INTRODUCTION

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Pathogenic Yersinia spp. have evolved a series of strategies for evasion and neutralization of host defense mechanisms. The ability to escape the host immune response largely depends on the presence of the 70-kb virulence plasmid pYV, which is common to the three Yersinia species that are pathogenic for rodents and humans. Y. pestis is the causative agent of plague, and Y. enterocolitica and Y. pseudotuberculosis cause gastrointestinal syndromes, lymphadenitis, and septicemia (6, 9). The Yersinia virulence plasmid pYV encodes a sophisticated bacterial virulence system for subverting eukaryotic cells (9). It encompasses the genes for a type III protein secretion machinery and for a set of at least six Yersinia effector proteins (Yersinia outer proteins [Yops] YopE, YopH, YopM, YopT, YopO/YpkA, and YopP/YopJ). The type III protein secretion system is activated upon host-cell contact and specifically mediates the delivery of the Yersinia effector proteins inside eukaryotic cell, there perturbing key cellular functions. By interference with the actin cytoskeleton dynamics, Yersinia blocks its phagocytosis by macrophages and polymorphonuclear neutrophils (4, 9). Furthermore, the action of Yops prevents killing of Yersinia by the phagocytic oxidative burst (4, 9). Besides these immediate effects on the phagocyte, Yersinia inhibits the production of the proinflammatory cytokine tumor necrosis factor alpha (TNF-) and triggers macrophage apoptosis (5, 23, 25, 29, 33, 35, 36). Both effects are conducted by YopP (Y. enterocolitica) or by its homologue, YopJ (Y. pseudotuberculosis and Y. pestis) (5, 23, 25, 29, 36). Yersinia suppresses the macrophage TNF- production by down-regulating the signaling cascades of transcription factor NF-B and of the mitogen-activated protein kinases (MAPK) (5, 29, 32, 33, 36). These signaling cascades synergistically control the TNF- production in response to bacterial infection and lipopolysaccharide (LPS) treatment. YopP/YopJ disrupts the MAPK pathways by binding and inhibiting MAPK kinases (MKK) 1 to 5 of the MKK superfamily, which function as upstream MAPK activators (27). Subversion of the NF-B cascade is accomblished by targeting YopP/YopJ to the NF-B-activating IB kinase- (IKK) (27).

NF-B acts as a key regulator of the inflammatory response. It rapidly upregulates the synthesis of cytokines, acute-phase proteins, and adhesion molecules and mediates cellular survival by the prevention of apoptosis (3, 15). Disruption of the antiapoptotic functions of NF-B plays a crucial role in the mechanism of apoptosis induction by Yersinia (32, 34). A number of extracellular stimuli, such as TNF- and ionizing radiation, activate pro- and antiapoptotic signaling pathways in eukaryotic cells. NF-B functions to up-regulate the synthesis of proteins that counteract the proapoptotic signals, such as inhibitor of apoptosis proteins and Bcl-2 family members. Hence, NF-B activation provides protection against apoptotic killing, otherwise induced by these stimuli (2, 30). Analogously, activation of NF-B is essential for self-defense and survival of macrophages when encountered with bacteria or LPS (1, 21, 32). The suppression of NF-B activation by YopP/YopJ and the simultaneous activation of LPS-induced signaling processes trigger severe apoptosis in macrophages (34). Thus, the impact of YopP/YopJ on NF-B and the activation of proapoptotic signals by LPS or bacterial infection crucially determine the fate of the Yersinia-infected macrophage. It was recently reported that the Yersinia-mediated proapoptotic process involves cleavage of Bid, a Bcl-2 family member that relays the apoptotic response (10).

NF-B activation depends on liberation of NF-B from its inhibitory proteins IB, IB, and IB, which sequester preformed NF-B in the cytoplasm (3, 15). Phosphorylation and degradation of IBs by the ubiquitin-proteasome pathway release NF-B, which translocates to the nucleus and activates transcription. The critical step in NF-B activation is phosphorylation of the IBs, which is conferred by the IKK complex (3, 15). This complex is composed of at least three proteins: IKK, IKK, and IKK. IKK and IKK encode the catalytic kinase subunits, mediating IB phosphorylation. After an initial NF-B activation, Y. enterocolitica downregulates NF-B activities in J774A.1 macrophages as little as 60 to 90 min after onset of infection, a lag time necessary for YopP to reach its targets and to exert its effects (32, 34). YopP selectively interacts with macrophage IKK, but not with IKK, and simultaneously suppresses IKK activities (34). This points out a strategy evolved by Yersinia that specifically targets IKK, which is the major LPS-responsive NF-B-activating kinase in monocytes/macrophages (26).

In this study, we analyzed the impact of the different pathogenic Y. enterocolitica serotypes on apoptosis in macrophages. We report that Y. enterocolitica serotype O8 exhibits an outstanding efficiency in apoptosis induction and NF-B suppression in comparison to other Y. enterocolitica serogroups. These features are specifically conferred by the serogroup O8 YopP. To localize the serotype-related effector domain of YopPO8, we conducted site-directed mutagenesis, by using an approach in which multiple or single amino acids between YopPO8 and YopPO9 from the well-characterized strains WA (serogroup O8) and E40 (serogroup O9) are interchanged. Our data show that an individual amino acid, the arginine-143 residue, plays a predominant role in determining YopP effector functions by impairing IKK activities.

	MATERIALS AND METHODS

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Bacterial strains, cell culture, and stimulation conditions. The Y. enterocolitica strains used in this study are listed in Table 1. The Y. enterocolitica serotype O9 wild-type strain E40 and the respective yopP-negative mutant E40-yopP were kindly provided by G. R. Cornelis (Microbial Pathogenesis Unit, Université Catholique de Louvain, Brussels, Belgium). Overnight cultures grown at 26°C were diluted 1:20 in fresh Luria-Bertani broth and grown for 2 h at 37°C as described previously (32). The bacteria were then washed once and resuspended in phosphate-buffered saline (PBS) at the desired concentration, which was adjusted by measuring the optical density at 600 nm and checked by plating serial dilutions of the samples on agar (32). The murine macrophage cell line J774A.1 was cultured in RPMI 1640 cell growth medium supplemented with 10% heat-inactivated fetal calf serum and 5 mM L-glutamine (32). Human blood monocytes were isolated from heparinized blood with magnetic anti-CD14 antibody beads and differentiated to macrophages as previously reported (22). Infections were performed at a ratio of 50 bacteria per cell. For incubation times longer than 90 min, bacteria were killed by addition of gentamicin (100 µg/ml) after 90 min of infection.

Construction of plasmids and complementation of Y. enterocolitica strains and mutants. The yopP-negative mutants WA-yopP and E40-yopP were complemented with plasmids encoding diverse yopP constructs. The yopP constructs were engineered by PCR. To obtain full-length yopP from serogroup O8 or O9, DNA fragments encompassing the entire yopP open reading frames from strain WA or E40 were amplified and subcloned in plasmid pCJYE138-G3 (20), replacing the gfp gene of pCJYE138-G3 in frame by yopP, as described previously (34). In the resulting plasmid, the yopP gene is located downstream of the yopE promoter and fused to regions encoding the first 138 amino acids of YopE. Induction of gene expression mediates production of a YopE138-YopP fusion protein that is intrabacterially stabilized by the YopE-specific chaperone SycE. Translocated YopE138 hybrid proteins do not exert a YopE effect (20). Complementation of WA-yopP and E-40yopP with these contructs generated Yersinia strains that selectively produce YopP from serogroup O8 (WA-yopP/+PO8 and E40-yopP/+PO8) or from serogroup O9 (WA-yopP/+PO9 and E40-yopP/+PO9). To enable complementation of the chloramphenicol-resistant strain WA-yopP, we disrupted the endogenous chloramphenicol resistance marker of the complementator plasmid by introducing a spectinomycin resistance fragment. For modulation and comparison of YopP effects by the different serogroups, we exchanged a central 514-bp BamHI fragment of the yopP gene between yopP from serogroups O8 and O9 [resulting in strains E40-yopP/+PO8(44-214PO9) and E40-yopP/+PO9(44-214PO8)]. The 514-bp DNA fragment encodes amino acid residues 44 to 214 of YopP. Mapping of the YopPO8-specific effector domain and site-directed mutagenesis of single amino acids were accomplished by generating mutated yopP BamHI fragments by the splicing and overlap extension method. Accordingly, two PCR fragments with homologous overlapping sequences at one terminus were engineered with appropriate complementary primers. For instance, for fusing a sequence from yopPO8 to the respective C-terminal coding region from yopPO9, one PCR was run on yopPO9 with a forward primer annealing outside the BamHI fragment and the reverse primer determining the fusion region within the BamHI fragment. The second PCR was run on yopPO8, with the forward primer being complementary to the reverse primer used for the PCR on yopPO9 and the reverse primer annealing outside the BamHI fragment. Both PCR products were fused by subsequent PCR. The resulting DNA fragment encompasses the yopPO9/yopPO8 chimeric DNA. Site-directed mutagenesis of amino acids was conducted by the same approach, except that both primary PCRs were performed with either yopPO9 or yopPO8, by using overlapping complementary mutation primers. The resulting PCR products, encompassing either mutagenized yopP or yopPO9/yopPO8 hybrid BamHI fragments, were introduced into the internal BamHI restriction sites of yopP, thereby replacing the endogenous BamHI fragment. The primers used for fusion of amino acids 44 to 129 from YopPO9 to amino acids 130 to 214 from YopO8 and vice versa were 5'-AAATGGGAAAACATCTCTGATATTGTTTGAACCAGC-3' (forward) and 5'-GCTGGTTCAAACAATATCAGAGATGTTTTCCCATTT-3' (reverse). The mutation primers for exchange of arginine to serine at position 143 in YopPO8 were 5'-GGCAATAAGTGCAAAAACGGCCATTGAACGTT-3' (forward) and 5'-AACGTTCAATGGCCGTTTTTGCACTTATTGCC-3' (reverse), for exchange of serine 143 to arginine in YopPO9 we used 5'-GGCAATAAGGACAAAAACGGCCATTGAACGTT-3' (forward) and 5'-AACGTTCAATGGCCGTTTTTGTCCTTATTGCC-3' (reverse); the mutations are underlined. The resulting strains, E40-yopP/+PO8(R143S) and E40-yopP/+PO9(S143R), produce the respective mutagenized YopP proteins. The same strategy was used to generate YopPO9(141-214PO8) hybrid protein and mutagenized YopPO8(A144T), YopPO8(RA143/144ST), YopPO9(T144A), and YopPO9(ST143/144RA). In an additional approach, E40-yopP was complemented by a 5.5-kb EcoRI-KpnI DNA fragment of the Yersinia pYV plasmid that encompasses the yopO/yopP operon encoding either wild-type or mutagenized yopPO8. Accordingly, a 4.6-kb EcoRI-KpnI DNA fragment of pYVO8, which encodes the yopO/yopP promoter region and the yopO gene, was subcloned. The KpnI restriction site of this fragment is located 60 bp upstream of the yopP start codon in the pYV plasmid (37). The 4.6-kb EcoRI-KpnI DNA fragment was extended in frame at the KpnI site by introduction of a 930-bp KpnI fragment that encodes the yopP gene and the preceding DNA sequence, including the endogenous KpnI restriction site. The 930-bp KpnI-yopP DNA fragments were engineered by PCR and by the splicing and overlap extension method with wild-type yopPO8 and mutagenized yopPO8(R143S) as described above. The resulting Yersinia strains, E40-yopP/+pYVyopPO8 and E40-yopP/+pYVyopPO8(R143S), produce YopPO8 or YopPO8(R143S) under control of the yopO/yopPO8 endogenous promoter. All PCR products were checked for sequence accuracy by sequencing. To generate Yersinia strains that secret and translocate the YopP constructs as unique effector Yops, the respective plasmids were introduced in strain WA-yop. WA-yop solely harbors a plasmid encoding the Y. enterocolitica secretion and translocation machinery and the gene for the adhesin YadA. It does not bear any effector Yop gene and thus does not produce any effector Yop (34).

EMSAs and IKK assays. To analyze nuclear translocation of NF-B, nuclear proteins were extracted and electrophoretic mobility shift assays (EMSAs) were performed as described previously (32, 34). In brief, the cells were washed with ice-cold PBS and lysed with hypotonic buffer on ice (5 mM HEPES [pH 7.9], 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM dithiothreitol [DTT] and protease inhibitors). Proteins were extracted from the nuclei by resuspension of the nuclear pellets in ice-cold extraction buffer (20 mM HEPES [pH 7.9], 25% glycerol, 1 M NaCl, 1.5 mM MgCl₂, 0.5 mM EDTA, 0.5 mM DTT and protease inhibitors). Seven micrograms of the extracted nuclear proteins was incubated with 2 to 5 ng of a radiolabeled oligonucleotide probe that encompasses the consensus binding site for NF-B dimeric complexes (Santa Cruz Biotechnology). The DNA-binding reactions were performed in the presence of a combination of 25 mM HEPES (pH 7.9), 0.5 mM EDTA, 0.5 mM DTT, and 5% glycerol for 30 min on ice. The DNA-protein complexes were subsequently separated by 5% polyacrylamide gel electrophoresis (PAGE) and analyzed by autoradiography (32, 34).

Coimmunoprecipitations and Western immunoblotting. Coimmunoprecipitations were performed as described previously (34). Briefly, 10⁸ J774A.1 cells were infected with bacteria for 60 min, washed, scraped, and lysed on ice in lysis buffer containing 10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl₂, 0.1 mM EDTA, 1% NP-40, and 1 mM DTT, and phosphatase and protease inhibitors. These conditions selectively lyse the cells, but not the bacteria. In addition, these conditions do not extract critical amounts of Yops from the bacteria. The cellular lysates were diluted 1/1 with precipitation buffer (20 mM Tris [pH 7.5], 200 mM NaCl, 1 mM DTT, and phosphatase and protease inhibitors) and precleared with an irrelevant polyclonal rabbit antibody and protein A-agarose (Santa Cruz Biotechnology). For immunoprecipitation, cell lysates were incubated with a polyclonal rabbit anti-YopE antibody (20) for 4 h at 4°C. Immune complexes were collected with protein A-agarose, washed with precipitation buffer, separated by SDS-PAGE, electrotransferred to PVDF membrane, and probed with either rabbit anti-YopE or polyclonal rabbit anti-IKK antibody (which exhibits partial cross-reactivity with IKK; Santa Cruz Biotechnology). Immunoreactive bands were visualized by incubation with goat anti-rabbit antibodies conjugated to horseradish peroxidase (Amersham Pharmacia) by using enhanced chemiluminescence reagents (Amersham Pharmacia). For quantification of translocated YopP constructs, cytoplasmic lysates of J774A.1 cells were produced 60 min after onset of infection. The lysates were separated by SDS-PAGE, electrotransferred to PVDF membrane, and probed with either rabbit anti-YopE, or polyclonal rabbit anti-MEK1 antibody (Santa Cruz Biotechnology). Nonsaturated immunoreactive bands were visualized as described above and processed for quantification by using Image-Master 1D software (Amersham Pharmacia).

Assessment of apoptosis by fluorescence microscopy. To quantify apoptosis of J774A.1 cells in response to bacterial infection, apoptotic cells were specifically labeled with fluorescein-conjugated annexin V (Boehringer, Mannheim, Germany) as described previously (35). Annexin V binds with high affinity to phosphatidylserine exposed on the outer leaflet of apoptotic cells and confers green fluorescence to cells undergoing apoptosis. The simultaneous application of the DNA stain propidium iodide (Sigma, St. Louis) allows the discrimination of apoptotic from necrotic cells. The rate of apoptosis was determined by counting a minimum of 200 cells per sample in a fluorescence microscope. Results are expressed as mean percentages of fluorescing apoptotic cells ± standard deviation from three independent experiments.

	RESULTS

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YopP from Y. enterocolitica serotype O8 is substantially more efficient in suppression of the NF-B pathway and mediation of apoptosis than serotype O9 YopP. In order to analyze the impact of Y. enterocolitica on macrophage apoptosis, we investigated a panel of wild-type strains from diverse human pathogenic Y. enterocolitica serotypes (listed in Table 1). Surprisingly, Y. enterocolitica strains from the serogroup O8 (strains WA and Y-8081) were by far more efficient at triggering apoptosis in murine J774A.1 macrophages than Yersinia strains from the serotypes O3 (strains Y-108-P and Y.e.-88) and O9 (strains E40, Y-96-P, and Y.e.-93). Accordingly, serogroup O8 Y. enterocolitica killed more than 90% of infected macrophages 6 h after onset of infection, whereas only 20 to 40% of the cells treated with serogroup O3 or O9 Y. enterocolitica were apoptotic within the same time. To reveal whether these discrepant apoptotic responses were due to more active YopPO8 versus YopPO9, we amplified and subcloned the yopP genes from the well-characterized strains WA (serotype O8) and E40 (serotype O9) in order to complement the yopP-negative knockout mutants. Accordingly, yopPO8 and yopPO9 were introduced in trans into the yopPO8-negative mutant WA-yopP. The resulting strains, WA-yopP/+PO8 and WA-yopP/+PO9, synthesize YopPO8 or YopPO9 as a fusion protein, with the first 138 amino acids of YopE controlled by the yopE promoter. This procedure allows efficient production and secretion of Yop hybrid proteins by Yersinia in comparable amounts. This is a useful method to complement yopP-negative mutants (34). yopP is located on an operon behind yopO; therefore, a complementation procedure using the endogenous yopP promoter is complicated (13). In all further approaches, we used this tool to introduce yopP constructs into Yersinia and to analyze their impact on the host cell.

View larger version (51K): [in this window] [in a new window]   FIG. 1.   YopPO8 efficiently triggers macrophage apoptosis and interferes with NF-B activation. (A) NF-B suppression and apoptosis induction. J774A.1 cells were left untreated (lane 1) or were stimulated with LPS (lane 2), virulence plasmid-cured WA-pYV (lane 3), wild-type WA from serogroup O8 (lane 4), yopPO8-negative WA-yopP (lane 5), YopPO8-producing WA-yopP/+PO8 (lane 6), YopPO9-producing WA-yopP/+PO9 (lane 7), WA-yop producing no effector Yops (lane 8), WA-yop/+PO8 producing only YopPO8 (lane 9), WA-yop/+PO9 producing only YopPO9 (lane 10), or wild-type E40 from serogroup O9 (lane 11). The NF-B activities were determined by EMSA 90 min after infection (upper panel). Only sections of the autoradiogram containing the NF-B-DNA complexes are shown. The EMSA shows data from one experiment representative of five performed. Apoptosis was assayed 6 h after onset of infection by staining cells with annexin V and counting apoptotic cells by fluorescence microscopy (lower panel). Results are expressed as mean percentages ± standard deviations from three independent experiments. (B) IKK activity assay. Cell extracts from 1.25 × 107 untreated cells (lane 1) or cells treated with LPS (lane 2), wild-type WA from serogroup O8 (lane 3), yopPO8-negative WA-yopP (lane 4), YopPO8-producing WA-yopP/+PO8 (lane 5), or YopPO9-producing WA-yopP/+PO9 (lane 6) for 30 min were incubated with anti-IKK antibodies to precipitate IKK. IKK activities were assayed by measuring the abilities of the immunocomplexes to radioactively phosphorylate recombinant GST-IB. Kinase reaction samples were subjected to SDS-PAGE and transferred to PVDF membrane. The upper part of the membrane was immunoblotted with anti-IKK antibodies (upper panel). The double band appearing in lanes 2 to 6 may reflect phosphorylated and nonphosphorylated forms of IKK. The lower part of the membrane including GST-IB was analyzed by autoradiography (lower panel). The results shown are from one representative experiment out of three performed. WB, Western blot; KA, kinase assay.

Arginine-143 is an essential residue of the YopPO8-specific effector domain. We next attempted to analyze the relationship between structural domains and the serotype-related functions of YopP isotypes. For that reason, we compared the deduced amino acid sequences of YopPO8 (GenBank accession no. AF336309) and YopPO9 (GenBank accession no. AF102990). YopPO8 from strain WA, which was investigated in our studies, displays a sequence corresponding to the published sequence of YopPO8 from strain Y-8081 (37). Figure 2 shows that the sequences of YopPO8 and YopPO9 are highly homologous (94% identity). Single amino acids that differ between these two serogroups are marked in boldface. In the first approach to localize the YopPO8 effector domain, we excised an internal 514-bp region located between two BamHI restriction sites of yopPO8 and yopPO9 and exchanged the fragment between the two yopP genes (Fig. 2; the corresponding amino acids of the BamHI restriction sites are underlined). The 514-bp DNA fragment encodes amino acids 44 to 214 of YopP. The respective yopPO8/yopPO9 chimeric DNA constructs were transferred into the yopPO9-negative mutant E40-yopP. We utilized strain E40-yopP instead of WA-yopP in this set of experiments, because the lack of any antibiotic resistance marker in E40-yopP facilitated the complementation procedure. Both E40-yopP and WA-yopP exhibited identical yopP-negative phenotypes. With the exchange of the 514-bp yopP BamHI region, the YopP effector functions were completely reversed in the resulting strains (Fig. 3). In contrast to the original strain E40-yopP/+PO8, strain E40-yopP/+PO8(44-214PO9) harboring the yopPO9 BamHI fragment, provoked a strong NF-B signal that was associated with substantial cellular survival (compare lanes 3 and 5). On the other hand, insertion of the yopPO8 BamHI fragment into E40-yopP/+PO9 strongly suppressed the NF-B response to the resulting strain E40-yopP/+PO9(44-214PO8), which led to fulminant apoptosis (compare lanes 4 and 6). Thus, the serogroup O8-specific effector domain of YopP and the corresponding phenotype are interchangeable between the two Y. enterocolitica serotypes.

View larger version (43K): [in this window] [in a new window]   FIG. 2.   Alignment of the deduced amino acid sequences from YopPO8 (GenBank accession no. AF336309) and YopPO9 (GenBank accession no. AF102990). Amino acid differences are marked in boldface. The corresponding amino acids of the internal yopP BamHI restriction sites are underlined. Dark shading denotes the proposed isotype-related YopP effector site (arginine-143, YopPO8; serine-143, YopPO9). Light shading indicates the amino acid residues of the proposed cysteine protease-related catalytic triad (histidine-109, glutamic acid-128, and cysteine-172).

View larger version (42K): [in this window] [in a new window]   FIG. 3.   Interchange of amino acids 44 to 214 reverses YopPO8 and YopPO9 isotype-dependent effector functions. J774A.1 cells were left untreated (lane 1) or were infected with yopPO9-negative E40-yopP (lane 2), YopPO8-producing E40-yopP/+PO8 (lane 3), YopPO9-producing E40-yopP/+PO9 (lane 4), E40-yopP/+PO8(44-214PO9) producing a YopPO8 construct in which the internal amino acids 44 to 214 were substituted for by the respective amino acids from YopPO9 (lane 5), and E40-yopP/+PO9(44-214PO8) producing a YopPO9 construct in which internal amino acids 44 to 214 were substituted for by the respective amino acids from YopPO8 (lane 6). The NF-B activities were determined 90 min after infection by EMSA (upper panel). Only sections of the autoradiogram containing the NF-B-DNA complexes are shown. The EMSA shows data from one experiment representative of three performed. Apoptosis was assayed 6 h after onset of infection by staining cells with annexin V and counting apoptotic cells by fluorescence microscopy (lower panel). Results are expressed as mean percentages ± standard deviations from three independent experiments.

View larger version (19K): [in this window] [in a new window]   FIG. 4.   Mapping of the YopPO8 isotype-related effector domain by interchange of amino acids between YopPO9 and YopPO8. Mutagenesis of YopPO9 (lane 1) was accomplished by replacement of an internal 514-bp yopPO9 DNA fragment by diverse yopPO9/yopPO8 hybrid sequences for this region (lanes 2 to 8). The DNA region encodes amino acids 44 to 214 of YopP. The fragments were generated by PCR and introduced into the internal yopPO9 BamHI restriction sites, which encompass the 514-bp region. In lanes 2 to 8, only the amino acid sequences introduced from YopPO8, but not the endogenous YopPO9 amino acid sequences, are displayed. Mutagenesis of YopPO8 (lane 9) was conducted by the same method, resulting in substitution of multiple or single amino acids of YopPO8 by residues from YopPO9 (lanes 10 to 13). Apoptosis was assayed 6 h after onset of infection by staining cells with annexin V and analyzing apoptotic cells by fluorescence microscopy [apoptosis: (+), 20 to 40%; +, 40 to 60%; ++, 60 to 80%; +++, >80%].

View larger version (24K): [in this window] [in a new window]   FIG. 5.   Arginine-143 critically determines YopP effector functions. (A) NF-B suppression and apoptosis induction. J774A.1 cells were infected with YopPO9-producing E40-yopP/+PO9 (lane 1), YopPO8-producing E40-yopP/+PO8 (lane 2), E40-yopP/+PO8(R143S) producing a YopPO8 construct in which arginine-143 was substituted for by serine (lane 3), or E40-yopP/+PO9(S143R) producing a YopPO9 construct in which serine-143 was substituted for by arginine (lane 4). The NF-B activities were determined 90 min after infection by EMSA (upper panel). Only sections of the autoradiogram containing the NF-B-DNA complexes are shown. The EMSA shows data from one experiment representative of three performed. Apoptosis was assayed 6 h after onset of infection by staining cells with annexin V and counting apoptotic cells by fluorescence microscopy (lower panel). Results are expressed as mean percentages ± standard deviations from three independent experiments. (B) IKK activity assay. Cell extracts from 108 cells infected with YopPO8-producing E40-yopP/+PO8 (lane 1), YopPO9-producing E40-yopP/+PO9 (lane 2), YopPO9(S143R)-producing E40-yopP/+PO9(S143R) (lane3), or YopPO8(R143S)-producing E40-yopP/+PO8(R143S) (lane 4) for 30 min were incubated with anti-IKK antibodies to precipitate IKK. IKK activities were assayed by measuring the abilities of the immunocomplexes to radioactively phosphorylate recombinant GST-IB. Kinase reaction samples were subjected to SDS-PAGE and transferred to PVDF membrane. The upper part of the membrane was immunoblotted with anti-IKK antibodies (upper panel). The lower part of the membrane including GST-IB was analyzed by autoradiography (lower panel). The results shown are from one representative experiment out of three performed. WB, Western blot; KA, kinase assay.

View larger version (32K): [in this window] [in a new window]   FIG. 6.   Targeting of macrophage IKK by the diverse YopP constructs. (A.) Interaction of YopP with macrophage IKK. J774A.1 cells were infected with yopPO9-negative E40-yopP (lane 1), YopPO8-producing E40-yopP/+PO8 (lane 2), YopPO9-producing E40-yopP/+PO9 (lane 3), WA-yop producing no effector Yops (lane 4), WA-yop/+PO8 producing only YopPO8 (lane 5), WA-yop/+PO9 producing only YopPO9 (lane 6), WA-yop/+PO8(R143S) producing only the YopPO8 construct with the mutation of arginine-143 to serine (lane 7), or WA-yop/+PO9(S143R) producing only the YopPO9 construct with the mutation of serine-143 to arginine (lane 8). After 60 min, cells were lysed, and YopP was immunoprecipitated with polyclonal anti-YopE antibody recognizing the YopE138-YopP fusion proteins. Immunocomplexes were subjected to SDS-PAGE and transferred to PVDF membrane. One part of the membrane was immunoblotted with anti-YopE antibodies, recognizing the YopP fusion proteins (lower panel), and the other part was immunoblotted with anti-IKK antibodies (upper panel). (B) Quantification of amounts of cytoplasmic YopP. J774A.1 cells were infected with WA-yop producing no effector Yops (lane 1), WA-yop/+PO8 producing only YopPO8 (lane 2), or WA-yop/+PO8(R143S) producing only the YopPO8 construct with the mutation of arginine-143 to serine (lane 3). After 60 min, cells were lysed under conditions that selectively lyse the cells. The lysates were subjected to SDS-PAGE and transferred to PVDF membrane. One part of the membrane was immunoblotted with anti-YopE antibodies, recognizing the YopP fusion proteins (upper panel), and the other part was immunoblotted with control anti-MEK1 antibodies to confirm equal loading with cellular lysates (lower panel). Nonsaturated immunoreactive bands were visualized with enhanced chemiluminescence reagents. The optical densities (OD) of the YopP bands were quantified, and the values obtained are indicated. The results shown are from one representative experiment out of three performed.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

This study demonstrates that YopP isotypes from diverse pathogenic Y. enterocolitica serogroups differ in their abilities to affect macrophage NF-B signaling and to trigger macrophage apoptosis. Yersiniae producing YopP from the Y. enterocolitica serotype O8 remarkably impaired IKK activities and nuclear translocation of NF-B, whereas serotype O9 YopP-producing yersiniae evoked substantial IKK and NF-B activation. The stronger inhibitory action of YopPO8 on IKK activities and NF-B activation coincided with a substantially enhanced apoptotic response. In contrast, apoptosis due to YopPO9 was remarkably less pronounced. This indicates that subversion of the NF-B pathway by YopP-producing yersiniae and the induction of macrophage apoptosis are tightly coupled. The close connection of these two effects supports the idea that macrophage apoptosis results from disruption of the NF-B survival pathway by Yersinia (32, 34). Indeed, overexpression of the transcriptionally active NF-B p65 subunit provides a protective effect against Yersinia-mediated apoptosis (34). These findings suggest that subversion of the NF-B pathway by YopP critically determines apoptosis induction by Y. enterocolitica, although direct action of YopP on cell death signaling was recently proposed (10).

To molecularly characterize the various effects of YopPO8 and YopPO9, we complemented yopP-negative mutants in trans by using a vector construct that mediates synthesis and translocation of distinct YopP proteins under identical conditions. The mutants then complemented by the respective yopPO8 and yopPO9 constructs injected comparable amounts of YopP in infected macrophages and restored the wild-type-related phenotypes. Both YopPO8 and YopPO9 interacted with macrophage IKK. To relate the different effects of the YopP isotypes to certain structural domains, we compared the amino acid sequences of YopPO8 and YopPO9. These sequences are highly homologous (94% identity). We gradually mutagenized YopPO8 and YopPO9, thereby replacing endogenous amino acids of YopPO8 with residues from YopPO9 and vice versa. Functional characterization of the resulting YopPO8/YopPO9 chimeric proteins identified arginine-143 of YopPO8 as crucially involved in both the suppression of the NF-B pathway and the mediation of apoptosis.

These data show that there exist functional differences between genetically highly homologous members of YopP from diverse Y. enterocolitica serotypes. Arginine-143 substantially enhances the inhibitory impact of YopP on NF-B signaling, leading to severe apoptosis. Interestingly, YopP from the Y. enterocolitica serotype O3 strain Y-108-P, which induced a serogroup O9-like, modest apoptotic response, displays serine at position 143 identical to YopPO9 (data not shown). On the contrary, YopPO8 from the strains WA and Y-8081, which both triggered intense apoptosis, are both bearing arginine-143 (37). The published sequences of the YopP homologues YopJ from Y. pseudotuberculosis (GenBank accession no. L33833) and Y. pestis (GenBank accession no. AF074612) also display arginine at position 143, similar to Y. enterocolitica serogroup O8. In correlation with the proposed critical role of arginine-143 in apoptosis induction, the corresponding Y. pseudotuberculosis strain YPIII (kindly provided by H. Wolf-Watz; Department of Cell and Molecular Biology, Umea, Sweden) induced a strong apoptotic response (data not shown). This suggests that YopPO8 shares structural and functional similarities with YopJ that differ from the effects observed for YopPO9 or YopPO3.

In fact, Y. enterocolitica of serogroup O8 is, with respect to pathogenicity, more related to Y. pestis and Y. pseudotuberculosis than Y. enterocolitica of the serogroups O3 and O9 (6, 7, 9, 16). Y. enterocolitica serotype O8, Y. pseudotuberculosis, and Y. pestis are well adapted to rodents as animal hosts and display high pathogenicity in the mouse infection model even at low bacterial doses. The high virulence phenotype, leading to mouse lethality, is predominantly attributed to the chromosomal Yersinia high-pathogenicity island (HPI), which encodes the yersiniabactin siderophore system (7). The Y. enterocolitica serotypes O3 and O9 lack the HPI and are therefore of low mouse virulence. Although the Yersinia virulence plasmid pYV is also essentially required for mouse virulence, the exchange of pYV between the Y. enterocolitica serotypes O8 and O9 does not significantly alter mouse lethality of the respective serogroups (12, 17). In view of these observations, distinct phenotypic profiles due to diverse YopP isotypes can probably not be expected from the mouse model of infection. Such clear differences may be even more unlikely, since the virulence phenotypes of yopJ/yopP-negative mutants in comparison to those of the respective wild-type strains have been shown to be subtle (13, 24, 38). Two studies could not attribute significant virulence functions to YopJ from Y. pseudotuberculosis and Y. pestis (13, 38). Preliminary results from our laboratory suggest that YopPO8 does not display an immediate obvious virulence phenotype. On the contrary, Monack et al. demonstrated that YopJ helps yersiniae in colonizing deeper lymphatic tissues and in the establishment of a systemic infection (24). These studies suggest that YopP/YopJ may play a discreet role in the infectious process. YopP/YopJ may predominantly be involved in the maintenance of prolonged colonization and asymptomatic latent infection, rather than in mediating severe infectious disease. This hypothesis is supported by the in vitro functions of YopP/YopJ, which primarily are intended to dampen the host immune response, thereby preventing overwhelming immune reactions and inflammation. In this context, a more active YopP/YopJ, carrying arginine-143 in the case of Y. enterocolitica serogroup O8, Y. pseudotuberculosis, and Y. pestis, may have evolved to establish an advantage to yersiniae for better colonization and adaptation to rodents as primary hosts.

The molecular mechanism by which YopPO8 exerts a stronger inhibitory effect on IKK is still not clear. It was suggested that binding of YopP/YopJ blocks phosphorylation and activation of its target proteins (27). However, a recent report indicates that YopP/YopJ may act as a cysteine protease on cellular proteins modified by the ubiquitin-like molecule SUMO-1 (28). Mutations of the proposed catalytic residues histidine-109 and cysteine-172 abolish the ability of YopP/YopJ to inhibit the NF-B pathway and to mediate apoptosis (10, 28). A link between reduced SUMO-1 conjugation of proteins and inhibition of IKK or MKKs is hitherto unclear. MKKs and IKK do not appear to be direct targets for SUMO-1 cleavage, and the proteins that are de-SUMOylated by YopP/YopJ still await identification (28). Orth and colleagues speculate that SUMO-1 conjugation may be an important posttranslational modification of signaling complexes, which directs their intracellular processing (28). This may involve the regulation of IKK- and MKK-dependent signaling pathways. The predicted secondary structure of YopP/YopJ perceives arginine-143 as a component of an -helix, implying mainly a structural role of arginine-143 in YopP constitution (28). On the other hand, the sequences of YopPO8 and YopPO9 both include the cysteine protease-related catalytic residues histidine-109, glutamic acid-128, and cysteine-172 (Fig. 2). Arginine-143 is located centrally within this catalytic domain, which suggests that arginine-143 may directly affect the proposed enzymatic activity of YopP/YopJ on SUMO-1-conjugated proteins. Together, these data point out a crucial role of arginine-143 in the down-regulation of IKK function by YopP, which critically determines the fate of the Yersinia-infected macrophage.