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Previous Article | Next Article 
Infection and Immunity, October 2004, p. 5646-5653, Vol. 72, No. 10
0019-9567/04/$08.00+0 DOI: 10.1128/IAI.72.10.5646-5653.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
A. David Ogunniyi,2, Moo-Hyun Choi,1 Suhk-Neung Pyo,1 Dong-Kwon Rhee,1* and James C. Paton2
College of Pharmacy, Sungkyunkwan University, Suwon, Korea,1 School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia2
Received 23 December 2003/ Returned for modification 6 May 2004/ Accepted 7 July 2004
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ABSTRACT |
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INTRODUCTION |
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The protein profiles of the heat shock response in pneumococci after exposure of the cells to several stresses were previously examined. Pulse-labeling of proteins with [35S]methionine revealed that a temperature shift from 30 to 37°C in vitro, similar to that encountered by S. pneumoniae after translocation from the nasal mucosa to the lungs, triggered the induction of DnaK and GroEL (6). The persistence of ClpL, DnaK, and GroEL upon return to 30°C indicated that HSPs do not appear to be actively degraded upon return to normal culture conditions (15). Moreover, ClpL contains two ATP-binding regions and was found to function as a chaperone and to modulate virulence gene expression (15). A clpP mutant of S. pneumoniae was recently shown to be sensitive to high temperatures, H2O2, and puromycin and was significantly attenuated for virulence in mice (15, 28). The specific roles of other heat shock genes, such as clpC, clpE, and clpX, have not been fully clarified (4, 5, 7, 32).
Recently, it was shown that heat shock induced the expression of pneumolysin (Ply) and modulated the expression of other virulence factors in wild-type pneumococci. A mutation in clpP resulted in an increase in ply mRNA expression but not in the level and hemolytic activity of Ply after heat shock (15). In this study, we investigated the underlying mechanism by which ClpP attenuates virulence and assessed whether immunization with ClpP could protect mice against challenge with virulent pneumococci.
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MATERIALS AND METHODS |
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Antisera, gel electrophoresis, and immunoblotting. Sera against PspA and PdB (the toxoid derivative of Ply) were prepared essentially as described previously (23). Bacteria for immunoblotting were grown in THY to an A600 of 0.3 and prepared as described previously (15). Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (15% polyacrylamide gel) was carried out as described by Laemmli (16). The proteins were electroblotted onto nitrocellulose membranes and then reacted with a 1:5,000 dilution of polyclonal mouse sera raised against PspA and PdB. The secondary antibody was a 1:2,000 dilution of goat anti-mouse immunoglobulin G conjugated to alkaline phosphatase (Bio-Rad).
Pneumolysin assay. Hemolytic activity was determined as previously described (19) with a minor modification. Briefly, pneumococci grown in THY to early log to mid-log phase (A600, 0.05 to 0.1) were harvested by centrifugation at 3,900 x g for 10 min at 4°C and resuspended in phosphate-buffered saline (PBS). Sodium deoxycholate was added to a final concentration of 0.1%, and the samples were incubated at 37°C for 10 min. After centrifugation of the samples, the supernatant was withdrawn and serially diluted. Hemolytic activity was determined by incubation with an equal volume of 1.5% washed human red blood cells (containing 0.001% 2-mercaptoethanol [Merck]) for 30 min at 37°C in 96-well microtiter plates. The hemolytic titer was determined as the reciprocal of the estimated dilution at which 50% of erythrocytes were lysed at A540.
RNA techniques. Aliquots of 1.5 ml of culture suspension were collected at intervals for extraction of total RNA. For measurement of mRNA half-lives, rifampin (100 µg/ml) was added. Total RNA was extracted by the hot acid-phenol method as described previously (24). Levels of mRNAs for cps(2)A and ply were quantified by one-step real-time reverse transcription (RT)-PCR by use of an Access RT-PCR system (Promega Biotech catalog no. A1250). The specific and internal control (16S rRNA) primers for these reactions were described elsewhere (24). RT-PCR preparations, cycling conditions, and analyses of data were essentially as described previously (15). All reactions were carried out by use of a Rotor-Gene 2000 real-time cycler (Corbett Research, Mortlake, New South Wales, Australia). Analysis of mRNA half-lives was performed with the SigmaPlot curve fitter program (nonlinear least-squares fitting to the sum of exponential functions). Two models were proposed, monophasic decay or biphasic decay. The model which fitted the data with the minimum deviation in each case was considered the more valid model.
Cloning, expression, and purification of ClpP in E. coli. The clpP open reading frame was PCR amplified with forward and reverse primers (5'-CGA ATT CAT GAT TCC TGT AGT TAT-3' and 5'-CGA GCT CTT AGT TCA ATG AAT TGT TG-3', which incorporate EcoRI and SacI sites, respectively) from CP1200 DNA. The PCR fragment was digested with the same enzymes and cloned into the corresponding restriction sites in pET30(a) (Novagen) to generate plasmid pET30(a)-clpP. Expression in E. coli BL21(DE3) was induced with 0.1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) exposure for 3 h. The cells were harvested by centrifugation at 6,000 x g for 10 min and then resuspended in lysis buffer (50 mM sodium phosphate [pH 8.0], 2 M NaCl, 40 mM imidazole) to which the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) had been added to a final concentration of 1 mM. The cells were lysed in a French pressure cell (SLM Aminco, Inc.) at 12,000 lb/in2, and the lysates were centrifuged at 100,000 x g for 1 h. The resultant supernatant, containing the His6-tagged protein, was loaded into a nickel-nitrilotriacetic acid column (Probond; Invitrogen), which was then washed with 10 column volumes of 10 mM sodium phosphate-20 mM imidazole-1 M NaCl (pH 6.0). Nickel-bound His6-tagged protein was eluted with a 30-ml gradient of 0 to 500 mM imidazole in 10 mM sodium phosphate buffer (pH 6.0) and dialyzed against 10 mM sodium phosphate buffer (pH 7.0). The protein was judged to be >95% pure by SDS-PAGE and staining with Coomassie brilliant blue R-250 (data not shown).
Isolation and localization of subcellular fractions. Exponentially growing cells were collected by centrifugation, and sucrose-induced protoplast formation was performed as described by Vijayakumar and Morrison (35). Briefly, cells were converted to protoplasts by incubation at 30°C for 1 h with 1 M sucrose buffer (1 M sucrose, 100 mM Tris-HCl [pH 7.6], 2 mM MgCl2, 1 mM PMSF). Centrifugation at 13,000 x g for 20 min separated the cell wall fraction (supernatant) from the protoplasts (pellet). The protoplasts were subjected to osmotic lysis by dilution with 19 volumes of hypotonic buffer (100 mM Tris-HCl [pH 7.6], 1 mM PMSF, 1 mM EDTA). Lysates were centrifuged initially at 5,000 x g for 5 min to remove unlysed cells and then at 50,000 x g for 30 min to obtain the cytoplasmic fraction (supernatant) and the membrane fraction (pellet).
Determination of MDH activity. The enzymatic activity of malate dehydrogenase (MDH) was determined by monitoring the rate of reduction of the A340 of 0.2 mM NADH at 25°C (A340, 6.22 mM–1 cm–1) in 0.15 M potassium phosphate (pH 7.6) containing 0.5 mM oxalacetate. After the sample was added, the reaction mixture was incubated at 25°C for 1 min 40 s, and the A340 was determined. The addition of the substrate was used to start the reaction. Initial slopes for the rate of oxidation of NADH from the first 1 min 40 s of the reaction were used to calculate MDH activity.
Adhesion and invasion assays. Invasion of human lung A549 cells by pneumococci was performed by a modification of the antibiotic protection assay described previously (31). A549 cells were grown to confluence in 24-well tissue culture plates and washed three times with PBS (pH 7.2), after which 1 ml of culture medium (without antibiotics) was added per well. Exponential-phase cultures of R-type CP1200 and its isogenic clpP mutant derivatives (A550, 0.3; 108 CFU/ml) were pelleted by centrifugation, washed once with PBS, and resuspended in Dulbecco modified Eagle medium. Monolayers were infected with 107 bacteria (bacterium/cell ratio, 10:1), and initial contact of the bacteria with the cell monolayers was aided by centrifugation at 800 x g for 10 min at 4°C followed by 2 h of incubation at 37°C. Fresh medium containing 10 µg of penicillin/ml and 200 µg of gentamicin/ml was added to each well, and this treatment was confirmed to be sufficient to kill all of the extracellular bacteria. After an additional 1 h of incubation, the monolayers were washed three times with PBS, and the cells were detached from the plates by treatment with 100 µl of 0.25% trypsin-0.02% EDTA and then lysed by the addition of 400 µl of Triton X-100 (0.025% in H2O). Appropriate dilutions were plated on blood agar to determine the numbers of viable bacteria.
To determine the total numbers of adherent and intracellular bacteria, infected monolayers were washed as described above and then trypsinized, lysed, and plated quantitatively without antibiotic treatment. All samples were assayed in triplicate, and each assay was repeated at least three times.
Survival in RAW264.7 cells. Cell monolayers were infected with 107 CFU of pneumococci (bacterium/cell ratio, 10:1) in RPMI 1640 culture medium without antibiotics and then incubated for 2 h at 37°C. The cells were washed three times with PBS, and fresh medium containing 10 µg of penicillin/ml and 200 µg of gentamicin/ml was added to kill the extracellular bacteria (time zero of the assay). To enumerate intracellular pneumococci at different times after infection, the supernatants were removed, and the cells were washed three times with PBS and then lysed with Triton X-100 as described above. Serial dilutions of the lysate from each well were plated on blood agar. The number of CFU was determined after 24 h of incubation at 37°C. Three independent assays were carried out (in triplicate) for each bacterial strain. Statistical analysis was performed by use of paired or unpaired Student's t tests.
Colonization studies. Before challenge, bacteria were cultured at 37°C overnight on blood agar (supplemented with erythromycin where appropriate) and then grown in THY for approximately 4 h at 37°C to give ca. 4 x 107 CFU/ml (A600, 0.1). Each bacterial culture then was adjusted in THY to ca. 109 CFU/ml, and 10 µl (ca. 107 CFU) of cells was inoculated into the nares of 5-week-old CD1 mice. At 1, 2, and 4 days postinfection, four mice from each group were sacrificed randomly to estimate the carriage of each strain.
Nasopharyngeal, blood, and lung samples were serially diluted as appropriate in sterile PBS and plated in duplicate on blood agar containing the appropriate antibiotic(s). Plates were incubated for approximately 16 h at 37°C in an atmosphere of 95% air-5% CO2, after which colonies were counted and averaged between replicates.
Immunization of mice and analysis of sera. Mice were immunized intraperitoneally as described previously (23). Four groups of 5- to 6-week-old female CBA/N mice (12 per group) were immunized intraperitoneally with either AlPO4 alone, genetically modified Ply toxoid (PdB) plus AlPO4, PspA plus AlPO4, or ClpP plus AlPO4. Each mouse received three doses of 10 µg of each protein antigen at 12- to 14-day intervals, and sera were collected from the mice by retro-orbital bleeding 1 week after the third immunization. The sera were pooled on a group-by-group basis and assayed for Ply-, PspA-, or ClpP-specific antibodies by an enzyme-linked immunosorbent assay. The sera were also subjected to Western immunoblot analysis with whole-cell lysates of S. pneumoniae D39 or purified Ply, PspA, or ClpP as the antigen.
Challenge. Intraperitoneal challenge of immunized mice with a highly virulent capsular type 2 strain (D39) was carried out 2 weeks after the last immunization. Before challenge, the bacteria were grown at 37°C overnight on blood agar and then inoculated into serum broth, consisting of 10% (vol/vol) horse serum in meat extract broth. They were then grown statically for 3 h at 37°C to give approximately 108 CFU/ml, and the inoculum was adjusted to 7.5 x 105 CFU per challenge dose. Serotype-specific capsule production was confirmed by the Quellung reaction with antisera obtained from Statens Serum Institut, Copenhagen, Denmark. After challenge, the mice were monitored every 4 h initially for 7 days and then daily up to 21 days, and the survival time for each mouse was recorded. Differences between the median survival times of groups were analyzed by the Mann-Whitney U test (one-tailed).
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RESULTS |
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Given that after heat shock, the half-lives of the ply and cps(2)A mRNA transcripts became shorter than those at 30°C in both the parent and the clpP mutant (the P value was <0.05, except for the ply mRNA in the clpP mutant), it is possible that the mRNA species are either liable to faster degradation at 42°C than is 16S rRNA or are subject to faster decay by some unknown mechanism(s). Therefore, we investigated whether heat shock itself could affect the half-life of ply mRNA. Cells cultured at 30°C were treated with rifampin and then heat shocked, and the decay kinetics of the mRNA were determined by RT-PCR analysis. Under these conditions, the levels of both cps(2)A and ply transcripts were stable or increased slightly over 20 min in the parent and the clpP mutant (Fig. 1C), a result which was in contrast to the scenario observed at 30°C (Fig. 1A). These results demonstrated that the transcripts synthesized at 30°C were indeed stabilized by heat shock at 42°C (Fig. 1C) but did not indicate that the transcripts synthesized at 42°C were stabilized by heat shock. The fact that the half-lives of the ply and cps(2)A mRNA transcripts after heat shock became shorter than those at 30°C in both the parent and the clpP mutant (Fig. 1B and Table 2) could partly explain the discrepancy between high RNA transcript levels and low protein levels.
The hemolytic activity of Ply is not activated by ClpP. The destabilizing function of ClpP could be responsible for the low stability of the ply processing product and that of the ply primary transcript. Even though the ply mRNA was stabilized by heat shock, there was no corresponding increase in the level of the Ply protein or its hemolytic activity. This discrepancy could be due to the activation of Ply directly by ClpP protease so that hemolytic activity could be increased in the parent but not in the clpP mutant. To test this hypothesis, S. pneumoniae cells were cultured at 30°C and then heat shocked at 42°C for 20 min. The cells were lysed with 0.1% sodium deoxycholate by incubation at 37°C for 10 min. Subsequently, cell lysates were incubated further at 37°C for 20, 40, and 60 min, and the hemolytic activity of Ply was determined. The hemolytic activities in both the wild type and the clpP mutant decreased over the period at 37°C, and the hemolytic titer of the clpP mutant was not significantly different from that of the parent; these data suggested that ClpP protease is not required for activation of the hemolytic activity of Ply (data not shown).
ClpP is translocated to the cell wall after heat shock. For Bacillus subtilis, immunogold labeling with antibodies revealed that ClpC and ClpX ATPases were detected at the cell envelope as well as inside the cells (14), suggesting that Hsp100, ClpC, and ClpX proteins are associated closely with protein aggregates during heat shock treatment. To localize ClpP in S. pneumoniae, fractionation of subcellular proteins was initially attempted with encapsulated strains. However, after incubation of the cells in 1 M sucrose buffer, centrifugation did not separate the cell wall from protoplasts due to the presence of a thick capsule (data not shown). Therefore, unencapsulated strains were used for localization experiments. Since the fractionation method may yield partial lysis or leakage of cytoplasmic contents during or after heat shock, MDH was used as an internal cytoplasmic marker. MDH activity in the cell wall was 11.8% of the total MDH activity at 30°C. In addition, MDH activity in the cell wall was 9.3% even after heat shock, suggesting that heat shock does not cause lysis or leakage of the cell membrane.
Exponentially growing S. pneumoniae CP1200 cells were exposed to 42°C, and cellular proteins were separated into cell wall, membrane, and cytoplasmic fractions by sucrose-induced protoplast formation followed by lysis in hypotonic buffer (see Materials and Methods). Subsequently, subcellular fractions were subjected to immunoblot analysis with polyclonal anti-ClpP serum. At 30°C, ClpP was detected predominantly in the cytoplasmic fraction (82.4% of the total ClpP); a smaller amount was detected in the cell wall fraction (11.8% of the total ClpP). However, after heat shock, the proportion of ClpP in the cell wall increased to 25.6%, whereas that in the cytosol decreased to 68.4%, although there was an increase in the absolute amount of ClpP in both fractions (Fig. 2). These results suggested that a significant amount of ClpP may be translocated to the cell wall after heat shock.
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Reduced survival of the clpP mutant in murine macrophage RAW264.7 cells. Alveolar macrophages are the primary elements in host defense against invasion by S. pneumoniae (13). Since it was observed in this study that the clpP mutant was defective in colonization of the nasopharynx compared to the parent, it was reasoned that this finding might be due to rapid clearance of the pneumoccoci in addition to the slower growth of the mutant. Therefore, the survival of the clpP mutant in murine macrophage RAW264.7 cells was examined (Fig. 4). The viability of the clpP mutant in RAW264.7 cells was significantly lower than that of the parent after 5 h (P < 0.01), 6 h (P < 0.05), and 8 h (P < 0.01) of infection (Fig. 4). The number of viable cells at 2 h after the addition of antibiotic would be expected to be approximately half the number present when the antibiotic was added. However, the actual number of CFU was much smaller, indicating that this result is not due to a growth defect of the clpP mutant but rather is due to a stress-sensitive phenotype or susceptibility of the clpP mutant to macrophages. These data suggested that ClpP is required for intracellular survival in RAW264.7 cells.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a previous report, the expression of ail was repressed in wild-type Yersinia enterocolitica, but in the respective clpP mutant, the expression of ail mRNA at 28°C was increased almost 20-fold, indicating that ClpP protease degrades a repressor of ail transcription. In contrast, the stability of ail mRNA did not increase in the clpP mutant (26). Also, in Salmonella enterica, ClpP was found to be a transcriptional repressor of the flagellar regulon (33). Recently, proteomic analysis revealed that ClpX, a substrate donor for ClpP protease, recognized transcriptional regulators such as the lexA (LexA repressor), rpoS (sigma S), rseA (negative regulator of sigma E), and rsd (regulator of sigma D) genes (9). Furthermore, an ATP-dependent Lon protease degraded a positive transcriptional regulator of capsule synthesis, RcsA, and induced the expression of capsular polysaccharide (colanic acid) synthesis genes in E. coli (30). Therefore, it is possible that S. pneumoniae ClpP acts as a transcriptional regulator of the ply and cps(2)A genes in some unknown way.
At 30°C, ply and cps(2)A mRNAs had longer half-lives in the clpP mutant than in the D39 parent. Furthermore, after heat shock, the half-lives of both cps(2)A and ply mRNAs in the clpP mutant were more than twofold longer than those in the wild type, indicating clearly that the half-lives of the mRNA species were increased in the absence of ClpP at 30 and 42°C. The hemolytic activity of Ply was not increased further by incubation of cell lysates at 37°C, indicating that ClpP is not directly responsible for activation of the hemolytic activity of Ply. We conclude from these findings that cps(2)A and ply mRNAs are subject to degradation in a ClpP-dependent fashion at the posttranscriptional level, but the specific mechanism by which this process occurs is unclear. This is the first demonstration that ClpP could affect mRNA stability.
Microarray analysis of the S. pneumoniae clpP mutant during exponential growth demonstrated the induction of genes that classically comprise the bacterial heat shock regulon (28). In contrast, the transcription of clpX and ftsH (ATP-dependent protease) was not induced even by heat shock (28). However, the levels of virulence factors after heat shock have not been determined. In this study, we demonstrated for the first time that ply transcript levels are much higher in the clpP mutant than in the wild type at both 30 and 42°C, whereas Ply protein levels are lower in the mutant than in the wild type (15). This paradox could be due to (i) rapid degradation of the ply mRNA after heat shock, (ii) decreased translation of the ply mRNA, and/or (iii) posttranslational modification of the ply transcript. Further studies are needed to distinguish among these possibilities.
In some pathogens, HSPs are present on the surface of cells and may mediate adherence to host cells (25, 27). In S. pneumoniae (4) and Listeria monocytogenes (21), ClpC was shown to be required for adherence and expression of virulence factors; S. pneumoniae clpC mutants displayed a deficiency in adherence to human type II alveolar cells and did not express Ply or the choline-binding protein CbpA, CbpE, CbpF, or CbpJ, suggesting that ClpC exerts a pleiotropic effect on adherence (4). In this study, we demonstrated that adherence and invasion of the clpP mutant of S. pneumoniae to host cells were not affected (data not shown).
Molecular chaperones of the Hsp70 and Hsp100 family have been shown to be associated with the translocation complex and to interact with translocation precursors (17, 36). Recently, B. subtilis ClpC and ClpX ATPases were detected at the cell envelope and in the cytoplasm (14). In those studies, translocation of the Clp protein after heat shock or by other stresses was not demonstrated. In this study, however, fractionation of S. pneumoniae revealed a substantial increase in the amount of ClpP in the cell wall after heat shock. Thus, ClpP is the first Clp protein shown to be mobilized into the cell wall fraction after heat shock; in this location it may interact with host cells or otherwise act by degrading pneumococcal proteins destined for transport or translocation.
Bacterial HSPs are induced during infection and mediate adherence and invasion in addition to playing a role in the proper folding of intracellular proteins (4, 21, 25). In this study, we demonstrated that immunization of mice with purified pneumococcal ClpP elicited protective immunity against systemic challenge with D39 to a level comparable to that achieved with the well-characterized pneumococcal protein vaccine candidates PspA and detoxified Ply (PdB). The fact that strong, antigen-specific antibody responses were generated in immunized mice prior to challenge suggests that the protection could, at least in part, be antibody mediated.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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H.-Y.K. and A.D.O. contributed equally to this work.
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