Role of YopP in suppression of tumor necrosis factor alpha release by macrophages during Yersinia infection

Abstract

The Yersinia plasmid-encoded Yop virulon enables extracellular adhering bacteria to deliver toxic effector proteins inside their target cells. It includes a type III secretion system (Ysc), at least two translocator proteins (YopB, YopD), and a set of intracellular Yop effectors (YopE, YopH, YopO, YopM, and YopP). Infection of macrophages with a wild-type strain leads to low levels of tumor necrosis factor alpha (TNF-alpha) release compared to infection with plasmid-cured strains, suggesting that the virulence plasmid encodes a factor impairing the normal TNF-alpha response of infected macrophages. This effect is correlated with the inhibition of the macrophage mitogen-activated protein kinase (MAPK) activities. To identify the Yop protein responsible for the suppression of TNF-alpha release, we infected J774A.1 and PU5-1.8 macrophages with a battery of knockout Yersinia enterocolitica mutants and we quantified the TNF-alpha released. Mutants affected in secretion (yscN), in translocation (yopB and yopD), or in synthesis of all the known Yop effectors (yopH, yopO, yopP, yopE, and yopM polymutants) were unable to block the TNF-alpha response of the macrophages. In contrast, single yopE, yopH, yopO, and yopM mutants behaved like the wild-type strain. A yopP mutant elicited elevated TNF-alpha release, and complementation of the yopP mutant or the yop effector polymutant strain with yopP alone led to a drop in TNF-alpha release. In addition, YopP was also responsible for the inhibition of the extracellular signal-regulated kinase2 (ERK2) and p38 MAPK activities. These results show that YopP is the Yop effector responsible for the Yersinia-induced suppression of TNF-alpha release by infected macrophages.

FIG. 1

TNF-α-like activity released by Y. enterocolitica-infected J774A.1 macrophages. J774A.1 cells were infected with various Y. enterocolitica strains at a multiplicity of 20 bacteria per cell: WT, wild-type E40; yscN, secretion mutant (S); yopB and yopD, translocation mutants (T); yopQ, yopE, yopH, yopO, yopM, and yopP, single yop mutants; yopP/P⁺⁺, yopP mutant complemented with the yopP overexpressing plasmid pMSK13. The infection procedure and the TNF-α assay were as described in Materials and Methods. Results are expressed as the percentage of the maximal response and are the mean values of three separate experiments. Error bars represent the standard deviation of the three experiments. Refer to Table 1 for the identification of the strains used.

FIG. 2

TNF-α-like activity released by J774A.1 cells upon infection with the Y. enterocolitica polymutant strains. Cells were infected with 20 bacteria per cell with the wild-type strain (WT), the yopB and yopP mutants as controls, the polymutant strains yopHOPEM and yopHOPEMB, and the complemented strains overexpressing yopP (yopP/P⁺⁺, yopHOPEM/P⁺⁺, and yopHOPEMB/P⁺⁺). NI, noninfected cells. TNF-α was assayed by the biological method. Results are expressed as the percentage of the maximal response and are the mean values of three separate experiments. Error bars represent the standard deviation of the three experiments. Refer to Table 1 for the identification of the strains used.

FIG. 3

YopP is involved in the inhibition of the ERK2 MAPK. After infection, the ERK2 MAPK was immunoprecipitated from the lysate of cells infected with the wild-type strain (WT), the secretion mutant (yscN), the translocation mutant (yopB), the yopP mutant, and the complemented yopP mutant (yopP/P⁺⁺). (A) Immunoblot showing immunoprecipitation of the ERK2 MAPK. The immunoprecipitated proteins were submitted to SDS-PAGE (12%), transferred to nitrocellulose membrane, and probed with the anti-ERK2 antibody. (B) MAPK activity of the immunoprecipitated ERK2 protein. The immunoprecipitated proteins were added to a kinase assay in the presence of [γ-³²P]ATP and with MBP as a substrate. Proteins were then separated by SDS-PAGE (15%), and the gel was subjected to autoradiography. + and −, presence or absence of immunoprecipitating anti-ERK2 antibodies in the sample. A strong labeling of the MBP indicates a high kinase activity of the immunoprecipitated ERK2 MAPK.

FIG. 4

YopP is involved in the inhibition of the p38 MAPK. After infection, the ERK2 or p38 MAPK was immunoprecipitated from the lysate of cells infected with the yopHOPEM/P⁺⁺ strain (lanes 1), the yopHOPEMB/P⁺⁺ strain (lanes 2), the yopHOPEM strain (lane 3), or the yopHOPEMB strain (lane 4). The immunoprecipitated proteins were added to a kinase assay in the presence of [γ-³²P]ATP and with MBP as a substrate. Proteins were then separated by SDS-PAGE (15%), and the gel was subjected to autoradiography. Panels A and B correspond to two separate experiments. ERK2 and p38, the antibodies used for immunoprecipitation; none, the control samples without immunoprecipitating antibodies. A strong labeling of the MBP indicates a high kinase activity of the immunoprecipitated ERK2 and p38 MAPKs.