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B signalling pathway
1 Institute of Veterinary Bacteriology, Vetsuisse Faculty, Universität Bern, Länggassstrasse 122, Postfach, CH-3001 Bern, Switzerland
2 Division of Molecular Pathology, Vetsuisse Faculty, Universität Bern, Länggassstrasse 122, Postfach, CH-3001 Bern, Switzerland
3 Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX, USA
Correspondence
Joachim Frey
joachim.frey{at}vbi.unibe.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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B) signalling pathways. AopP inhibits the NF-B pathway downstream of IB kinase (IKK) activation, while a catalytically inactivated mutant, AopPC177A, does not possess this inhibitory effect. Unlike other effectors of the YopJ family, such as YopJ and VopA, AopP does not inhibit the MAPK signalling pathway.
B kinase; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase; NF-B, nuclear factor kappa B; TNF-
, tumour necrosis factor alpha; TTSS, type three secretion system; wt, wild-typeThe GenBank/EMBL/DDBJ accession number for the plasmid pAsal1 sequence reported in this paper is AJ508382.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), found within the 
subclass of the Proteobacteria. During the past two decades, the number of phenotypically defined species and DNA hybridization groups within the genus Aeromonas has grown rapidly, and, at present, the genus comprises 17 well-defined genomic species. Aeromonas species are widespread throughout the environment, occurring in fresh, brackish and marine waters. Many of the species are pathogenic, causing disease in both human and animal hosts. Human isolates of Aeromonas are primarily associated with gastrointestinal disease. However, these isolates are also associated with extraintestinal disorders, such as wound infections and septicaemia. In animals, Aeromonas species are associated with disease in both warm- and cold-blooded vertebrates, including fish, frogs, snakes and birds.
The species Aeromonas salmonicida is included among the aeromonad fish pathogens and comprises both typical and atypical isolates. Typical isolates, which belong to the subspecies salmonicida, are a homologous group that are primarily associated with systemic infections in salmonid fish. Atypical isolates, however, form a biochemically and genotypically heterogeneous group that also vary in terms of their host and disease symptoms (Belland & Trust, 1988; Umelo & Trust, 1998; Gudmundsdóttir, 1998, 2003; Lund & Mikkelsen, 2004). While the significance of atypical A. salmonicida infections in both wild and cultivated fish stocks is now becoming clear (Gudmundsdóttir, 1998), the majority of the work carried out on A. salmonicida in the past has concerned typical isolates. In fact A. salmonicida subsp. salmonicida is probably the most extensively studied bacterial fish pathogen, largely due to its widespread distribution and impact on aquaculture.
Much of the work carried out on A. salmonicida has focused on the identification of potential virulence factors expressed by the bacterium. To date, several such factors have been reported. These include bacterial surface structures, such as the surface layer protein (Noonan & Trust, 1997) and type IV pili (Masada et al., 2002), as well as extracellular proteins, including serine protease, glycerophospholipid : cholesterol acyltransferase and several haemolysins (Nomura et al., 1988; Lee & Ellis, 1990; Hirono & Aoki, 1993; Vipond et al., 1998).
A. salmonicida isolates have also been shown to harbour genes encoding a type III secretion system (TTSS) (Burr et al., 2002, 2005). Such secretion systems are virulence mechanisms, common among Gram-negative bacteria, which enable the secretion and translocation of effector proteins, produced in the bacterial cytoplasm, directly into the cytosol of target eukaryotic cells (Ghosh, 2004). Once within the eukaryotic cytosol, effector proteins are able to disrupt the cytoskeleton or interfere with cell signalling cascades (Cornelis & Van Gijsegem, 2000).
Studies carried out with a typical A. salmonicida isolate, strain JF2267, have found that the TTSS is responsible for the translocation of the ADP-ribosylating toxin AexT into target fish cells (Burr et al., 2003a). To date, AexT is the only type III effector protein produced by members of the genus Aeromonas to have been described. In this study, we show that A. salmonicida produces a second type III effector protein, which we have termed AopP.
AopP was found to belong to the YopJ family of type III effector proteins, whose members inhibit specific cell signalling cascades. YopJ, the first member of the YopJ family of effector proteins to be characterized, is expressed by pathogenic Yersinia species and prevents the activation of the superfamily of mitogen-activated protein kinase (MAPK) kinases, which include MAPK kinases (MKKs) and the IB kinase (IKK) complex (Orth, 2002). As a result of these activities, signalling via the MAPK and nuclear factor kappa B (NF-B) pathways is prevented. In this way, YopJ is able to block cytokine production and promote apoptosis in the target host cell. While AopP shares sequence homology with YopJ, it differs in its spectrum of inhibitory activities. We show that AopP inhibits the NF-B signalling pathway downstream of IB phosphorylation but does not affect MAPK signalling.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Escherichia coli strains were routinely grown in Luria–Bertani (LB) agar or broth at 37 °C. Pseudomonas aeruginosa strain ATCC 27853 was grown on LB plates at 37 °C. A. salmonicida strains were grown in tryptic soy broth or on LB agar at 18 °C, unless otherwise indicated. When indicated, antibiotics were added to the culture media at the following final concentrations: 100 µg ampicillin ml–1, 40 µg kanamycin ml–1 and 40 µg chloramphenicol ml–1.
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). An ordered set of nested deletions was then obtained using the Erase-a-Base system (Promega) according to the manufacturer's instructions.
Sequencing was performed using the dRhodamine Terminator Cycle Sequencing kit (Applied Biosystems) according to the manufacturer's protocol, with either T3 or T7 primers flanking the cloned insert in pBSK– or synthesized internal primers (Microsynth). Reaction products were analysed on an ABI Prism 3100 genetic analyser (Applied Biosystems). Sequence alignment and editing was performed using the software Sequencher (Gene Codes Corp.). Identification of potential ORFs was carried out using the ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Comparison of DNA sequences and their corresponding amino acid sequences was performed using BLAST (Altschul et al., 1990).
Southern blot analysis.
The aopP gene of A. salmonicida subsp. salmonicida strain JF2267 was amplified by PCR in the presence of 20 µM DIG (Roche Diagnostics) in order to generate a labelled probe. PCR was carried out using the following primer pair: AsORF28, 5'-GAGAGTTGGCTAGCGGTGAG-3', and AsORF38, 5'-TCCTCATGGAGCGCATCCAG-3', and PCR conditions were 3 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 30 s at 58 °C and 30 s at 72 °C.
Plasmid DNA was prepared using the Qiaprep Spin Miniprep kit (Qiagen) and was digested with the restriction enzyme BamHI. The DNA was separated by gel electrophoresis, and Southern blotting was then performed by alkaline transfer onto positively charged nylon membranes (Roche Diagnostics). Hybridization was carried out overnight at 60 °C and the membrane was washed with 2x saline sodium citrate (SSC), 0.1 % SDS, followed by a second wash with 0.2x SSC, 0.1 % SDS. Hybridization reactions were detected using anti-DIG antibodies and the chemiluminescent reagent CDP-Star (Roche Diagnostics) according to the manufacturer's protocol. DNA from A. salmonicida subsp. salmonicida strain JF2267 and P. aeruginosa strain ATCC 27853 served as positive and negative controls, respectively.
Complementation.
In order to complement the 
ascV mutation in A. salmonicida subsp. salmonicida strain JF2747, the ascV gene and its ribosome-binding site were amplified from the wild-type (wt) strain JF2267 by PCR using primers ascVSalI, 5'-GTCGACATGTCGATCAGCTGATCAG-3', and ascVHindIII, 5'-AAGCTTCCAGATAGTGAGAGGCAACC-3' (underlined nucleotides indicate recognition cut sites for restriction endonucleases SalI and HindIII, respectively). PCR was carried out using a primer-annealing temperature of 58 °C and an extension time of 3.5 min. The resulting PCR product was then cloned into the broad-host-range plasmid pMMB66EH (Fürste et al., 1986) and transformed into E. coli strain S17.1 (Simon et al., 1983). The plasmid was introduced into A. salmonicida subsp. salmonicida strain JF2747 by filter mating (Simon et al., 1983) for 2 days at 15 °C. Clones containing the recombinant plasmid were selected for on media containing 100 µg ampicilin ml–1 and 40 µg kanamycin ml–1, and the presence of the wt gene was verified by PCR. Expression of the cloned gene was induced by the addition of IPTG to a final concentration of 1 mM.
AopP secretion.
A. salmonicida strains were grown in tryptic soy broth containing protease inhibitor (Complete, Roche Diagnostics), for 18 h at 18 °C. Equivalent amounts of cells and supernatants were harvested and separated on 12 % SDS-PAGE gels according to the method of Laemmli (1970). Following separation, Western blot analysis was performed using anti-AopP antibodies. Antibody binding was visualized using ECL plus Western Blotting Detection reagents (Amersham Biosciences).
Site-directed mutagenesis.
A catalytically inactivated AopP mutant, AopPC177A, was constructed by recombinant PCR using the method of Vallette et al. (1989) with mutagenesis primers aopPC177A-fwd, 5'-GAAGCAATTCAGAAGCAGGGATATTCAGCG-3', and aopPC177A-rev, 5'-GCTGAATATCCCTGCTTCTGAATTGCTTC-3' (mutations underlined). The PCR conditions were as follows: 3 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 58 °C, and up to 90 s at 68 °C. An extension step of 2 min at 68 °C was carried out following the last cycle in order to ensure full-length synthesis of the fragments. The final product was sequenced to ensure the correct mutation and was cloned into plasmids pSFFV or pcDNA3.1/V5-HIS.
Production of polyclonal anti-AopP antibodies.
To generate polyclonal antibodies directed against AopP, we first amplified the aopP gene using primers HISaopPSpeI, 5'-GAACTAGTTATGAATATACCCCCCATCC-3', and HISaopPNotI, 5'-GGGCGGCCGCAAACGGATTTTCCGTCATCAG-3' (underlined nucleotides indicate recognition cut sites for restriction endonucleases SpeI and NotI, respectively). The PCR conditions were as follows: 3 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 60 °C and 2 min at 68 °C. An extension step of 5 min at 68 °C was carried out following the last cycle. The resulting PCR product was cloned into the pGEM-T Easy vector (Promega) and transformed into E. coli strain XL-1 Blue (Bullock et al., 1987). Recombinant plasmids were then digested with the restriction enzymes SpeI and NotI, and the resulting DNA fragment was ligated into the expression vector pETHIS-1 in order to generate polyhistidine tags at both the N- and C-terminal ends of the recombinant protein. The cloned aopP insert was sequenced to ensure correct fusion with the vector poly-HIS codons before being transformed into E. coli BL21(DE3) cells (Studier et al., 1990) for expression of the fusion protein. LB broth (50 ml) containing ampicillin was inoculated with E. coli BL21(DE3) cells harbouring plasmid pETHIS-aopP and incubated at 37 °C to an OD600 of 0.3. Cells were then induced by the addition of 1 mM IPTG and grown for an additional 3 h. Following induction, the fusion proteins were purified from cell extracts under denaturing conditions, using Ni2+ chelate affinity chromatography columns (Qiagen) according to the manufacturer's instructions. The bound protein was eluted by decreasing the pH from 8.0 to 5.0 with 50 mM potassium phosphate buffer, 300 mM NaCl, 6 M guanidine hydrochloride. The eluted protein was then dialysed against 50 mM phosphate buffer, 300 mM NaCl, pH 8.0, before being electro-eluted from 12 % SDS-PAGE gels using an Elutrap (Schleicher & Schuell). Polyclonal antibodies against AopP were obtained by immunization of a rabbit with purified recombinant AopP–HIS protein mixed 1 : 1 with GERBU Adjuvant 10 (GERBU Biotechnik). IgG was isolated from the anti-AopP antisera using HiTrap affinity columns (Amersham Pharmacia, Biotech) according to the manufacturer's instructions.
MAP kinase assay.
Approximately 7.5x105 HEK293 cells were seeded and transfected using 7 µl FuGENE 6 reagent (Roche Diagnostics) with 100 ng pHA-ERK haemagglutinin (HA)–extracellular signal-regulated kinase (ERK) plasmid and 100 ng pSFFV vector, YopJ-pSFFV, YopJC172A-pSFFV, AopP-pSFFV or AopPC177A-pSFFV, and were allowed to rest for 24 h. The cells were starved overnight to reduce background or stimulus-independent phosphorylation. The whole-cell lysates harvested after epidermal growth factor (EGF) treatment (100 ng ml–1) for 5 min at room temperature were then separated by SDS-PAGE and transferred to nitrocellulose membranes for Western blotting with anti-phospho-ERK and anti-HA antibodies.
IB phosphorylation assay.
Approximately 1x105 HeLa cells were seeded and transiently transfected using the PolyFect reagent (Qiagen) according to the manufacturer's instructions, and incubated at 37 °C in 5 % CO2 for 18 h to allow for expression. DNA quantities used were as follows: 1 µg each of pIB-FLAG and MEKK1-pcDNA3.1; 0.5 µg each of YopJ-pSFFV, YopJC172A-pSFFV, AopP-pcDNA3.1/V5-HIS and AopPC177A-pcDNA3.1/V5-HIS. The proteasome inhibitor MG-262 (Affiniti) was then added to a final concentration of 250 nM, and cells were incubated for a further 4 h. For tumour necrosis factor alpha (TNF-) stimulation, HeLa cells were transiently transfected with 1 µg each of YopJ-pSFFV, YopJC172A-pSFFV, AopP-pcDNA3.1/V5-HIS or AopPC177A-pcDNA3.1/V5-HIS only, and stimulated with TNF- (100 ng ml–1) for 3 min. Cell lysates were prepared and separated on 12 % SDS-PAGE gels according to the method of Laemmli (1970). Once separated, the proteins were electroblotted onto nitrocellulose membranes (Bio-Rad Laboratories). Immunoreactive proteins were detected with antibodies to IB (C-21, sc-371; Santa Cruz Biotechnology), phospho-IB (Cell Signalling Technology), MEK kinase-1 (C-22, sc-252; Santa Cruz Biotechnology), V5 (Invitrogen) or tubulin (Sigma) using horseradish peroxidase (HRP)-conjugated anti-rabbit (Cell Signalling Technology) or anti-mouse (Dako) secondary antibodies. Antibody binding was visualized using ECL plus Western Blotting Detection reagents (Amersham Biosciences).
Immunofluorescence microscopy.
Approximately 2x104 HeLa cells, grown on 12 mm glass coverslips, were transiently transfected using PolyFect reagent (Qiagen). DNA quantities used were as follows: 1 µg of AopP-pcDNA3.1/V5-HIS, AopPC177A-pcDNA3.1/V5-HIS or pcDNA3.1/V5-HIS only. Cells were allowed 22 h for expression and were stimulated with TNF- (100 ng ml–1) for 30 min. Cells were washed twice with PBS and then fixed for 20 min in 4 % formaldehyde, 10 % fetal calf serum (FCS) in PBS. Cells were then permeabilized with 0.1 % Triton X-100 for 10 min and washed twice in PBS. Fixed samples were incubated in blocking solution (10 % FCS in PBS) for 10 min. Coverslips were incubated for 1 h with either rabbit anti-p65 antibody (C-20, sc-372; Santa Cruz Biotechnology) diluted 1/50, mouse anti-V5 antibody (Invitrogen) diluted 1/500 or anti-YopP/J antibodies (kindly provided by Guy Cornelis, Biozentrum, Basel) diluted 1/1000 in blocking solution. Coverslips were then washed three times and subsequently incubated with anti-rabbit Alexa Fluor 488 antibody (Molecular Probes) diluted 1/1500 or anti-mouse Texas red antibody (Vector Laboratories) diluted 1/800. The cell nuclei were stained with Hoechst. The coverslips were mounted on glass slides and stained cells were observed using a Nikon Eclipse 800 microscope. Images were produced by digital confocal imaging using Openlab software (Improvision).
Nucleotide sequence accession numbers.
The nucleotide sequence of plasmid pAsal1 has been submitted to the EMBL Nucleotide Sequence Database under accession number AJ508382. The accession numbers of the aopP gene from all A. salmonicida strains studied are AM181130–AM181138.
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RESULTS AND DISCUSSION |
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; Hanninen et al., 1995; Giles et al., 1995), A. salmonicida subsp. salmonicida strain JF2267 was found to harbour three small plasmids, which we have termed pAsal1 (6371 bp), pAsal2 (5424 bp) and pAsal3 (5259 bp). As previously reported, plasmid pAsal2 is identical to plasmid pAsa1 found in the virulent A. salmonicida subsp. salmonicida isolate, strain A449, whereas plasmid pAsal3 is virtually identical to plasmid pAsa2 of strain A449 (6 base pair differences) (Boyd et al., 2003). Plasmid pAsal1 is not found in strain A449.
Sequence analysis of plasmid pAsal1 revealed the presence of five significant ORFs (Fig. 1A, Table 2). Of particular interest was the identification of a gene encoding a potential type III effector protein, which we have termed aopP. The aopP gene is 900 bp in length and encodes a predicted protein, AopP, of 299 aa. The amino acid sequence of AopP reveals sequence similarity to YopJ/P proteins of the pathogenic Yersinia sp. (46 % identity, 64 % similarity), AvrA of Salmonella enterica Typhimurium (40 % identity, 59 % similarity) and VopA of Vibrio parahaemolyticus (37 % identity, 55 % similarity). Like these YopJ family members, amino acid sequence alignment revealed that AopP contains a catalytic triad, formed by a histidine, a glutamic acid and a cysteine residue, which is necessary for the activity of these proteins (Fig. 1B) (Denecker et al., 2001; Orth, 2002).
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). No other potential type III effector genes were found on plasmid pAsal1, or on the other small plasmids found in A. salmonicida subsp. salmonicida strain JF2267, pAsal2 and pAsal3.
Detection of aopP in A. salmonicida field isolates
A number of A. salmonicida field isolates, including both typical and atypical strains, were investigated for the presence of aopP. Plasmid DNA was isolated from each strain and examined for the presence of aopP by Southern blot hybridization (Fig. 2A). The results indicated that all the typical A. salmonicida strains investigated harboured the aopP gene, while three out of five atypical isolates possessed the gene (Fig. 2B). Strains ATCC 27013T and As51, the two atypical strains that did not give a positive signal for aopP, were also negative when the Southern blot was repeated using genomic DNA (data not shown).
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Secretion of AopP
As AopP showed sequence similarity to type III secreted proteins, we examined secretion of AopP into the external environment by all 12 A. salmonicida isolates. The results indicated that four typical A. salmonicida isolates, strains JF2267, CC-27, CC-72 and JF3224, were able to secrete AopP. Previous studies have also shown that all four of these strains possess the gene cluster that encodes the type III secretion apparatus in A. salmonicida (Burr et al., 2003b, 2005). Only one typical isolate that is known to possess type III secretion genes and that gave a positive signal for aopP by Southern blot hybridization was unable to secrete AopP, strain CC-29 (Fig. 3A). The remaining typical A. salmonicida isolates, strains ATCC 33658T and Austin 98, do not harbour a functional TTSS (Stuber et al., 2003; Burr et al., 2005) and were not able to secrete AopP (Fig. 3A), even though both strains expressed the AopP protein (results not shown).
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). In agreement with these findings, strains NCIMB 1110T and F-265/87 are known to possess a TTSS, while strain As209 does not (Burr et al., 2005).
To confirm whether the AopP protein is secreted via the TTSS, we examined secretion of AopP in two derivatives of the wt A. salmonicida subsp. salmonicida strain JF2267, an isogenic type III secretion mutant, strain JF2747, and a complemented strain, JF3239. Strain JF2747 possesses a mutation in the gene ascV, which encodes a structural inner-membrane component of the TTSS. As a result, this strain is unable to secrete proteins via the TTSS (Burr et al., 2003a, 2005). In strain JF3239, the ascV mutation has been complemented in trans with plasmid pMMB66EHascV+. All three strains were grown overnight in tryptic soy broth, and the cell pellets and culture supernatants were then subjected to Western blot analysis using polyclonal anti-AopP antibodies. The results indicated that AopP is found in the culture supernatant of the wt strain JF2267 but not in the supernatant of the ascV mutant, strain JF2747. This finding indicates that AopP is secreted into the external environment via the TTSS pathway. In trans complementation of the ascV mutation restored the ability to secrete AopP (Fig. 3B), confirming that secretion of AopP is type III-secretion dependent.
Lack of inhibition of MAPK signalling pathway upstream of ERK
The AopP homologues YopJ/P and VopA are able to inhibit the MAPK signalling pathway of target eukaryotic cells (Palmer et al., 1999; Orth et al., 1999; Trosky et al., 2004; Zhou et al., 2005). We therefore assayed whether AopP also possesses this ability. As substitution of the cysteine residue within the catalytic triad is enough to abolish the enzymic activity of YopJ and its homologues (Orth et al., 2000; Trosky et al., 2004), we also constructed a catalytically inactivated mutant of AopP, AopPC177A, whereby the cysteine at position 177 was changed to an alanine. HEK293 cells were transfected with plasmids expressing AopP, YopJ or the catalytically inactivated forms of these effectors (AopPC177A and YopJC172A), together with pHA-ERK. Cells were then stimulated with EGF to induce ERK activation. wt YopJ, but not wt AopP or the catalytically inactive effectors, was able to inhibit EGF-induced ERK activation (Fig. 4A). We conclude that, in contrast to YopJ and VopA, AopP is unable to inhibit the MAPK signalling pathway. As in the case of AopP, the S. enterica Typhimurium homologue AvrA has also been found to be unable to inhibit MAPK signalling in mammalian cells (Trosky et al., 2004).
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B signalling pathway downstream of IKKB signalling pathway by assaying the effect of AopP on IB phosphorylation. HeLa cells were transiently transfected with plasmids expressing IB-FLAG, MEKK1 (as an internal inducer of endogenous IKK), and the wt or catalytically inactivated forms of AopP or YopJ. Western blot analysis of cell lysates using antibodies against the phosphorylated form of IB showed that expression of wt AopP and the catalytically inactive forms of both effectors had no effect on the phosphorylation of IB (Fig. 4B). In contrast, expression of wt YopJ resulted in significantly reduced phosphorylation of IB (Fig. 4B). These data suggest that AopP does not interfere with IKK activation per se. To further rule out an inhibitory activity of AopP on the NF-B pathway upstream of IKK, we next tested whether AopP affected IKK activity triggered by stimulation of the TNF receptor. HeLa cells were transiently transfected with plasmids encoding wt or catalytically inactivated forms of AopP or YopJ, and stimulated for 3 min with TNF-. Western blot analysis using anti-phospho-IB antibodies showed that neither AopP nor its catalytically inactive mutant inhibited TNF--induced IKK activation; on the contrary, in AopP-expressing cells, a slight increase in levels of phospho-IB could be observed (Fig. 4C, top panels, lane 4). Expression of YopJ, on the other hand, led to a reduction in IB phosphorlyation by 
50 % compared to the catalytically inactive YopJC172A mutant (Fig. 4C, top panels, lanes 2 and 3). Interestingly, YopJ expression also further reduced the low levels of constitutive IKK activity that can be detected in unstimulated HeLa cells (Fig. 4C, lower panels, lane 1).
The lack of inhibition by AopP cannot be attributed to low levels of AopP expression. Immunofluorescence analysis of HeLa cells transfected with either plasmid AopP-pcDNA3.1/HIS-V5 or YopJ-pSFFV revealed comparable transfection efficiencies and confirmed that both proteins, AopP and YopJ, were sufficiently expressed (Fig. 4D). Our results are consistent with previous findings that indicate that YopJ blocks IKK activation (Orth et al., 1999; Collier-Hyams et al., 2002), and indicate that AopP does not interfere with IKK-mediated IB phosphorylation.
As the Salmonella homologue of AopP, AvrA, is also unable to prevent phosphorylation of IB, yet is capable of inhibiting the NF-B pathway downstream of IKK
activation (Collier-Hyams et al., 2002), we used immunolocalization of the p65 subunit of NF-B to further study the potential interference of AopP with the NF-B pathway. HeLa cells were transiently transfected with plasmids expressing wt AopP, the catalytically inactive mutant AopPC177A or the vector only. After 22 h incubation, the transfected cells were stimulated with TNF- for 30 min. As can be seen in Fig. 5, TNF- induced nuclear translocation of p65 in cells transfected with the vector only. However, expression of wt AopP in HeLa cells greatly reduced the nuclear translocation of p65 following stimulation with TNF-. In contrast, the catalytically inactive mutant AopPC177A did not prevent translocation of p65 from the cytoplasm into the nucleus.
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B pathway at a point downstream of IB phosphorylation (Fig. 6). Furthermore, AopP was found to have no inhibitory effect on the MAPK signalling pathway. It has recently been shown that YopJ functions as a deubiquitinating enzyme that negatively regulates the MAPK and NF-B signalling pathways by removing lysine63- and lysine48-linked ubiquitin moieties from regulatory proteins (Zhou et al., 2005). In the NF-B pathway, YopJ impedes the lysine63-linked ubiquitination processes required for IKK activation, and proteasomal degradation of IB is prevented by cleaving lysine48-linked ubiquitin chains. Our findings indicate that AopP does not show the same promiscuity as YopJ. AopP blocked neither the MAPK pathway nor IB phosphorylation. The fact that p65 translocation is blocked, however, might suggest that the activity of AopP is restricted to preventing IB degradation. AopP is therefore another example of how members of the YopJ family, despite relatively high sequence similarity, display marked diversity in their signalling pathway inhibitory profiles (Fig. 6).
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B signalling cascade by repressing translocation of the eukaryotic transcription factor NF-B into the nucleus, we anticipate that this protein has the potential to regulate the host inflammatory response. However, the exact role of AopP in the natural disease process may be more complex and will require further investigation, particularly as some virulent A. salmonicida subsp. salmonicida strains do not possess the aopP gene (Boyd et al., 2003).
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ACKNOWLEDGEMENTS |
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B-FLAG, and to Guy Cornelis for anti-YopP antibodies. This work was supported by the Swiss National Science Foundation (grant no. 3100A0-101595). |
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Received 1 February 2006;
revised 22 May 2006;
accepted 9 June 2006.
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