Proteolytic cleavage of pertussis toxin S1 subunit is not essential for its activity in mammalian cells
© Carbonetti et al; licensee BioMed Central Ltd. 2005
Received: 23 September 2004
Accepted: 03 February 2005
Published: 03 February 2005
Pertussis toxin (PT) is an exotoxin virulence factor produced by Bordetella pertussis, the causative agent of whooping cough. PT consists of an active subunit (S1) that ADP-ribosylates the alpha subunit of several mammalian G proteins, and a B oligomer (S2–S5) that binds glycoconjugate receptors on cells. PT appears to enter cells by endocytosis, and retrograde transport through the Golgi apparatus may be important for its cytotoxicity. A previous study demonstrated that proteolytic processing of S1 occurs after PT enters mammalian cells. We sought to determine whether this proteolytic processing of S1 is necessary for PT cytotoxicity.
Protease inhibitor studies suggested that S1 processing may involve a metalloprotease, and processing does not involve furin, a mammalian cell protease that cleaves several other bacterial toxins. However, inhibitor studies showed a general lack of correlation of S1 processing with PT cellular activity. A combination of replacement, insertion and deletion mutations in the C-terminal region of S1, as well as mass spectrometry data, suggested that the cleavage site is located around residue 203–204, but that cleavage is not strongly sequence-dependent. Processing of S1 was abolished by each of 3 overlapping 8 residue deletions just downstream of the putative cleavage site, but not by smaller deletions in the same region. Processing of the various mutant forms of PT did not correlate with cellular activity of the toxin, nor with the ability of the bacteria producing them to infect the mouse respiratory tract. In addition, S1 processing was not detected in transfected cells expressing S1, even though S1 was fully active in these cells.
S1 processing is not essential for the cellular activity of PT. This distinguishes it from the processing of various other bacterial toxins, which has been shown to be important for their cytotoxicity. S1 processing may be mediated primarily by a metalloprotease, but the cleavage site on S1 is not sequence-dependent and processing appears to depend on the general topology of the protein in that region, indicating that multiple proteases may contribute to this cleavage.
Pertussis toxin (PT) is a complex exotoxin and an important virulence factor produced by Bordetella pertussis, a bacterial pathogen of the human respiratory tract that causes the disease whooping cough. PT holotoxin is a multi-subunit complex with an AB5 structure [1, 2]: the enzymatically active A subunit (S1) is an ADP-ribosyltransferase that modifies the alpha subunit of several heterotrimeric G proteins (primarily Gi proteins) in mammalian cells [3, 4], and the B oligomer (S2, S3, 2 copies of S4, and S5) binds unidentified glycoconjugate receptors on cells [5, 6]. The events in the intracellular trafficking of PT between surface binding and ADP-ribosylation of target G proteins on the cytoplasmic side of cellular membranes are relatively obscure. Electron microscopy studies and experiments with inhibitors suggest that the holotoxin is internalized by endocytosis [7–9]. Subcellular fractionation experiments and inhibition of cytotoxicity by Brefeldin A (BFA), which disrupts the Golgi apparatus , provide evidence for subsequent retrograde transport of PT to the Golgi apparatus [7–9]. Trafficking of PT beyond the Golgi apparatus is relatively uncharacterized, though it has been hypothesized that further retrograde transport of PT through the secretory pathway to the endoplasmic reticulum (ER) occurs [11–13]. After dissociation of S1 from the holotoxin, the liberated S1 subunit is then proposed to traverse the ER membrane to gain access to its target G proteins in the cytosol . Evidence supporting this ER-to-cytosol translocation was obtained from transfection studies with constructs expressing S1 with a signal peptide for ER localization .
Another observation that may bear on the cell biology and cytotoxicity of PT is that the S1 subunit appears to be proteolytically processed to a lower molecular weight form upon interaction of PT with mammalian cells . This processing was shown to be dependent upon entry of PT into cells and seemed to involve an early endosome function. The size of the processed form of S1 (approximately 22 kDa versus 26 kDa for the full-length S1) suggested that processing may be targeted at a protease-sensitive loop near the C-terminus of S1 that contains primary sites for trypsin and chymotrypsin cleavage . However, evidence for the location of the cellular cleavage site on S1 was not presented. In addition, a link between processing of S1 and activity of PT in cells was not established. Proteolytic processing is a common theme in the activation of bacterial toxins upon interaction with mammalian cells. For example, anthrax toxin, diphtheria toxin, Pseudomonas exotoxin A and shiga toxin are all activated after cleavage by the endogenous eukaryotic protease furin , a subtilisin-like protease residing in the secretory pathway of eukaryotic cells , or by closely-related proteases . Cholera toxin (CT) and Escherichia coli heat-labile toxin (LT) A subunits are cleaved at a protease-sensitive loop to promote maximal activity [19, 20], and CT A subunit was found to be cleaved upon interaction of CT with T84 epithelial cells, by an unidentified protease .
In this study we extend the analysis of proteolytic processing of cell-associated S1 and conclude that S1 processing is not essential for the cellular activity of PT.
Results and discussion
Processing and fractionation of S1 in PT-treated CHO cells
Effects of protease and cell trafficking inhibitors on S1 processing in CHO cells
We also determined the inhibitory activity on S1 processing by CHO cells of two inhibitors of cellular trafficking and secretion, bafilomycin A1, which inhibits vacuolar proton ATPase and therefore prevents endosome acidification , and BFA, which disrupts the Golgi apparatus . Bafilomycin A1 had a significant inhibitory effect on S1 processing, with 51% of cell-associated S1 in the unprocessed form (Fig. 2B), consistent with the hypothesis that S1 processing occurs in the endosomal compartment of CHO cells and demonstrating a role for endosome acidification in this processing event. However, BFA had no inhibitory effect on S1 processing (Fig. 2B), consistent with previously reported results  and with the hypothesis that S1 processing occurs prior to the Golgi apparatus in the putative retrograde trafficking pathway.
Effects of S1 processing inhibitors on cellular activity of PT
Proteolytic processing of bacterial toxins is a common theme in their activation within mammalian cells , but whether cellular processing of S1 plays a role in the activity of PT had not been previously addressed. As a preliminary investigation of this question, we sought to determine whether the inhibitors of S1 processing by CHO cells had any effect on the ability of PT to ADP-ribosylate target G proteins in CHO cells. CHO cells were preincubated with inhibitors before addition of PT (1 nM) as before. Controls were cells to which either PT or PT* (which has no ADP-ribosylation activity) was added in the absence of inhibitor. Cells were recovered after 3 h, and lysates were prepared and tested in the ADP-ribosylation assay. In this assay, active PT within cells will ADP-ribosylate available G proteins, so that when a lysate is prepared from these cells and used in an in vitro ADP-ribosylation assay with PT and 32P-labelled NAD, there is no labeling of G proteins in the lysate, since they were already modified by the PT added to the cells. If PT activity within cells is inhibited, then a proportion of the G proteins in the lysate from these cells will be unmodified and therefore labeled in the in vitro reaction with PT. The assay was repeated twice and the result of one experiment is shown in Fig. 2C. The mean percent inhibition for the various inhibitors (in decreasing order of their inhibitory effect) was as follows: pefabloc SC – 95%, BFA – 79%, bafilomycin A1 – 49%, EDTA – 44%, phenanthroline – 28%, 3,4-DCI – 9%, pepstatin – 8%. Therefore there was no strong correlation between the extent of inhibition of S1 processing by the inhibitors and their inhibitory activity on ADP-ribosylation of G proteins by PT in CHO cells. The cell trafficking/secretion inhibitors had a significant inhibitory effect (BFA has previously been shown to inhibit cellular activity of PT [7–9], presumably due to its disruption of PT trafficking in cells, despite its lack of inhibition of S1 processing), but the metalloprotease inhibitors had only a mild inhibitory effect. Of the other protease inhibitors, pefabloc SC had the greatest inhibitory effect, as it did on S1 processing, whereas pepstatin had no significant inhibitory effect on ADP-ribosylation (or S1 processing). To rule out an effect of the inhibitors on the enzymatic activity of PT (independent of its cellular activity), we also performed in vitro ADP-ribosylation assays with PT in the presence of the various inhibitors at the concentrations used on the CHO cells. No significant inhibitory effect was seen with any of the inhibitors (data not shown) with the exception of 3,4-DCI, which was also somewhat toxic to the CHO cells but did not significantly inhibit processing of S1. Altogether, these data are inconclusive with regard to the hypothesis that cellular processing of S1 plays a role in the ADP-ribosylation of G proteins in CHO cells by PT.
Location of processing site on S1
Effect of processing site mutations on PT activity
We also tested several strains expressing mutant PT constructs in our mouse infection model of B. pertussis virulence , and none of these strains was significantly defective in colonization compared to the parental wild type strain (data not shown). Although these data suggest that S1 processing is not essential for the virulence of B. pertussis, it is possible that S1 could be processed in vivo by alternative proteases absent from CHO cells.
Processing in stable CHO cell transfectants expressing S1
The data from these transfectants do not rule out the possibility that S1 processing may contribute to the trafficking of PT to the ER. However, the data are consistent with the idea that S1 processing is not essential for its activity in mammalian cells, and therefore that translocation of the full-length S1 across the ER membrane occurs. This would distinguish PT from several other bacterial toxins of similar subunit structure, such as cholera toxin, heat-labile toxin and shiga toxin, for which processing of the A subunit is apparently important for cellular activity . The difference may be in the association of the A subunit with the B oligomer. The other toxin A subunits have a relatively long helix that protrudes through a central pore in the B oligomer , and cleavage of the A subunit is required to release the enzymatic domain from this complex. S1 has a relatively short helix associated with the B oligomer , and therefore cleavage may be unnecessary for its release from the holotoxin complex in the ER, or whichever compartment translocation occurs from.
In this study we have further characterized the cellular processing of the S1 subunit after PT interacts with mammalian cells. Our major conclusion is that this processing event is not essential for PT activity in mammalian cells, based on several lines of evidence: (1) protease inhibitor studies showed a general lack of correlation between S1 processing and PT cellular activity; (2) mutant forms of PT in which S1 processing was apparently abolished retained significant (>75% of wild type) cellular activity; and (3) no S1 processing was apparent in transfected cells expressing active S1. Although we have not definitively ruled out a contribution of S1 processing to the cellular activity of PT due to the imperfect nature of our unprocessed mutants, it is possible that the processing event is completely unrelated to PT cytotoxicity and instead is an irrelevant activity occurring, possibly in lysosomes, on the large majority of the intracellular pool of PT molecules that do not enter the putative retrograde transport pathway to the ER and then on to the cytosolic target proteins.
Bacterial strains and growth conditions
The B. pertussis strain used in this study was a streptomycin- and nalidixic acid-resistant derivative of W28 (Wellcome). The PT9K/129G (PT*) derivative of this strain was constructed as previously described . The other PT mutant derivatives used in this study were constructed as described below. B. pertussis was grown on Bordet-Gengou agar (Difco) plates containing 15% defibrinated sheep blood and the following antibiotics at the indicated concentrations where necessary: streptomycin 400 μg ml-1, nalidixic acid 20 μg ml-1, gentamicin 10 μg ml-1; or in Stainer-Scholte liquid medium  containing heptakis-dimethylcyclodextrin (Sigma). Escherichia coli strains used were DH10B  for standard cloning experiments and S17.1  for conjugation with B. pertussis, and these were grown on LB agar plates containing 10 μg ml-1gentamicin where necessary or in LB broth containing 100 μg ml-1ampicillin.
Plasmid and strain construction
Plasmid pJ-PT was obtained by subcloning a 2.3 kb Eco RI-Bsr GI fragment (containing the PT S1 and S2 genes) into Eco RI/Acc 65I-digested allelic exchange vector plasmid pJHC1 . Mutations were engineered into the S1 sequence of this plasmid, which was confirmed by DNA sequencing, transformed into E. coli S17.1 and introduced into the B. pertussis chromosome by conjugation and allelic exchange as described previously . Deletion, insertion and substitution mutations were constructed by overlap extension PCR , and the 210–218/R1 and R2 replacement mutations were constructed by using a degenerate oligonucleotide (CARBON 1097; 5'-GATAAGAGCTCCVNNVNNVNNVNNVNNVNNVNNVNNVNNGCCGGCGAGGCCTCGCC-3' where V = G, A, or C and N = any nucleotide) which was allowed to anneal, extended with DNA polymerase Klenow fragment, digested with SacI and BglI and inserted into SacI- and BglI-digested pJ-PT. The GST-αC20 construct was obtained by inserting annealed complementary oligonucleotides (encoding the C-terminal 20 amino acids of human Giα3) into the plasmid pGEX-2T (Pharmacia) to derive pGEX-αC20. The PT*-CSP/N construct was obtained as previously described .
Samples were run on 12% SDS-PAGE gels and transferred to nitrocellulose filters. To detect S1, blocked filters were incubated with S1-specific monoclonal antibody X2X5 or 1C7, followed by peroxidase conjugated anti-mouse IgG secondary antibody (Amersham). Blots were developed using ECL Plus (Amersham) and exposed to X-ray film.
PT and mutant derivatives were prepared from B. pertussis culture supernatants by the fetuin affinity method of Kimura et al. , resuspended in PBS and stored at -80°C until use. The protein concentration was determined by BCA assay (Pierce). GST-αC20 protein was purified from a culture of E. coli DH10B containing the plasmid pGEX-αC20. For induction of the fusion protein, the strain was grown in LB to A600 1.0 and then IPTG was added to a concentration of 0.5 mM. 2 h after IPTG addition, cells were centrifuged, lysed in BPER lysis reagent (Pierce), cleared by centrifugation, and passed through a GSTrap column (Amersham-Pharmacia). Fusion protein was eluted in reduced glutathione buffer, dialyzed against PBS and analyzed by SDS-PAGE and BCA assay (Pierce) to determine protein concentration.
Cell lysis and fractionation
CHO cells were grown in 6-well plates to near confluency and then PT was added and incubated for the indicated times at 37°C. Cells were then collected by trypsinization, washed in PBS, resuspended in 50–100 μl of either NET/Triton lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.4, 0.01% NaN3, and 0.5% Triton X-100) or RIPA buffer (10 mM Tris, pH 7.4, 0.1% SDS, 1% sodium deoxycholate, 1% NP-40, 150 mM NaCl) and incubated 30 min on ice. The lysate was then centrifuged 15 min at 13,000 rpm at 4°C in a microfuge, the supernatant was removed to a fresh tube and the pellet was resuspended in sample buffer. Samples were boiled 5 min and loaded onto an SDS-PAGE gel.
Inhibitors were added to CHO cells 30 min prior to the addition of PT. For protease inhibitors we followed the suggestions of Barrett  to determine the catalytic type of protease involved. Protease inhibitors (BMB/Roche) and concentrations used were: aprotinin (0.15 μM), 3,4-dichloroisocoumarin (3,4-DCI, 1 mM) and 4-(2-aminoethyl)-benzenesulfonyl-fluoride, hydrochloride (pefabloc SC, 1 mM) for inhibition of serine proteases; trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64, 1 mM) and leupeptin (1 μM) for inhibition of cysteine proteases; pepstatin (1 μM) for inhibition of aspartic proteases; and EDTA (0.5–1 mM) and 1,10-phenanthroline (0.5–1 mM) for inhibition of metalloproteases. Other inhibitors used were BFA (Sigma, 5 μg/ml) and bafilomycin A1 (ICN, 0.5 μM). After SDS-PAGE and western blotting of samples, band intensities were measured by densitometry and used to calculate the extent of inhibition of S1 processing.
Trypsin digestion of PT
PT (100 or 200 ng) was digested with trypsin (Sigma) in a volume of 20 μl at room temperature for 1 h in 50 mM Tris, pH 8, and 2 mM CaCl2. Trypsin was present at 70 μg/ml. Sample buffer was added and the samples were then boiled 5 min before loading onto an SDS-PAGE gel.
Samples were run on 12% SDS-PAGE gels and transferred to nitrocellulose filters. To detect S1, blocked filters were incubated with S1-specific monoclonal antibody X2X5 (3) (a generous gift from Drusilla Burns) or 3CX4, followed by peroxidase conjugated anti-mouse IgG secondary antibody (Amersham). Blots were developed using ECL Plus (Amersham) and exposed to X-ray film.
To determine the cellular activity of PT samples, PT (0.5–2 nM) was added to near confluent CHO cells in 12-well culture plates, and after 3 h at 37°C cells were recovered from plates, washed in PBS and lysed in NET/Triton lysis buffer 30 min on ice. The lysate was then centrifuged 15 min at 13,000 rpm at 4°C and the supernatant was stored at -20°C until the assay. The ADP-ribosylation assay contained, in 25 μl, 0.1 M Tris, pH 7.5, 20 mM dithiothreitol (DTT), 0.5 mM ATP, 1 μM 32P-NAD (specific activity 30 Ci/mmol; NEN), 10 ng PT, and an aliquot of the lysate sample. For assessment of in vitro enzymatic activity of PT samples by ADP-ribosylation assay, reactions contained, in 25 μl, 0.1 M Tris, pH 7.5, 20 mM DTT, 0.5 mM ATP, 1 μM 32P-NAD (specific activity 30 Ci/mmol; NEN), 10–50 ng PT, and 0.5 μg GST-αC20 protein as substrate. The mixture was incubated 90 min at room temperature, sample buffer was added, and the sample was boiled 5 min and loaded onto 15% SDS-PAGE gels. After electrophoresis, gels were fixed, dried and exposed to X-ray film. Band intensities were measured by densitometry and used to calculate the extent of ADP-ribosylation of target proteins.
Mass spectrometry analysis
CHO cells were incubated overnight with PT and then lysates were made using Triton X-100 buffer at room temperature (to maximize the proportion of S1 in the soluble fraction). Purified 1C7 monoclonal antibody was allowed to bind to a PS10 chip (Ciphergen Biosystems) for 2 h at room temperature, which was then washed with PBS. The CHO cell lysates (soluble fraction) were diluted 1:1 with PBS and 100 μl of each lysate was added to a spot on the chip and incubated at 5°C for 4 days. The chip was washed 3 times with PBS containing 0.1% Triton X-100, 3 times with PBS, and twice with 5 mM HEPES (pH 7.0). After the chip was air-dried, matrix (Sinapinic acid) was added to each spot and allowed to dry. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analysis of these samples was performed in a model PBS-II machine (Ciphergen).
Six-week-old female BALB/c mice (Harlan) were used for infection experiments. Bacterial inocula were prepared and intranasal inoculation of mice was performed as previously described . Seven days after inoculation, mice were sacrificed by carbon dioxide inhalation and the respiratory tract (trachea + lungs) was removed, homogenized in 2 ml PBS, and dilutions were plated on BG-blood agar plates containing streptomycin to determine the number of colony forming units (CFU) per respiratory tract.
We thank Drusilla Burns for antibodies, Steve Leppla for the FD11 cells, Joe Barbieri for advice on ADP-ribosylation assays, Pat Campbell for performing the Ciphergen analysis, and Ulrike McNamara, Susan Kinnear and Monica Castro for performing some of the initial experiments. This project was supported by NIH grant AI50022.
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