Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Center, 3330 Hospital Dr. NW, Calgary, Alberta, Canada T2N 4N1
Correspondence
P. A. Sokol
psokol{at}ucalgary.ca
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ABSTRACT |
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The GenBank accession number for the B. cepacia zmpA and B. pseudomallei zmpA sequences reported in this paper are AY143552 and AY143551, respectively.
Present address: Genomics Institute for the Novartis Research Foundation, San Diego, CA 92121, USA.
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INTRODUCTION |
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B. cepacia is naturally found in soil and water. Strains originally identified as B. cepacia have been classified into at least nine genomovars, which are referred to as the B. cepacia complex (Vandamme et al., 1997; Vermis et al., 2002
). Colonization of cystic fibrosis (CF) patients with B. cepacia complex organisms can lead to chronic airway infection and increase morbidity and mortality in these patients. These infections occasionally result in a rapid pulmonary decline associated with septicaemia, which may result in death, often referred to as cepacia syndrome (reviewed by Mohr et al., 2001
).
Sixty-nine to 88% of clinical B. cepacia isolates produce proteases (Gessner & Mortensen, 1990; Gilligan, 1991
; McKevitt & Woods, 1984
; Nakazawa et al., 1987
). A recent study by Gotschlich et al. (2001)
reported that strains of B. cepacia genomovars I and III, and Burkholderia stabilis are positive for extracellular protease activity, whereas strains of B. cepacia genomovar VI, Burkholderia multivorans and Burkholderia vietnamiensis do not have detectable extracellular protease activity (Gotschlich et al., 2001
). In Canada, approximately 80 % of CF isolates are classified as genomovar III and 9·3 % are classified as B. multivorans (Speert et al., 2002
). In the United States 50 % of CF isolates are classified as genomovar III organisms, 38 % are classified as B. multivorans and 5 % are classified as B. vietnamiensis (LiPuma et al., 2001
).
A 36 kDa zinc metalloprotease, originally designated PSCP (Pseudomonas cepacia protease), has been described in B. cepacia genomovar III strain Pc715j (McKevitt et al., 1989). McKevitt et al. (1989)
demonstrated that this zinc metalloprotease is capable of cleaving gelatin, hide powder and human collagen types I, IV and V (McKevitt et al., 1989
). Biochemical evidence indicates that PSCP is a zinc metalloprotease since it is inhibited by 0·1 mM EDTA and 0·1 mM 1,10-phenanthroline. This inhibition is reversible by the addition of zinc and calcium salts (McKevitt et al., 1989
). PSCP may play a role in the virulence of B. cepacia. McKevitt et al. (1989)
demonstrated that purified PSCP induces bronchopneumonia in rat lungs characterized by polymorphonuclear leukocyte infiltration and proteinaceous exudate in the airways. Immunization with a peptide corresponding to a conserved zinc metalloprotease epitope significantly decreased the severity of experimental B. cepacia lung infections (Sokol et al., 2000
). Further study of the role of extracellular proteases in the pathogenesis of B. cepacia infections has been limited by the lack of isogenic protease mutants.
Bacillus thermoproteolyticus thermolysin was the first zinc metalloprotease for which the three-dimensional structure was determined (Colman et al., 1972). Consequently, thermolysin has become a model for the zinc metalloproteases belonging to the M4 peptidase family, also known as the thermolysin-like metalloproteases (Rawlings & Barrett, 1995
). In this study the thermolysin sequence was used to search for a homologue in the B. pseudomallei K96243 genome sequence (www.sanger.ac.uk/projects/B_pseudomallei/blast_server.shtml) in an effort to identify a zinc metalloprotease gene in B. pseudomallei. The B. pseudomallei zinc metalloprotease gene was then used to identify a homologue in B. cepacia. The role of this B. cepacia zinc metalloprotease in virulence was investigated.
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METHODS |
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Cloning zmpA from B. pseudomallei.
The B. thermoproteolyticus thermolysin sequence (accession no. X76986) was used to identify a thermolysin-like zinc metalloprotease gene (zmpA) homologue in the B. pseudomallei K96243 genome sequence at the Wellcome Trust Sanger Institute (www.sanger.ac.uk/projects/B_pseudomallei/blast_server.shtml). The zmpA gene from B. pseudomallei 1026b was amplified by PCR with the oligodeoxyribonucleotide primers NP5' (5'-CGGGATCCGTTCGAAGGTACCTCTCACG-3') containing a BamHI linker and NP3' (5'-GCTCTAGAATCGTCACGTGCGCTTATCGG-3') containing an XbaI linker. The PCR products were cloned into pCR2.1-TOPO (Invitrogen Life Technologies).
Cloning zmpA from B. cepacia.
A 1·9 kb BamHIXbaI fragment derived from plasmid pTOPOZMPA containing B. pseudomallei zmpA was radiolabelled with [32P]dCTP and hybridized to a Southern blot of B. cepacia Pc715j PstI-digested chromosomal DNA fractionated by using a sucrose density gradient. The DNA fraction that hybridized to the probe was ligated into the PstI site of pEX18Tc (Hoang et al., 1998). The plasmid containing the zmpA gene was identified by colony hybridization (Woods, 1984
) and designated pCC12.
DNA sequencing and sequence analysis.
The nucleotide sequences of the zmpA genes were determined using the T7 and M13R universal primers and primers designed to the partially determined sequence. Custom oligonucleotides were synthesized by Invitrogen Life Technologies. Nucleotide sequencing was conducted using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit with Ampli-Taq DNA polymerase (Perkin-Elmer) and analysed with an ABI373A DNA sequencer by University Core DNA Services (University of Calgary). Sequences were analysed using the gapped BLASTX and BLASTP programs (Altschul et al., 1997) and DNAMAN software (Lynnon Biosoft). SignalP V1.1 (www.cbs.dtu.dk/services/SignalP/#submission) (Nielsen et al., 1997
) was employed for the identification of putative signal sequence cleavage sites. Alignments were conducted using the CLUSTALX program (www.ebi.ac.uk/clustalw/index.html).
N-terminal sequencing of PSCP.
B. cepacia PSCP was purified as described previously (Kooi et al., 1994; McKevitt & Woods, 1984
) and partial N-terminal amino acid microsequencing of PVDF-electroblotted PSCP was performed using an ABI sequencer at the Department of Biochemistry, University of Victoria (UVic-Genome BC Proteomics Center).
zmpA mutant construction.
A trimethoprim cassette was inserted into the BsiWI restriction site of B. cepacia zmpA, resulting in plasmid pCC12T. Triparental matings were performed using E. coli DH5(pRK2013) (Figurski & Helinski, 1979
) to mobilize pCC12T from E. coli DH10b into B. cepacia Pc715j or K56-2. The insertional inactivation of zmpA was confirmed by PCR or by Southern hybridization analysis. Mutants were confirmed to have the same enzymic profiles as their parent strain using the API20E system (Analytab Products).
Extracellular protease analysis.
Twenty-hour cultures were centrifuged at 10 000 g for 25 min at 4 °C. The cell-free supernatants were precipitated with trichloroacetic acid (TCA) (10 % final concentration) and electrophoresed on SDS-12·5 % polyacrylamide gels by the method of Laemmli (1970). Protein profiles were also analysed by two-dimensional gel electrophoresis. Protein was resuspended in isoelectric focusing (IEF) rehydration buffer (8 M urea, 0·2 % Biolyte ampholytes 5/8, 1 % CHAPS, 30 mM DTT, 0·001 % bromophenol blue) and quantified by using the RC DC protein assay (Bio-Rad). Approximately 400 µg extracellular protein was desalted using Micro Bio-Spin 6 chromatography columns (Bio-Rad) and the volume was adjusted to 200 µl with rehydration buffer. The resuspended protein was used to rehydrate 7 cm ReadyStrip IPG strips pH 58 and electrofocused using Protein IEF (Bio-Rad) as per the manufacturer's recommendations (Bio-Rad). The focused strips were equilibrated in each of the following buffers; Buffer 1 (6 M urea, 0·375 M Tris, pH 8·8, 2 % SDS, 20 % glycerol, 2 % DTT) and Buffer 2 (6 M urea, 0·375 M Tris, pH 8·8, 2 % SDS, 20 % glycerol, 2·5 % iodoacetamide). Two-dimensional gels were silver-stained using the PlusOne silver staining kit (Amersham Pharmacia Biotech) or transferred to polyvinylidene difluoride membrane (Millipore) by the method of Towbin et al. (1979)
. Western immunoblots were reacted with mAb 36-6-6 to PSCP as described by Kooi et al. (1994)
.
Protease activity was determined using skim milk or hide blue azure (Sigma) as substrates. Mid-exponential phase cultures were normalized to the same optical density at 600 nm and spotted (3 µl) in triplicate onto dialysed 1·5 % brain heart infusion agar containing 10 % skim milk (Sokol et al., 1979). The plates were incubated at 37 °C for 24 h and examined for clear zones surrounding the colonies. The hide blue azure assays were based on the method of Rinderknecht et al. (1968)
and performed as described by Kooi et al. (1994)
.
Animal studies.
The relative virulence of the B. cepacia zmpA mutants was determined using an agar bead model of chronic lung infection as described by Cash et al. (1979). On days 7 and 14 post-infection (p.i.) lungs from 35 animals per group were removed and fixed in 10 % formalin. Saggital slices of the left lung were mounted and stained with haemotoxylin and eosin. The percentage of the lung infiltrated with inflammatory exudate was quantified as described previously using a point counting method (Dunnil, 1962
; Sokol et al., 2000
). The lungs of 34 animals in each group were removed and homogenized (Polytron Homogenizer; Brinkman Instruments) in 3 ml PBS (10 mM sodium phosphate pH 7·2, 150 mM NaCl). Serial dilutions of the homogenates were plated to quantify bacteria.
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RESULTS |
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Recently, the genomic sequence of B. cepacia J2315 has been completed by the Wellcome Trust Sanger Institute (www.sanger.ac.uk/Projects/B_cepacia/). The J2315 zmpA gene is 99·5 % identical to Pc715j zmpA. There is a single amino acid change at residue 489 from threonine to serine in Pc715j. Although the K56-2 zmpA gene was not sequenced it belongs to the same ET12 clone of genomovar III as J2315 (Mahenthiralingam et al., 2000). A putative transcriptional start site was identified upstream of zmpA in the J2315 and the Pc715j nucleotide sequence (Neural Network Promoter Prediction; www.fruitfly.org/seq_tools/promoter.html). This suggests that the transcription of zmpA is under the control of a promoter immediately upstream of the gene.
Effects of zmpA mutations on protease expression
Mutations in the zmpA genes were constructed by insertional inactivation of zmpA with a trimethoprim cassette, followed by allelic exchange into strains Pc715j or K56-2, resulting in Pc715j-72 and K56-2-9, respectively. SDS-PAGE analysis of the extracellular protein profiles of K56-2(pUCP26), K56-2-9(pUCP26) and K56-2-9(pCC13) revealed a decrease in a 36 kDa protein, corresponding in mass to PSCP, in the zmpA mutant, which was restored in K56-2-9(pCC13) (Fig. 2a). An approximately 20 kDa protein was also present in the parent and K56-2-9(pCC13), but absent in K56-2-9. This protein has the predicted mass of the 20·6 kDa pro-region of PSCP. On both SDS-PAGE and Western Immunoblots a 36 kDa protein was present in the zmpA mutant that reacted with antibodies to PSCP (Fig. 2b
). Similar results were observed in Pc715j (data not shown). Interestingly, the insertion of the Tp cassette at base 1066 results in translation of a protein with a predicted molecular mass of 36 268 Da. This consists of the propeptide and the truncated mature protein to the stop codon within the trimethoprim cassette. This fusion protein has a predicted pI (6·8) that is higher than that of the mature PSCP enzyme (6·3). (http://ca.expasy.org/cgi-bin/pi_tool). Therefore, two-dimensional gel electrophoresis was used to examine the extracellular protein profiles of Pc715j and Pc715j-72 (Fig. 3
a, b). In the Western immunoblot of Pc715j extracellular proteins a 36 kDa protein corresponding to PSCP was detected by mAb 36-6-6 (Fig. 3c
) whereas a 36 kDa protein with a higher isoelectric point than PSCP was detected with mAb 36-6-6 in the immunoblot of Pc715j-72 (Fig. 3d
). We hypothesize that this peptide is the unprocessed propeptide with the truncated mature peptide that has a predicted pI of 6·8 compared to the predicted pI of 6·3 for the mature PSCP. The mAb 36-6-6 reacts with epitopes surrounding the active site of P. aeruginosa elastase (Kooi et al., 1997
). Although the strongest reacting epitope (peptide 15) of elastase is absent in this truncated fusion protein, some of the other reactive epitopes are present. This explains why we observed an extracellular protein in the zmpA mutant strains of B. cepacia that migrated at 36 kDa on one-dimensional SDS-PAGE and reacted with mAb 36-6-6. This fusion protein has no proteolytic activity since the active site has been interrupted. A 40 kDa protein was also detected in Western immunoblots of Pc715j, Pc715j-72 (Fig. 3
), K56-2 and K56-2-9 (Fig. 2
) as previously observed by Kooi et al. (1994)
. In Pc715j a 20 kDa protein was also detected on the immunoblots. Although this corresponds in size to the 20 kDa protein present in supernatants of K56-2, the K56-2 protein did not react with mAb 36-6-6 and was predicted to be the pro-region of PSCP (Fig. 2
). It is not clear if the 20 kDa protein in Pc715j is a degradation product of PSCP or an immunologically related protein.
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The growth of the zmpA mutants was compared to their respective wild-type strains in a rich medium, PTSB, and in M9 minimal medium supplemented with 0·1 % casein (data not shown). There were no growth rate differences between the B. cepacia zmpA mutants and their wild-type strains in either medium, indicating that the decrease in proteolytic activity observed in the zmpA mutants is not due to decreased growth. AP120 E systems were used to examine the enzymic activities of the parent and mutant strains. The profile of the Pc715j zmpA mutant was identical to Pc715j, with the exception that it did not cleave gelatin. Neither K56-2 nor K56-2-9 cleaved gelatin in this system.
Relative virulence of the B. cepacia zmpA mutants
Rats were infected with B. cepacia Pc715j, K56-2 and their respective mutants, and on days 7 and 14 p.i. quantitative histopathologic and bacteriologic analyses were performed on the lungs. On days 7 and 14 p.i. the number of bacteria (c.f.u. ml-1) recovered from the lungs of rats infected with K56-2-9 was approximately 4 logs lower than the parent strain (Table 2). Interestingly, the K56-2-9 zmpA mutant was only recovered from one of three animals on day 7 p.i. and two of four animals on day 14 p.i. These results indicate that the B. cepacia K56-2-9 zmpA mutant is less able to persist in the lung than the B. cepacia K56-2 parent strain, which had similar numbers of bacteria recovered on both days 7 and 14 p.i. There was no difference between the numbers of B. cepacia Pc715j and Pc715j-72 recovered from the lungs of rats on day 7 or 14 p.i. (Table 2
). This indicates that both Pc715j and Pc715j-72 were able to persist within the lung and the production of PSCP was not necessary for persistence in this strain.
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DISCUSSION |
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The prediction that B. cepacia PSCP is secreted via the general secretory system agrees with the findings of Nakazawa & Abe (1996). They conducted transposon mutagenesis and found that protease mutant KFT1008 could be complemented with a fragment of DNA harbouring gspF, a homologue of P. aeruginosa xcpS encoding a component of the general secretory system (Nakazawa & Abe, 1996
). Furthermore, they determined that protease production was dependent on DsbB, suggesting that B. cepacia protease is secreted via the general secretory pathway and involves disulfide bond formation (Abe & Nakazawa, 1996
; Nakazawa & Abe, 1996
).
Anti-PSCP mAbs react with PSCP and a 40 kDa protein present in B. cepacia Pc715j cell-free supernatants (Kooi et al., 1994). The 40 kDa protein preparation demonstrated weak proteolytic activity and could potentially be a precursor of PSCP or an immunologically related protein (Kooi et al., 1994
). Abe & Nakazawa (1996)
predicted that a 43 kDa protein (likely to be the 40 kDa described by Kooi et al., 1994
) was perhaps a precursor to a 37 kDa protease secreted by B. cepacia KF1. In this study we determined that the precursor to PSCP has a predicted molecular mass of approximately 59·8 kDa and therefore it is unlikely that the 40 kDa protein is a precursor to PSCP. Also, the extracellular protein profiles of the B. cepacia zmpA mutant strains still contain the 40 kDa protein, indicating that it is not a precursor to PSCP.
B. cepacia Pc715j has more extracellular protease activity than B. cepacia K56-2. The Pc715j zmpA mutant still produces protease, suggesting that this strain produces more than one extracellular protease. In contrast, the zmpA mutant of B. cepacia K56-2 elicits minimal protease activity, suggesting that under the conditions employed in this study, PSCP may be the major extracellular protease in this strain.
Thermolysin-like metalloproteases have been implicated in bacterial pathogenesis (Morihara, 1995). In vitro assays demonstrate that PSCP is capable of cleaving biologically relevant substrates, including collagen (McKevitt et al., 1989
), human IgA, IgG, IgM, transferrin and lactoferrin (C. Kooi & P. A. Sokol, unpublished observation). Previously, we have demonstrated a possible role for the extracellular zinc metalloprotease, PSCP, in B. cepacia virulence (Sokol et al., 2000
). In this study, we demonstrate that the expression of a thermolysin-like protease by B. cepacia K56-2 contributes to virulence in an agar bead model of lung infection. The K56-2-9 zmpA mutants were less able to persist in rat lungs than the parental K56-2 strain, indicating that PSCP contributes to the persistence of B. cepacia K56-2 within the lung. PSCP may directly degrade host tissue allowing the organisms to replicate in the lung or disrupt the host immune response by degrading immunoglobulins or other host proteins involved in the inflammatory response. This may lead to a reduced ability of the animals to clear the bacteria. The virulence of the Pc715j zmpA mutant was not decreased; however the Pc715j zmpA mutant continues to produce extracellular protease. This suggests that at least some strains of B. cepacia produce more than one protease. Due to this possible redundancy, the loss of a single protease may not compromise the virulence of a strain in a particular infection model and, therefore, the contributions of this extracellular protease to virulence may vary among B. cepacia strains.
Immunization with a conserved zinc metalloprotease peptide was previously shown to decrease the severity of B. cepacia Pc715j infection. Antibodies to this peptide, however, also react with additional proteins in B. cepacia supernatants, including a 40 kDa protein. It is possible that the reduced lung injury observed in immunized animals is due to the abilities of the induced antibodies to neutralize the proteolytic activity of PSCP as well as react with the 40 kDa protein and possibly inactivate its activity. Further studies are in progress to identify additional proteases in B. cepacia and to determine the role of the 40 kDa protein since we have determined it is not a precursor of PSCP.
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ACKNOWLEDGEMENTS |
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Received 17 January 2003;
revised 14 April 2003;
accepted 25 April 2003.