Institut für Molekulare Biowissenschaften (IMB), Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria
Correspondence
Günther Koraimann
guenther.koraimann{at}uni-graz.at
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ABSTRACT |
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INTRODUCTION |
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In contrast to structurally and functionally related lysozymes, the reaction catalysed by LTs is not a hydrolysis but rather a transglycosylase reaction which results in formation of an internal 1,6 anhydro bond in the MurNAc residue (Höltje et al., 1975). Experimental data strongly suggest that the cleavage reaction is triggered by a conserved glutamyl residue in the active site of LTs (van Asselt et al., 2000
). A natural breakdown product produced by LTs, the muropeptide GlcNAc-(1,6 anhydro)MurNAc-tetrapeptide has been shown to induce inflammatory cytokines in human monocytes (Dokter et al., 1994
). An intracellular receptor and component of the human innate immune system, Nod1, has been shown to specifically recognize Gram-negative PG and PG breakdown products (Girardin et al., 2003
). Recognition of PG and PG breakdown products from Gram-negative and Gram-positive bacteria has been acknowledged as a key part of the innate immune system present in diverse organisms (Girardin & Philpott, 2004
), yet the source and structural composition of PG breakdown products that elicit a defence reaction in the host of a pathogen are unknown. A potential link between the innate immune system recognizing PG from Gram-negative bacteria and the activity of specialized LTs could be the finding that the delivery of PG to epithelial cells by the human pathogen Helicobacter pylori is mediated by the T4SS, which is an essential virulence determinant of this organism (Viala et al., 2004
).
Here we focus on biochemical characterization of specialized LTs from diverse T3SS and T4SS. We cloned and overexpressed specialized LTs from both types of secretion systems. From T3SS: IpgF from Shigella sonnei, which is 98 % identical to IpgF, encoded within the entry region of the virulence plasmids pWR100 and pCP301 of Shigella flexneri serotypes 5a and 2a, respectively (Allaoui et al., 1993; Buchrieser et al., 2000
; Jin et al., 2002
); and IagB, which is encoded by the Salmonella enterica pathogenicity island SPI-1 (Miras et al., 1995
). From T4SS: P19 encoded by the conjugative resistance plasmid R1 (Koraimann et al., 1993
), VirB1 encoded by the Ti plasmid of Agrobacterium tumefaciens (Thompson et al., 1988
), VirB1 encoded by the small chromosome of Brucella suis (O'Callaghan et al., 1999
), TrbN from the IncP plasmid RP4 (Pansegrau et al., 1994
) and HP0523 encoded by the cag pathogenicity island (cag-PAI) of H. pylori (Tomb et al., 1997
). Inactivation of the genes encoding these proteins in the respective system mostly resulted in virulence-attenuated phenotypes which could have arisen from the observed redundancy of LTs in bacteria (Koraimann, 2003
). Whereas mutations in ipgF and iagB were reported to have no obvious effects on pathogenicity or needle complex formation (Allaoui et al., 1993
; Sukhan et al., 2001
), a deletion of the corresponding LT gene in the PAI of Citrobacter rodentium, rorf3, led to decreased T3SS assembly, effector translocation and virulence (Deng et al., 2004
). Mutations inactivating P19 of the derepressed F-like plasmid R1-16 reduced the conjugation frequency five- to tenfold (Bayer et al., 1995
, 2001
). Mutations inactivating VirB1 encoded by the Ti plasmid of A. tumefaciens led to attenuated tumour formation in plants (Mushegian et al., 1996
). Notably, HP0523, the cag-PAI-encoded LT from H. pylori was found to be essential for both CagA translocation into the human host cells and IL-8 induction (Fischer et al., 2001
).
The main aim of the work presented here was to unambiguously demonstrate the muralytic activity of these enzymes, which up to now has not been shown for any of the specialized LTs encoded by T3SS or T4SS. This goal was reached by combined in vivo studies, zymogram analyses and in-solution muramidase assays, including a novel test system employing Cy3-labelled PG.
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METHODS |
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DNA manipulations and recombinant plasmids.
Recombinant DNA techniques were performed as described by Sambrook et al. (1989) or Ausubel et al. (1987)
or according to the manufacturer's protocols. DNA fragments amplified by PCR using the primers given below were cloned into pMAL-p2X (New England Biolabs) for periplasmic and into pMAL-c2X (New England Biolabs) for cytoplasmic expression of fusion proteins; the inserted sequences were verified by DNA sequencing in each case. Signal sequences, when present, were removed during the cloning procedure. All constructs and their relevant features are listed in Table 1
. For gene 19 amplification the forward primer BamP19_T20 (5'-ACGCGGATCCACTGATTGCTTTGATCTTGC-3') was used. Reverse primers were P19end_Hind (5'-ACCCAAGCTTTTAATTATTCTGCAGCGTG-3'), 19Hind150G (5'-CATTAAGCTTTTATTAACCTTTACTGCTTTTTATCCC-3'), 19Hind144G (5'-CCTAAAGCTTTATTACCCGGTATAAATCC-3') and 19Hind140R (5'-CATTAAGCTTTTATTACCGGTAAACCTCTG-3'). Primers for site-specific PCR mutagenesis for the creation of the P19_E44A mutant were P19E44A_1 (5'-CATGGAAAGCATCCCGTTAC -3') and P19E44A_2 (5'-GTAACGGGATGCTTTCCATG-3'), which were used in combination with the primers BamP19_T20 and P19end_Hind. Plasmids pCK217 (Koraimann et al., 1993
) and pETBla19E44QHis+ (Bayer et al., 2001
) were used as templates for the PCR amplifications. hp0523 was amplified from a DNA clone containing the cag-PAI of H. pylori using primers Bam_HP0523 (5'-CTTAGCCGGATTCTGCCAAAAGGTAACCATTAGTTTC-3') and HP0523_end_Hind (5'-GGGAAAGCTTACTACTCGTTATATCGCACTTGAG-3'). All gene 19 fragments and the hp0523 fragment were digested with BamHI and HindIII and ligated into the pMAL vectors. Oligonucleotides used to amplify ipgF from a clinical isolate of Sh. sonnei were IpgF-EcoRI (5'-GCTTGAATTCGATTGTTGGGATAAGGCTGG-3') and IpgF_HindIII (5'-GAGCAAGCTTTTATTATATCCTTCGATTATTCTGCTTGCTC-3'). Site-specific mutagenesis resulting in the IpgF_E42Q mutant was done with the mutagenic primers IpgF_EQ_fw (5'-GCGATTGCGGAAAAACAGTCCGGATTTA-3') and IpgF_EQ_rev (5'-ATTAAATCCGGACTGTTTTTCCGCAATCG-3') in combination with the two ipgF-specific primers above. The iagB gene was amplified from Sal. enterica using primers IagB_EcoRI (5'-GATTGAATTCGATTGCTGGCTTCAGGC-3') and IagB_HindIII (5'-CCGCAAGCTTTTATTATTTGTTTACCGCGATAGAAAGTC-3'). The B. suis virB1 gene was amplified using primers Bruc_EcoRI (5'-GTACGAATTCGTGCCATTCCTTGTCCTCGC-3') and Bruc_HindIII (5'-CAGTAAGCTTTTATTAGAAAACAACTACGCCGTC-3'). ipgF, iagB and virB1 fragments were digested with EcoRI and HindIII and ligated into the pMAL vectors. Primers for the amplification of virB1 from A. tumefaciens were VirB1_BamHI (5'-CTAAGGATCCGAGTTCGACCATGTTGCTCG-3') and VirB1_NotIa (5'-GATTGCGGCCGCTTATTAGTATAAGTCGAATAAGAC-3'); finally, trbN primers were TrbN_BamHI (5'-CTAAGGATCCCCCGATCTGTCGGAACAGATGG-3') and TrbN_NotI (5'-CTAAGCGGCCGCTCCTTATTATGGCGTGTTGTTG-3'). The amplified fragments were digested with BamHI and NotI, cloned into the vector pET-32a(+) (Novagen), and subsequently BamHIXhoI fragments were ligated into the BamHI and SalI restriction sites of the pMAL vectors.
DNA and protein sequence analyses.
The Wisconsin Package Version 10.3 (Accelrys) was used for the analysis and in silico manipulations of DNA and protein sequences. The calculation of similarity scores was performed using the BESTFIT program with the BLOSUM 62 matrix (gap weight 6; length weight 2, gaps longer than 5 not penalized).
Expression and affinity chromatography purification of fusion proteins.
E. coli SF100 cells (ompT) harbouring recombinant plasmids for cytoplasmic expression were cultivated at 30 °C to an OD600 of 0·40·7. Expression of fusion proteins was induced by addition of 0·3 mM IPTG. After 1·5 h cells were harvested, resuspended in 2 ml chromatography buffer (20 mM Tris/HCl pH 7·4, 200 mM NaCl, 1 mM EDTA) and lysed using a French pressure cell. Cell debris was removed by low-speed centrifugation at 500 g; the supernatants were diluted to 10 mg total protein ml1 and applied to an amylose resin (New England Biolabs) column of 2 ml bed volume. The column was washed first with 20 ml chromatography buffer and subsequently with 10 ml chromatography buffer supplemented with complete protease inhibitor (Roche Molecular Biochemicals). Fusion proteins were eluted with 4 ml elution buffer (20 mM Tris/HCl pH 7·4, 200 mM NaCl, 1 mM EDTA, 10 mM maltose). Protein concentrations in the samples were determined using the Bio-Rad Protein Assay.
Anion-exchange chromatography.
Affinity-purified fractions of MalE-IpgF and the active-site mutant MalE-IpgF_E42Q were dialysed against 200 vols 20 mM Tris/HCl pH 7·4 at 8 °C overnight. To allow dissociation of GroEL from the fusion protein, the dialysed fractions were incubated with 1 mM ATP, 1 mM MgCl2 and 0·1 % Triton X-100 for 30 min at 25 °C (Keresztessy et al., 1996; Mattingly et al., 1995
). The fractions were centrifuged at 10 000 g to remove any insoluble material. The supernatant corresponding to 8 mg fusion proteins was applied to a Mono Q HR 5/5 column (Amersham Pharmacia) using FPLC and eluted with a linear gradient from 0 to 2 M NaCl in 20 mM Tris/HCl pH 7·4 containing 0·1 % Triton X-100 at 8 °C. Fractions were collected and analysed by SDS-PAGE, Western blotting, zymogram analyses, in-solution muramidase assay, and the Cy3-PG spot assay.
MALDI-TOF analysis.
Samples (12 µg) of affinity-purified fractions of MalE-P19_153L and MalE-IpgF were separated electrophoretically on a 12·5 % SDS polyacrylamide gel. Proteins were Coomassie stained and the bands corresponding to the unknown protein were excised. The gel slices were destained with 500 µl 50 % methanol/50 % acetic acid in water at room temperature overnight. After a second destaining procedure, the gel slices were dehydrated in 200 µl acetonitrile. The samples were then reduced in 50 µl 10 mM DTT for 30 min at room temperature. After removal of DTT, proteins were alkylated with 50 µl 50 mM iodoacetamide for 30 min at room temperature and then washed with 100 µl 100 mM ammonium bicarbonate for 10 min. Subsequently, the gel slices were dehydrated as described above. The gel slices were then rehydrated by swelling in 100 µl 100 mM ammonium bicarbonate for 10 min. Ammonium bicarbonate was removed and the gel dehydrated as above. Acetonitrile was removed and 200 µl acetonitrile was added. Subsequently, the gel slices were dried in vacuo. Reswelling of the gel slices was achieved by incubation with 50 µl 20 ng µl1 trypsin (Promega) in ice-cold 50 mM ammonium bicarbonate for 10 min on ice. Excess trypsin solution was removed and 10 µl ammonium bicarbonate was added. Tryptic digestion of the proteins in the gel slices was performed overnight at 37 °C. Peptides were first extracted with 30 µl 100 mM ammonium bicarbonate. After collection of the supernatant, peptides were extracted twice with 30 µl formic acid in 50 % acetonitrile. The three supernatant fractions were pooled and the sample volume was reduced to 15 µl in a vacuum centrifuge. MALDI-TOF analysis was performed by piChem, Graz. The GroEL chaperone of E. coli was identified using the mass fingerprint data obtained by MALDI-TOF and the PeptIdent software (http://ca.expasy.org/tools/peptident.html).
Complementation assays.
Complementation of the gene 19 defect was performed with donor strains carrying either the resistance plasmid R1-16 or R1-16/mut1 together with a complementation plasmid. The latter plasmids, pMalpP19 wt, pMalpP19_150G, pMalpP19_144G, pMalpP19_E44Q, pMalpP19_E44A, pMalpIpgF, pMalpIpgF_E42Q, pMalpIagB, pMalpVirB1_AT, pMalpVirB1_BS, pMalpHP0523 and pMalpTrbN, are described in Table 1. Induction of transcription from the tac promoter with IPTG was not necessary for complementation. Overnight cultures from single colonies grown in 2 ml 2x TY, supplemented with kanamycin and ampicillin in case of the donors, were used for mating. Forty microlitres of the donor cells, i.e. E. coli MC1061 harbouring R1-16 or R1-16/mut1 and the different complementation plasmids, were pipetted into 0·9 ml prewarmed 2x TY medium and incubated for 60 min at 37 °C. Then 100 µl of an overnight culture of recipient cells, i.e. E. coli J5, was added, and the mixture was incubated for 60 min at 37 °C without shaking. Conjugation was interrupted by vigorously mixing for 1 min and placing the tubes on ice. Dilutions of 103 to 107 in 0·9 % NaCl were plated on lactose MacConkey agar plates containing kanamycin. The conjugation frequency was determined by counting white donor and red transconjugant colonies and is expressed as the number of transconjugants per 100 donor cells.
PG isolation and purification.
PG was isolated from 1 litre of E. coli J5 cells in stationary phase according to Rosenthal & Dziarski (1994). Cells were harvested and washed with 40 ml 10 mM Tris/HCl pH 6·8. After washing, cells were resuspended in 30 ml 10 mM Tris/HCl pH 6·8. This suspension was added dropwise to 300 ml boiling 4 % SDS. After an additional 45 min of boiling, PG sacculi were collected by ultracentrifugation at 200 000 g for 20 min at 20 °C. The pellet was resuspended in 150 ml 2 M NaCl and incubated overnight at room temperature. After ultracentrifugation as described above, sacculi were washed with 60 ml water, collected by ultracentrifugation and resuspended in 20 ml water containing 0·1 mM MgCl2. The suspension was incubated first with 50 µg ml1 DNase I, 50 µg ml1 RNase A and 200 µg ml1
-amylase (Roche Molecular Biochemicals) for 90 min at 37 °C and second, after addition of 200 µg ml1 Pronase (Roche Molecular Biochemicals) at 60 °C for 60 min. Enzymes were inactivated by addition of SDS to a final concentration of 8 % and 15 min boiling. PG was collected by ultracentrifugation and washed twice with 20 ml water. The final pellet was resuspended in 5 ml water and stored at 20 °C. The amount of PG was estimated by evaporation and weighing an aliquot of the dried material.
Zymogram analyses.
Zymogram analyses for the determination of PG-degrading activity was performed according to Bernadsky et al. (1994). Purified PG was incorporated into 12·5 % and 16 % polyacrylamide gels to a final concentration of 0·05 % (w/v). Protein samples were separated electrophoretically on polyacrylamide gels containing 0·02 % SDS. After electrophoresis, the gels were incubated with water for 60 min at 4 °C, then transferred to 25 mM potassium phosphate buffer pH 5·2 containing 0·1 % Triton X-100 (renaturation buffer) and incubated for 60 min at 4 °C. After this equilibration step, the gels were incubated with fresh renaturation buffer for 72 h. Peptidogylcan degradation was visualized by staining of gels with 0·1 % methylene blue/0·01 % KOH for 60 min at 4 °C. Gels were destained with water for 4560 min.
Muramidase assay.
Muramidase activity was determined by measuring solubilization of radiolabelled murein polymer (Engel et al., 1991). [3H]A2pm-labelled murein sacculi (2·5 µg; 5000 c.p.m.) were incubated in the presence of enzyme samples (36 µM) in a total volume of 100 µl 20 mM Bistris buffer pH 5·3 for 30 min at 37 °C. To precipitate the insoluble substrate, 100 µl of 1 % cetyltrimethyl ammonium bromide solution was added. The samples were kept on ice for 30 min. After centrifugation for 2 min at 17 000 g in an Eppendorf centrifuge at 4 °C, 100 µl of the supernatant was added to 1·5 ml scintillation cocktail, and radioactivity was measured in a Beckman LS 6500 scintillation counter. Inhibition of muramidase activity was assayed by addition of 1 µM GroEL (20 µl) to the reaction mixture prior to incubation at 37 °C.
Cy3 labelling of PG.
A 500 µl sample (approx. 1 mg) of PG purified as described above was incubated with 50 µl 1 M borate buffer pH 9·6 and 5 µl Cy3 NHS ester (1 mg ml1 in dimethylformamide, Amersham Biosciences) for 60 min at room temperature. Excess Cy3 was removed by dialysis against 500 vols water using Slide-A-Lyser dialysis cassettes (MWCO 10 000, Pierce). Cy3-labelled PG was stored at 20 °C.
Cy3-PG spot assay.
Eight-well glass slides (ICN) were coated with poly-L-lysine (Sigma-Aldrich) for 30 min at room temperature. After removal of unbound poly-L-lysine by washing with water, the slides were dried. Then 10 µl water and 1 µl Cy3-labelled PG were spotted onto the coated wells and incubated at room temperature for 45 min in the dark. To remove residual SDS and low-molecular-mass material the slides were rinsed with deionized water for 2 min and then dried at room temperature. Ten microlitres of buffer (10 mM or 25 mM sodium phosphate or ammonium acetate, pH 6) was pipetted on each of the Cy3-PG spots. Before and after addition of 1 µl enzyme, fluorescence microscopy images were taken using a Zeiss Axioskop and Zeiss filter set #15 (BP 546/12, FT 580, LP 590). Fluorescence microscopy images were taken using a cooled CCD camera and analysed with the Metamorph 5.0 software package (Visitron Systems). To determine the pH optimum of MalE-IpgF the Cy3-PG spot assay was performed as described above using different buffers for incubation (pH 4 and 5, 10 mM sodium citrate buffer; pH 6 and 7, 10 mM sodium phosphate buffer; pH 6·8 and 8, 10 mM Tris/HCl). After addition of 1 µl MalE-IpgF the slides were incubated at room temperature for 15 min. To remove the digested material, the slides were rinsed with deionized water for 2 min and dried at room temperature. To quantitate the fluorescence signals, dry slides were scanned before and after incubation with the enzyme using an array scanner (Axon GenePix 4000B, PMT setting 320, scan power 33 %) and images were obtained with the GenePix Pro 4.1 program; the 16 bit tiff files were subsequently analysed using ImageQuant 5.1 software. PG-degrading activity of IpgF was calculated as the ratio of fluorescent PG spot values before and after treatment minus one. To test the effects of bulgecin A a gift of A. J. Dijkstra (HoffmannLa Roche, Basel, Switzerland) and hexa-N-acetylchitohexaose (Seikagaku, Japan), various amounts of the compounds were added to the reaction mixture prior to the addition of the enzyme. The stocks of the various enzyme preparations had the following concentrations: MalE-IpgF, 6 µg µl1; MalE-IpgF_E44Q, 4 µg µl1; lysozyme from chicken egg white (Sigma), 20 µg µl1; GroEL, 1·6 µg µl1.
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RESULTS |
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Interaction of MalE-LT fusion proteins with GroEL
SDS-PAGE of affinity-purified MalE-LT fusion proteins revealed that in all cases except MalE-P19_140R (data not shown), a protein with a molecular mass of approximately 60 kDa co-purified with the MalE-LT fusion protein (arrowhead in Fig. 3b). The protein corresponding to that band was identified by MALDI-TOF analysis of peptide fragments produced by an in-gel tryptic digest. Six prominent peptide fragments were detected which gave an 86·6 % match to the E. coli chaperonin GroEL (data not shown). The results obtained using peptide mass fingerprinting were confirmed by Western blotting and detection using a commercially available anti-GroEL antibody (Fig. 3c
). GroEL did not co-purify with a preparation of MalE alone; thus, GroEL specifically interacted with the LT part of the fusion protein. Several attempts to remove GroEL from MalE-LTs originating from T4SS failed. MalE-P19 turned out to be unstable and precipitated as soon as GroEL was removed. Removal of GroEL was only accomplished in the case of affinity-purified MalE-IpgF (Fig. 3
) and MalE-IpgF_E42Q (data not shown) after addition of ATP and Mg2+ and subsequent anion-exchange chromatography. In this way, MalE-IpgF and MalE-IpgF_E42Q proteins with an estimated purity of >95 % suitable for further enzymic characterizations were obtained.
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DISCUSSION |
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One intriguing observation during our efforts to purify MalE-LT fusion proteins was that they strongly interacted with the chaperone GroEL in the cytoplasm. Since LTs normally possess signal sequences and are transported into the periplasm like P19 (Bayer et al., 2000), further investigations are currently being performed in this laboratory to elucidate whether the binding of GroEL to the LT is solely an artifact created by overexpression of the fusion proteins with concomitant accumulation of misfolded protein in the cytoplasm or serves a physiological role. Interestingly, the interaction of GroEL with the MalE-IpgF fusion protein also inhibited the PG-degrading activity of this enzyme. Presumably, the LT proteins are instable or misfolded in the cytoplasm and are recognized by the GroEL/ES complex unless they are transported to the periplasm by the Sec machinery. At least in the case of the 169 aa P19 protein, residues between R140 and G144 (RIYTG) are mainly responsible for the strong interaction with GroEL. Substrate recognition by the GroEL/ES complex is not well defined; the molecular basis of substrate recognition and binding by GroEL is supposed to be the presentation of hydrophobic amino acids on the surface of misfolded proteins (Wang et al., 1999
).
It has been recently recognized that PG fragments are specifically recognized by NOD proteins, which have been identified as key constituents of the innate immune system of animals and humans (for reviews see Dziarski, 2003; Girardin & Philpott, 2004
; Royet & Reichhart, 2003
). Human Nod1 is a cytosolic receptor in epithelial cells and detects a unique diaminopimelate-containing GlcNAc-MurNAc tripeptide found in Gram-negative bacterial PG, resulting in activation of the transcription factor NF-
B pathway (Girardin et al., 2003
). Only very recently has it been found that non-invasive H. pylori elicits this innate immune response by delivering bacterial PG fragments into epithelial cells via the H. pylori cag-PAI (Viala et al., 2004
). It is tempting to speculate that in this case HP0523, the specialized LT encoded by the H. pylori cag-PAI, could produce the PG fragments that are then translocated via the T4SS into epithelial host cells. HP0523 from H. pylori was shown here to be capable of degrading PG in vitro and it was found earlier to be essential for both CagA translocation and IL-8 induction (Fischer et al., 2001
). Future studies will be required to address the question whether HP0523 or other LTs like IpgF can produce PG fragments that are recognized by Nod1 and thus can trigger an innate immune response.
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ACKNOWLEDGEMENTS |
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Received 21 April 2005;
revised 27 June 2005;
accepted 29 June 2005.
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