1 INSERM U411, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France
2 Laboratoire Mixte PasteurNecker de Recherche sur les Streptocoques et Streptococcies, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France
3 INSERM U345, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France
Correspondence
Shamila Nair
nair{at}necker.fr
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ABSTRACT |
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The GenBank accession number for the Streptococcus agalactiae clpP gene sequence reported in this paper is AJ413168.
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INTRODUCTION |
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Group B Streptococcus (GBS), also known as Streptococcus agalactiae, is part of the normal human microflora colonizing the respiratory, gastrointestinal and urogenital tracts. This extracellular bacterium is one of the leading causes of invasive infections, bacterial sepsis and meningitis in neonates (Schuchat, 1998). The physiopathology of GBS infections implies that this bacterium can adapt rapidly to various growth conditions, including pH, osmolarity and temperature variations (Tamura et al., 1994
). In this study, we investigated the contribution of oxidative stress due to heat exposure in GBS. We identified the clpP gene of S. agalactiae and showed that its product, the serine protease ClpP, is involved in the regulation of growth at high temperatures and survival under stress conditions. During heat shock, an S. agalactiae
clpP mutant was growth-arrested and displayed important modifications of its total protein content, including a decreased level of essential metabolic enzymes such as the alcohol dehydrogenase. Under these conditions, ClpP also contributed to cell division and septum formation. Finally, we demonstrated that in the absence of the ClpP protease, the level of carbonylated DnaK was increased. Our results suggest that, during heat shock, GBS ClpP might play an important role in the synthesis of functional proteins in the cell.
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METHODS |
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Cloning and sequencing of ClpP.
Based on the alignment of the ClpP amino acid sequences of Streptococcus pyogenes, Streptococcus salivarius, Streptococcus mutans and Streptococcus pneumoniae, we designed two degenerate oligonucleotide primers, designated P1 and P2. These primers were used to amplify a 573 bp DNA fragment internal to the streptococcal clpP gene. PCR was carried out in a final volume of 50 µl containing 50 ng S. agalactiae DNA, 0·1 µM each primer, 200 µM each dNTP and 2 U AmpliTaq Gold DNA polymerase (Applied Biosystems) in 1x times; amplification buffer. DNA was sequenced with an ABI model 310 automated DNA sequencer using the ABI PRISM dye terminator cycle sequencing kit (Applied Biosystems). Sequence analysis of the cloned PCR fragment revealed that it was highly related to known clpP genes present in the databases (data not shown). Southern blot analysis of S. agalactiae NEM316 chromosomal DNA using the cloned PCR fragment as a probe revealed that the clpP gene was located on a 1500 bp SspI DNA fragment (data not shown). Therefore, the remaining portion of the clpP gene was obtained by inverse PCR of chromosomal DNA digested with SspI. Self-ligated DNA was used as a template in PCRs carried out with primers P3 and P4. The resulting 950 bp PCR fragment was cloned into pUC18 and sequenced using custom-synthesized oligonucleotides. Assembly of the sequences of the DNA fragments amplified with the primer pairs P1/P2 and P3/P4 yielded a 1458 bp SspI DNA fragment containing clpP. To verify that we had correctly assembled the PCR fragments, primers P5 and P6 were used to amplify a 1316 bp fragment from S. agalactiae NEM316 chromosomal DNA; three clones were sequenced in their entirety on both strands.
Construction of an allelic GBS ClpP mutant.
To construct the S. agalactiae clpP strain, we inserted a promoterless and terminatorless kanamycin-resistance cassette, aphA-3, within DNA segments internal to clpP. This was done by ligating, after digestion with the appropriate enzymes, the amplicons P7P8, KanKKanB and P9P10. The corresponding EcoRIPstI fragments were cloned into pG+host5; the resulting recombinant vector, pSN600, was introduced into S. agalactiae NEM316 by electroporation. The double-crossover events leading to the expected gene replacements were screened and obtained as described previously (Biswas et al., 1993
; Rouquette et al., 1998
). In the resulting S. agalactiae mutant NEM1968 (
clpP), the promoterless kanamycin-resistance cassette used to inactivate clpP is likely to be transcribed from PclpP. Southern analysis of restriction-enzyme-digested chromosomal DNA revealed that the mutant strain was devoid of sequences related to pG+host5 and that insertion of the resistance cassette had occurred at the expected location (data not shown).
For complementation analysis, we used the following strategy. Primer pair P5/P6 was used to amplify clpP together with its upstream promoter region (409 bp), PclpP. The corresponding 1316 bp fragment was cloned into pAT113/Sp to give pAT113/SpclpP. This vector was conjugatively transferred (Poyart et al., 2001b
) from HB101/pRK24 to S. agalactiae
clpP/pTCV-int to restore the ClpP activity in this mutant strain. The plasmid insertion site was characterized in three integrants harbouring a single copy of pAT113/Sp
clpP inserted at different loci. This was done by inverted PCR as described previously (Poyart et al., 2001b
). The complemented strain NEM1969 (
clpPc) was chosen for further studies because in this strain pAT113/Sp
clpP was not inserted within a protein-coding sequence (data not shown).
RNA preparations and Northern blot analysis.
Total RNAs were extracted as described previously (Gaillot et al., 1997) from exponential phase (OD600 0·6) cultures of S. agalactiae grown in BHI broth at 37 or 41 °C without agitation. For Northern blot analysis, 40 µg RNA were separated through a 1·3 % formaldehyde/agarose gel (Sambrook et al., 1989
) and transferred to a Hybond-N+ membrane (Amersham). The filters were baked for 2 h at 80 °C in an oven. Pre-hybridization and hybridization were performed under stringent conditions as described by Sambrook et al. (1989)
. The DNA probe used was a PCR fragment obtained from NEM316 genomic DNA by using primers P1 and P2. DNA fragments were labelled with [
-32P]dCTP by using the Nick Translation Kit (Amersham).
Flow cytometry analysis.
GBS strains were cultivated in BHI broth, at either 37 or 41 °C, and collected during the exponential and stationary phases of growth. Samples were fixed by the addition of cold ethanol to a final concentration of 70 % and stored for up to one week at 4 °C in ethanol. When appropriate, fixed cultures were centrifuged and resuspended in PBS (10 mM potassium phosphate; 150 mM sodium chloride; pH 7·0). Dilutions were mixed with 10 µM propidium iodide (PI) and cells were analysed on a Becton Dickinson Calibur System. Excitation was at 458 nm and fluorescence was measured at 495 nm. Flow cytometry data were collected and analysed using CELLQUEST software (Becton Dickinson).
Carbonylation assays.
Exponential and stationary phase cultures (20 ml) were pelleted and crude protein extracts were prepared using the Fast Prep BIO101 machine and kit (Ozyme). The carbonyl groups in the protein side chains were derivatized, using the OxyBlot kit (Oncor), to 2,4-dinitrophenylhydrazone (DNP-hydrazone) by reaction with 2,4-dinitrophenylhydrazine as described by Dukan & Nystrom (1998). The DNP-derivatized crude protein extracts were separated by SDS-PAGE and subsequently transferred to PVDF membranes by using a semi-dry blotting system. The filters were incubated with primary antibody, specific to the DNP moiety of the proteins, and subsequently incubated with a secondary (goat-anti-rabbit) horseradish peroxidaseantibody conjugate directed against the primary antibody. For detection, the filters were treated with the ECL+ chemiluminescence blotting substrate (Amersham Pharmacia Biotech).
Protein analysis.
Protein extracts were prepared from cultures of the wild-type, clpP and
clpPc strains grown at 37 or 41 °C using the Fastprep kit (Ozyme) and the BIO 101 machine. The concentration of protein in each soluble extract was measured using the Bradford assay and equal amounts of protein were loaded into each lane. SDS-PAGE and Western blot analyses were done as described previously (Nair et al., 2000a
). Polyclonal antibodies raised against pneumococcal DnaK (Kim et al., 1998
) were diluted 1 : 1000 in PBS. Comparative two-dimensional gel analysis of proteins was performed in a Protean II xi 2D-cell apparatus (Bio-Rad). IEF was prepared with ampholines covering pH 310.
The Panvera Protease Activity Detection kit was used to calculate overall protease activity. This kit uses an FTC-labelled casein as a substrate, which decreases in size as a result of degradation causing a change in spectrophotometric absorbance and fluorescence. Experiments were repeated at least three times on different protein extracts.
Protein sequencing was done by Edman degradation at the Protein Sequencing Laboratory, Pasteur Institute.
Electron microscopy.
Exponential or stationary phase growing bacteria (37 and 41 °C) were processed for thin-sectioning and examined under the electron microscope as described by Frehel & Leduc (1987).
Mouse virulence assays.
Six- to eight-week-old pathogen-free ICR female Swiss mice (Janvier, Le Geneset St Isle) were used in this study. Groups of 10 mice were inoculated intravenously with increasing doses of the wild-type, ClpP mutant and complemented strain. The LD50 was determined by the probit method. For estimation of bacterial numbers in organ homogenates, groups of four mice were inoculated intravenously with 106 bacteria diluted in 0·9 % NaCl. Bacterial numbers in homogenates of spleen, liver, brain and blood were determined at various intervals by plating onto BHI agar plates supplemented, when possible, with the appropriate antibiotic(s) as described previously (Poyart et al., 2001b). Mice were killed by cervical dislocation in accordance with the policies of the Animal Welfare Committee of the Faculte Necker (Paris), and the experiment was performed twice.
Oligonucleotides.
The sequences (5' to 3') of the relevant oligonucleotides used in this study were: P1, ATGATTCCTGTWGTWATTGAACAAAC; P2, CCATRATTTCATCRATRAARCC; P3, CATAAGAGCGTTCACCACGAC; P4, CACTTGATTATGGCTTCATCG; P5, GTTACCTGAAGATATTGATCAACG; P6, CCTGATCAATATCATCAATTGC; P7, ATGAATTCTGTTGTTATTGAACAAACAAGTCG; P8, AGCGGTACCAGCCGATACTGAACCACCTGG; P9, GTGGATCCATGAACTTTATTAAATCGGACG; P10, ATGCTGCAGTCGATGAAGCCATAATCAAGTG. The sequences of the restriction sites added for molecular cloning are shown in bold.
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RESULTS |
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ClpP is required for growth under different stress conditions
The ClpP proteins of various prokaryotes, including B. subtilis (Gerth et al., 1998) and the facultative intracellular pathogen L. monocytogenes (Gaillot et al., 2000
), have been shown to play an essential role in stress tolerance. To determine the role of ClpP in GBS stress tolerance, we constructed an S. agalactiae strain lacking a functional ClpP (
clpP) and an S. agalactiae
clpP complemented strain (
clpPc) (see Methods). Stress tolerances of the
clpP and
clpPc strains were compared to those of the wild-type. At 37 °C, the growth rate of
clpP was reduced when compared to that of the wild-type and
clpPc strains (Fig. 2
a); in the presence of NaCl or H2O2, the growth rate of the
clpP strain was severely impaired (Fig. 2c, d
). During heat shock at 41 °C, the mutant grew very slowly and after reaching an OD600 value of approximately 0·4 stopped growing, even after overnight incubation (Fig. 2b
). Bacterial growth was fully restored in the complemented strain. Taken together, these results indicate that ClpP is required for growth of S. agalactiae under heat-shock, salt- and oxidative-stress conditions. However, in the presence of ethanol, which is supposed to induce a heat-shock response (Bochner et al., 1984
), we observed that growth of the mutant was not as affected by this stress as compared to growth in the presence of NaCl or H2O2 (data not shown).
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Lack of ClpP protease induces DnaK protein carbonylation
As shown previously, the growth of the clpP mutant was blocked during heat shock. In E. coli and Saccharomyces cerevisiae, oxidative stress is involved in heat-induced cell death (Davidson et al., 1996
; Dukan & Nystrom, 1998
, 1999
). Therefore, we looked at the levels of oxidized proteins during heat shock and growth arrest of the
clpP mutant as compared to the wild-type and complemented strains. This was done by using an immunochemical assay to detect protein carbonyl groups (Tamarit et al., 1998
) in crude protein extracts obtained after overnight growth at 37 or 41 °C. This analysis revealed a similar low level of protein carbonylation in the wild-type and complemented strains grown at both temperatures (Fig. 6
a, lanes 11' and 22'). In contrast, higher levels of carbonylated proteins were detected in the clpP mutant and the patterns obtained clearly indicated that, in this strain, carbonylation is heat-induced (Fig. 6a
, lanes 33'). Using SDS-PAGE, we further analysed the pattern of carbonylation of proteins extracted from the wild-type and mutant strains grown at 41 °C. Three carbonylated protein bands were detected in all strains (Fig. 6b
). This is in contrast to the many carbonylated proteins observed in E. coli by Dunkan & Nystrom (1998)
. We took advantage of this situation to sequence carbonylated proteins directly off one-dimensional gels and in all cases, HPLC analysis of Edman degradation products clearly revealed single peaks corresponding to a single protein. Two of these bands were identified as DnaK (XKIIGIDLGTTNSAV) and glyceraldehyde-3-phosphate dehydrogenase (VVKVGINGFGRIGRLAFRRIQNV) (Glaser et al., 2002
). It is worth noting that, at 41 °C, the levels of oxidized DnaK were higher in the mutant than in the wild-type strain, indicating that in the absence of the ClpP protease there is an accumulation of oxidized DnaK (Fig. 6b
). This result cannot be explained by an increased production of DnaK since a Western blot analysis demonstrated that the amount of this protein was very similar in the wild-type and mutant background (Fig. 6c
).
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DISCUSSION |
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During heat shock, an S. agalactiae clpP mutant was growth-arrested and displayed important modifications to its protein content, which included a decreased level of essential metabolic enzymes such as the alcohol dehydrogenase. Pyruvate can be converted to ethanol via acetaldehyde, a reaction requiring alcohol dehydrogenase and generating NAD+. If NAD+ were not regenerated, glycolysis could not proceed beyond glyceraldehyde 3-phosphate, which means that no ATP would be generated (Lehninger, 1982
; Lodish et al., 2000
). Thus, the absence or availability of decreased levels of metabolic enzymes, such as the alcohol dehydrogenase, may be responsible for the growth arrest of the
clpP mutant observed at 41 °C. As a major effect, heat shock may cause cellular oxidation and many of the heat-shock proteins may function to protect against oxidation (Bochner et al., 1984
). Interestingly, we showed that there is an accumulation of oxidized GBS DnaK in the absence of ClpP. This might indicate that DnaK is a preferred target for oxidation and/or that oxidized DnaK is a preferred substrate for ClpP. Oxidized proteins lose their structural integrity and catalytic activity, and the levels of oxidized proteins increase exponentially with age (Stadtman, 1992
). Mutations inactivating the genes encoding the chaperone proteins GroESL and DnaK are known to have pleiotropic effects on host metabolism, including defects in DNA and RNA synthesis, proteolysis and cell division. E. coli cells lacking DnaK die rapidly during stasis and fail to develop resistance to heat and oxidation (Georgopoulos et al., 1994
). In particular, DnaK is important in the resurrection of the activity of heat-inactivated RNA polymerase (Georgopoulos et al., 1994
). These findings are consistent with our proposal that, at 41 °C, the
clpP GBS mutant synthesizes numerous aberrant and prematurely terminated peptides made from truncated mRNAs probably due to oxidized/inactive DnaK. However, the lack of ClpP, which, together with ClpX and ClpC, has to fulfil general quality control functions and possible regulatory functions, could also be the more-direct cause of these phenotypes. It has recently been shown that the S. pneumoniae
clpP mutant is also sensitive to H2O2 (Robertson et al., 2002
). In addition, micro-array analysis of this mutant showed regulatory phenotypes that included downregulation of the oxidative-stress response. Thus, the phenotypes we observed in the S. agalactiae
clpP mutants were probably due to pleiotropic regulatory effects caused by the lack of ClpP.
The role of chaperone proteins in the cell division cycle is unknown, yet many of the division components are peripheral or integral inner-membrane proteins. Defects in cell division as a result of mutations in the clp genes have also been observed in B. subtilis and L. monocytogenes, suggesting that proteins involved in cell morphology or cell division are controlled either directly or indirectly by the ClpXP protease complexes (Gerth et al., 1998; Nair et al., 1999
). In C. crescentus, ClpXP is required in vivo for the cell-cycle-dependent degradation of the regulatory protein CtrA (Jenal & Fuchs, 1998
). Common strategies in cell-cycle control exist in prokaryotes and eukaryotes, suggesting that specific proteolysis/degradation events play a key role in the cell cycle. Similarly, we observed that, during heat shock, an S. agalactiae
clpP mutant displayed important morphological alterations, indicating that, just like in eubacteria, the ClpP in GBS could play an essential role in the checkpoint mechanism of cell-cycle control. Under these growth condition, the mutant also showed altered basic cell functions, as exemplified by the absence of the alcohol dehydrogenase and DnaK oxidation, which are likely to be responsible for the dramatic phenotypic modifications observed, including the growth arrest. Since the cytoplasm becomes pro-oxidant during lethal heating, a role for GBS ClpP as an anti-oxidant in the protection of DnaK cannot be excluded.
Finally, our results showed that ClpP does not play a major role in the virulence of S. agalactiae, at least in our murine infection model. The slight difference observed between the kinetics of elimination of the wild-type and mutant strains is likely to reflect the growth defect of the clpP mutant observed in vitro at 37 °C, i.e. the temperature of the bodies of the mice. Our results apparently conflict with those of Jones et al. (2000)
, who identified, by using signature-tagged mutagenesis, a GBS Clp regulatory subunit as an essential virulence factor. However, it should be noted that, whereas the proteolytic activity sensu stricto resides in the ClpP subunit, the regulatory subunits also act as molecular chaperones involved in the folding and assembly of proteins (Schirmer et al., 1996
). The Clp chaperonin activity might thus be more important than its proteolytic activity for the proper development of the GBS infectious process. Thus, we have concluded that, as opposed to the Gram-positive intracellular pathogen L. monocytogenes (Gaillot et al., 2000
), ClpP is not critical for the virulence of the extracellular pathogen S. agalactiae. This difference might reflect the difference in the lifestyles (extracellular versus intracellular) of these two pathogens.
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ACKNOWLEDGEMENTS |
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Received 3 June 2002;
revised 8 October 2002;
accepted 8 October 2002.
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