria into T cells primarily binds to LAT and SLP-76. Furthermore, among the proteins of the TCR signaling pathway, the tyrosine phosphorylation levels of LAT and SLP-76 are the most affected in T cells exposed to low numbers of Yersinia pseudotuberculosis. This is the first example showing that a pathogen targets these adaptor proteins in the TCR signaling pathway, suggesting a novel mechanism by which pathogens may efficiently alter T cell-mediated immune responses.

Figure 1.

TCR-stimulated calcium flux in 5C.C7 T cell blasts exposed to different MOIs of wild-type Y. pseudotuberculosis . (A) Calcium flux of 5C.C7 T cell blasts either unexposed (unexp.) or exposed to wild-type Y. pseudotuberculosis at MOIs of 1, 2, 5, 10, 20, and 50 or to yopHC403A-HA (YopHC/A) at an MOI of 50, loaded with the calcium-sensitive dye indo-1 and activated through the TCR by the addition of anti-CD3ɛ at time 0, was measured by flow cytometry. The calcium flux kinetics are plotted as the median of the intracellular calcium levels in the population measured in three independent experiments versus time. (B) Relative number of cells at the peak of the population's calcium flux (as marked by asterisks in A) that show no significant calcium flux or intracellular calcium levels of 1.5-fold or 2-fold higher than the initial levels.

Figure 2.

Antigen-stimulated calcium flux and PI3K accumulation in Yersinia -exposed T cells. (A) 5C.C7 T cell blasts expressing PH(AKT)-YFP were exposed to wild-type Y. pseudotuberculosis or left unexposed, loaded with the calcium sensitive dye fura-2, mixed with MCC peptide presenting CH27 cells, and analyzed by video microscopy. The differential interference contrast (DIC), the fura-2 ratio in ratio-dependent false color (calcium), and a midplane section of the YFP z-stack (PH[AKT]-YFP) are shown over a time frame of 6 min and at 10 min after contact. The full antigen-stimulated response in calcium flux and PI3K activity was observed in unexposed 5C.C7 T cell and the sparking response was observed in Y. pseudotuberculosis–exposed cells. Image quality of the PH(AKT)-YFP accumulation was improved by using a blind deconvolution algorithm to remove out-of-focus light from the z-stack. (B) Images of 15 cells of each phenotype were analyzed using the Metamorph program. Averages of intracellular calcium levels and PH(AKT)-YFP front-accumulation were plotted relatively to their corresponding base levels (-fold) versus time. Error bars represent standard deviation. Data were acquired at intervals of 30 s over a time frame of 10 min.

Figure 3.

Inhibition of tyrosine phosphorylation in Jurkat T cells exposed to wild-type Y. pseudotuberculosis . Jurkat T cells were exposed to Y. pseudotuberculosis at MOIs of 1.5, 5, and 15 for 1 h or left unexposed (−). Total lysates of 4 × 10⁵ resting (−) or OKT3-activated (+) T cells were analyzed by antiphosphotyrosine blotting (pTyr). Subsequently, blots were stripped and reprobed with antibodies reacting with phosphotyrosine-394-Lck (Lck-pY394), YopH, and actin. Labeled proteins were identified by reprobing with the corresponding antibodies. A representative result of three independent experiments is shown. (B) Amounts of tyrosine phosphorylated proteins were quantified in three independent experiments using the NIH Image. Error bars represent standard deviation. (C) Kinase activity of Lck in E6.1 Jurkat T cells unexposed or exposed to wild-type Y. pseudotuberculosis at MOIs of 1.5, 5, 15, and 50 or to yopHC403A-HA (YopHC/A) at an MOI of 50 was measured by ELISA in three independent experiments. Lck-deficient J.CaM1 Jurkat cells were used as negative control. Error bars represent standard deviation.

Figure 4.

Analysis of the effects of YopH variants on TCR-stimulated tyrosine phosphorylation. Immunoblot analysis of resting (−) and OKT3-activated (+) Jurkat T cells that were either unexposed (−) or exposed for 1 h at an MOI of 50 of the following Y. pseudotuberculosis strains: WT, YopH-deficient (ΔH), yopHC403A-HA (yopHC/A-HA), and yopH-HA (yopH-HA). Total lysates of 4 × 10⁵ T cells were first probed with antiphosphotyrosine antibodies (pTyr). Subsequently, the blot was stripped and reprobed with anti-YopH and anti-actin antibodies. Labeled proteins were identified by reprobing with the corresponding antibodies. A representative result of three independent experiments is shown.

Figure 5.

Substrate trap YopH associates with multiple components of the LAT/SLP-76 signalosome in activated Jurkat T cells. Detergent lysates of OKT3-activated Jurkat T cells exposed to yopHC403A-HA Yersinia at an MOI of 2 for 1 h, or left unexposed, were subjected to immunoprecipitation with affinity-purified anti-YopH antibodies (α-YopH) or protein G alone (−). (A) Antiphosphotyrosine blot of 4 × 10⁵ T cell equivalents of the lysates before (L) and after (dL) immunoprecipitation and the immunoprecipitated complexes (IP) from 3 × 10⁷ T cell equivalents. A representative result of at least three independent experiments is shown. (B) Blots were stripped and reprobed with a panel of antibodies to identify the precipitated proteins. Anti–SLAP-130, SLP-76, LAT, and Gads antibodies bound to proteins coprecipated with YopHC403A-HA (lane 2). The phosphorylated proteins of 50-kD and 65-kD size (A, lane 2) could not be identified. The white line indicates that intervening lanes have been spliced out. (C) Blots were reprobed with anti–HA-tag antibodies (to detect YopHC403A-HA) and actin antibodies.

Figure 6.

YopH precipitated from T cell lysates is YopH that had been injected. (A) Antiphosphotyrosine blot of total lysates of 4 × 10⁵ OKT3-activated Jurkat T cells exposed to yopH-HA or a YopH-HA–expressing lcr mutant strain (lcr yopH-HA) at an MOI of 50 for 1 h. (B) Anti-HA blot of the anti-YopH immunoprecipitated proteins from lysates of 4 × 10⁶ Jurkat cells was exposed and activated as described in A. (C) Anti-HA blot of lysates of 4 × 10⁵ T cells was exposed as in A, but lysed by boiling in SDS sample buffer, which also lyses the bacteria.

Figure 7.

YopH substrate trap-associated tyrosine phosphorylated proteins in LAT- and SLP-76–deficient Jurkat cells. (A) Antiphosphotyrosine blot of anti-YopH immunoprecipitated proteins from total lysates of OKT3-activated Jurkat T cells (E6.1) and the E6.1-derived mutants J14 (SLP-76 deficient) and J.CaM2 (LAT deficient), exposed to yopHC403A-HA Yersinia at an MOI of 2 for 1 h. Lysates of 4 × 10⁵ T cells before (L) and after (dL) immunoprecipitation and the precipitated immunocomplexes (IP) of 3 × 10⁷ T cell equivalents were analyzed. A representative result of three independent experiments is shown. Blots were reprobed with actin antibodies and anti–HA-tag antibodies (to detect YopHC403A-HA). (B) Tyrosine-phosphorylated proteins associated with YopHC403A in per-vanadate–stimulated E6.1, J14, J.CaM2, and J.CaM1 (Lck deficient) Jurkat cells were analyzed as in A. A representative result of three independent experiments is shown.