Interaction of Yersinia pestis with macrophages: limitations in YopJ-dependent apoptosis - PubMed (original) (raw)
Interaction of Yersinia pestis with macrophages: limitations in YopJ-dependent apoptosis
Ayelet Zauberman et al. Infect Immun. 2006 Jun.
Abstract
The enteropathogenic Yersinia strains are known to downregulate signaling pathways in macrophages by effectors of the type III secretion system, in which YopJ/YopP plays a crucial role. The adverse effects of Yersinia pestis, the causative agent of plague, were examined by infecting J774A.1 cells, RAW264.7 cells, and primary murine macrophages with the EV76 strain and with the fully virulent Kimberley53 strain. Y. pestis exerts YopJ-dependent suppression of tumor necrosis factor alpha secretion and phosphorylation of mitogen-activated protein kinases and thus resembles enteropathogenic Yersinia. However, Y. pestis is less able to activate caspases, to suppress NF-kappaB activation, and to induce apoptosis in macrophages than the high-virulence Y. enterocolitica WA O:8 strain. These differences appear to be related to lower efficiency of YopJ effector translocation by Y. pestis. The efficiencies of effector translocation and of apoptosis induction can be enhanced either by using a high bacterial load in a synchronized infection or by overexpressing exogenous YopJ in Y. pestis. Replacing YopJ with the homologous Y. enterocolitica effector YopP can further enhance these effects. Overexpression of YopP in a yopJ-deleted Y. pestis background leads to rapid and effective translocation into target cells, providing Y. pestis with the high cytotoxic potential of Y. enterocolitica WA O:8. We suggest that the relative inferiority of Y. pestis in triggering cell death in macrophages may be advantageous for its in vivo propagation in the early stages of infection.
Figures
FIG. 1.
Induction of apoptotic cell death by Y. pestis EV76 and Y. enterocolitica WA O:8 and their derivatives. Cell death and apoptosis were monitored upon infection by overlay of macrophages with various Yersinia strains (bacterial suspensions were added to the cell monolayers and washed off after incubation as described in Materials and Methods). The death of J774A.1 cells, as well as RAW264.7 cells, infected with the Yersinia strains was determined by LDH release 6 h postinfection (A), and detachment of cells (J774A.1) from culture wells was examined 20 h postinfection by light microscopy of Gimenez-stained monolayers (B). (C) The onset of J774A.1 cell apoptosis was evaluated 3 h postinfection by annexin-V staining (left panels). In the right panels the same fields visualized by phase-contrast microscopy are shown. (D) Nuclear morphology was determined by DAPI staining (magnification, ×1,000) at 4 h postinfection. Y. entero., Y. enterocolitica.
FIG. 2.
NF-κB DNA-binding activity in J774A.1 macrophages infected with Y. pestis or Y. enterocolitica. J774A.1 cells were exposed by using the overlay procedure to different Yersinia strains and their plasmid-cured derivatives (in panel A, Y. enterocolitica O:8 and Y. pestis EV76 and their pCD1/pYV-cured derivatives, and in panel B, Y. pestis Kimberley53 and its pCD1- and pPCP1-cured derivative). Nuclear lysates were tested for interaction with an NF-κB-specific DNA probe by EMSAs. The specificity of binding was demonstrated by a supershift assay using antibodies directed against the p65 NF-κB subunit (panel A, lane 7) and by competition with a 100-fold molar excess of the unlabeled oligonucleotide (panel A, lane 8). Nuclear lysate of uninfected cells was used as a control (panel A, lane 2). Y. entero., Y. enterocolitica.
FIG. 3.
Induction of caspase 3/caspase 7 activity in macrophages infected with Y. pestis or Y. enterocolitica. J774A.1 cells infected by using the overlay procedure with Y. pestis strains EV76 and Kimberley53 were compared to cells infected with Y. enterocolitica WA O:8. Caspase 3/caspase 7 activity was measured 3 h after infection as described in Materials and Methods and is expressed in relative fluorescence units (RFLU). Y. entero., Y. enterocolitica.
FIG. 4.
Enhancing the cytotoxic effect of Y. pestis EV76 by forcing bacteria onto cells. (A) RAW264.7 cells were infected with Y. enterocolitica, Y. pestis, and Y. pestis with a knockout in yopJ (Δ_yopJ_ [ΔJ]), using two different infection techniques, forcing bacterial contact by centrifugation (impact procedure) (striped bars) and overlay of a bacterial suspension (solid bars), and examined for LDH release 6 h postinfection; bone marrow macrophages (BMM) were infected only by the impact procedure and were evaluated for LDH release 5 h postinfection. (B) Caspase 3/caspase 7 activity was determined 3 h postinfection by the impact procedure. RFLU, relative fluorescence units; Y. entero., Y. enterocolitica.
FIG. 5.
Levels of expression of YopJ/YopP in Y. pestis EV76 and Y. enterocolitica O:8 and amounts of effector translocated into infected macrophages. (A) For evaluation of bacterial YopJ/YopP expression, Y_ersinia_ strains were grown for 3 h at 37°C. Then 3 × 107 bacteria were subjected to SDS-PAGE and developed with polyclonal rabbit anti-peptide YopP/YopJ antibodies as described in Materials and Methods. (B and C) For evaluation of effector translocation following infection by the overlay procedure (B) or the impact procedure (C), infection was arrested at the times indicated, and cytosolic fractions of infected RAW264.7 cells were obtained by lysis with 0.1% Triton X-100. Aliquots equivalent to 1 × 106 cells (normalized by protein measurement) were subjected to SDS-PAGE. Gels were blotted and developed with rabbit polyclonal anti-peptide YopP/YopJ antibodies. Bands were visualized by ECL. Note that the blot in panel B is overexposed compared to the blot in panel C. Y. entero., Y. enterocolitica.
FIG. 6.
Overexpression of YopJ and YopP in Y. pestis background: effect on translocation. (A) Translocation of effectors YopJ and YopP was evaluated by infecting RAW264.7 cells by the impact procedure and monitoring for the presence of the effectors in cell lysates by SDS-PAGE at different times after infection. Blots were developed as described in the legend to Fig. 5, except that the anti-peptide YopP/YopJ antibodies were purified by antigen affinity chromatography to reduce the nonspecific background. (B) In parallel, translocation of YopH was examined 60 min postinfection. YopH was visualized as described in Materials and Methods.
FIG. 7.
Overexpression of YopJ and YopP in Y. pestis background: effects on cytotoxicity to macrophages. Cytotoxicity was evaluated by monitoring LDH release 6 h following infection of RAW264.7 cells by the impact procedure (A) and by the overlay procedure (B) with different amounts of bacterial strains (MOIs, 10 to 100). □, infection by Y. pestis (EV76); ▴, infection by Y. pestis Δ_yopJ_ (Y. pestis_ΔJ); •, infection by Y. pestis Δ_yopJ+yopJ (Y. pestis_ΔJ+J); ▪, infection by Y. pestis Δ_yopJ+yopP (_Y. pestis_ΔJ+P); ⧫ and dashed line, infection by Y. enterocolitica (Y. entero.).
FIG. 8.
Suppression of TNF-α secretion and p38 phosphorylation in _Yersinia_-infected macrophages. (A) To assay TNF-α secretion, J774A.1 cells were infected by the overlay procedure (solid bars) or by the impact procedure (striped bars) with different Yersinia strains. The secretion of TNF-α into cell medium was monitored 2 h after initiation of infection by an enzyme-linked immunosorbent assay. (B and C) Activation and suppression of p38 phosphorylation were examined in cells infected by the overlay procedure for different times. Macrophage lysates were analyzed by immunoblotting with phospho-specific p38 antibodies (P-p38). Y. entero., Y. enterocolitica.
References
- Ben-Gurion, R., and A. Shafferman. 1981. Essential virulence determinants of different Yersinia species are carried on a common plasmid. Plasmid 5:183-187. - PubMed
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