An extracellular zinc metalloprotease gene of Burkholderia cepacia

C. R. Corbett, M. N. Burtnick{dagger}, C. Kooi, D. E. Woods and P. A. Sokol

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Burkholderia cepacia produces at least one extracellular zinc metalloprotease that may be involved in virulence. A B. cepacia zinc metalloprotease gene was cloned using a Burkholderia pseudomallei zinc metalloprotease gene as a probe. The predicted amino acid sequences of these B. cepacia and a B. pseudomallei extracellular zinc metalloproteases indicate that they are similar to the thermolysin-like family of metalloproteases (M4 family of metalloendopeptidases) and they are likely to be secreted via the general secretory pathway. zmpA isogenic mutants were constructed in B. cepacia genomovar III strains Pc715j and K56-2 by insertional inactivation of the zmpA genes. The zmpA mutants produced less protease than the parent strains. The B. cepacia strain K56-2 zmpA mutant was significantly less virulent than its parent strain in a chronic respiratory infection model; however, there was no difference between the virulence of B. cepacia strain Pc715j and a Pc715j zmpA mutant. The results indicate that this extracellular zinc metalloprotease may play a greater role in virulence in some strains of B. cepacia.


Abbreviations: CF, cystic fibrosis; PSCP, Pseudomonas cepacia protease; PTSB, peptone-trypticase soy broth; Ap, ampicillin; Tc, tetracycline; Tp, trimethoprim

The GenBank accession number for the B. cepacia zmpA and B. pseudomallei zmpA sequences reported in this paper are AY143552 and AY143551, respectively.

{dagger}Present address: Genomics Institute for the Novartis Research Foundation, San Diego, CA 92121, USA.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Burkholderia cepacia and Burkholderia pseudomallei are Gram-negative opportunistic pathogens belonging to the {beta}-subclass of the phylum Proteobacteria (Yabuuchi, 1992). B. pseudomallei is a soil saprophyte and the causative agent of melioidosis, a glanders-like disease of humans and animals (Dance, 1991). Infection is most likely due to soil or water contamination of skin abrasions or inhalation of contaminated sources. Individuals more susceptible to B. pseudomallei infection include diabetics, alcoholics and those with chronic renal failure (Chaowagul et al., 1989).

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.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and culture conditions.
Strains and plasmids used in this study are listed in Table 1. For genetic manipulations, bacterial cultures were grown at 37 °C in Luria–Bertani (LB) (Life Technologies) or Bacto-terrific broth (Difco). For the virulence studies cultures were grown overnight at 32 °C in dialysed-trypticase soy broth (Difco) treated with Chelex-100 (Bio-Rad) (Ohman et al., 1980). Trypticase soy agar (TSA) (Difco) and Burkholderia cepacia selection agar (Henry et al., 1997) were used to quantify bacteria in lung homogenates. For protease production, cultures were grown in 0·25 % trypticase soy broth (Difco) with 5 % Bacto-peptone (PTSB). For growth curves, cultures were grown in PTSB or M9 (Sambrook, 1989) supplemented with 0·1 % casein. When required, antibiotics were used at the following concentrations: for B. cepacia tetracycline (Tc) 250 µg ml-1, trimethoprim (Tp) 100 µg ml-1; and for Escherichia coli, 100 µg ampicillin ml-1 (Ap), 15 µg Tc ml-1 and 1·5 mg Tp ml-1.


View this table:
[in this window]
[in a new window]
 
Table 1. Bacterial strains and plasmids used in this study

 
DNA manipulations.
Molecular biology techniques were generally performed as described by Sambrook et al. (1989). Genomic DNA was isolated from B. cepacia as described by Ausubel et al. (1989) and from B. pseudomallei using the Wizard Genomic DNA Purification Kit (Promega). Recombinant plasmids were electroporated into B. cepacia K56-2 as described previously (Dennis & Sokol, 1995). Recombinant plasmids were transformed into B. cepacia Pc715j by triparental mating using E. coli DH5{alpha} (pRK2013) (Figurski & Helinski, 1979) to mobilize plasmids from E. coli DH10b into B. cepacia Pc715j.

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 BamHI–XbaI 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{alpha}(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 5–8 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 3–5 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 3–4 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.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning and sequence analysis of the extracellular zinc metalloprotease genes
An ORF with homology to thermolysin was identified in the B. pseudomallei K96243 genome sequence using the TBLASTN search tool (Madden et al., 1996). The gene was cloned from B. pseudomallei 1026b via PCR and used as a probe to identify the gene encoding an extracellular metalloprotease of B. cepacia. The product of the B. cepacia zmpA ORF was predicted to be 565 aa in length and to have a molecular mass of 59 806 Da. The deduced amino acid sequence revealed a putative signal peptide cleavage site at aa 24 and a 13 aa region, 218-AAATGTGRSLYYG-230, identical to the partial N-terminal amino acid sequence determined for mature PSCP. Processing at this site would yield a peptide of 36 857 Da with a predicted pI of 6·3 (http://ca.expasy.org/cgi-bin/protparam). This is consistent with the molecular mass of B. cepacia PSCP (36 000 Da) observed by SDS-PAGE analysis (Kooi et al., 1994). Similar features were observed for the deduced amino acid sequence of B. pseudomallei ZmpA as illustrated in Fig. 1. The above properties indicate that the proteases have a preproenzyme structure similar to other zinc metalloproteases such as Pseudomonas aeruginosa elastase, Bacillus thermoproteolyticus thermolysin and Vibrio cholerae HA/protease (Bever & Iglewski, 1988; Hase & Finkelstein, 1991; O'Donohue & Beaumont, 1996).



View larger version (74K):
[in this window]
[in a new window]
 
Fig. 1. Alignment of the deduced amino acid sequences of the B. cepacia (B.cep) and B. pseudomallei (B.pse) extracellular zinc metalloproteases. The predicted signal peptidase cleavage site is underlined with a dotted line and the determined mature N-terminal amino acid sequence of PSCP is boxed (solid line). The conserved zinc metalloprotease motif, HExxH, is boxed with a dashed line and a second consensus sequence, GGLNESTSD, is underlined (solid line). Bold letters represent strictly conserved residues of the thermolysin-like metalloproteases. ‘*’ indicates identical residues; ‘:’ indicates conserved residues; ‘.’ indicates semi-conserved residues.

 
Analysis of the deduced amino acid sequences of the B. cepacia and B. pseudomallei proteases reveals two conserved sequence characteristics of the thermolysin-like family of metalloproteases (M4 family of metallopeptidases) (Rawlings & Barrett, 1995), the zinc metalloprotease active-site motif (HExxH) at aa residues 376-HEMSH-380 and 396-GGLNESTSD-404, which contains the third zinc ligand (aa residue 400-E). The presence of these conserved residues suggests that these proteases belong to the thermolysin family of zinc metalloproteases. Interestingly, biochemical evidence demonstrates that PSCP is inhibited by the metalloprotease inhibitors EDTA and 1,10-phenanthroline (McKevitt et al., 1989), but it is not inhibited by phosphoramidon, an inhibitor of thermolysin (data not shown). The B. cepacia and B. pseudomallei zinc metalloproteases are 86 % identical at the amino acid level and approximately 30 % identical to Staphylococcus epidermidis elastase (Teufel & Gotz, 1993), Staphylococcus aureus aureolysin (Banbula et al., 1998) and Bacillus thermoproteolyticus thermolysin (O'Donohue et al., 1994). They are approximately 26 % identical to P. aeruginosa elastase (LasB) (Bever & Iglewski, 1988).

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. 3a, 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.



View larger version (61K):
[in this window]
[in a new window]
 
Fig. 2. Extracellular protein profiles of K56-2(pUCP26), K56-2-9(pUCP26) and K56-2-9(pCC13). (a) SDS-PAGE (12·5 %) of TCA-precipitated cell-free supernatants. Lanes: 1, pre-stained molecular mass markers; 2, K56-2(pUCP26); 3, K56-2-9(pUCP26); 4, K56-2-9(pCC13); 5, PSCP; 6, 40 kDa protein. (b) Western immunoblot of TCA-precipitated cell-free supernatants. Lanes: 1, pre-stained molecular mass markers; 2, K56-2(pUCP26); 3, K56-2-9(pUCP26); 4, K56-2-9(pCC13); 5, PSCP.

 


View larger version (73K):
[in this window]
[in a new window]
 
Fig. 3. Extracellular protein profiles of B. cepacia Pc715j and Pc715j-72. (a, b) Two-dimensional 12·5 % SDS-polyacrylamide gels of the TCA-precipitated extracellular proteins of B. cepacia Pc715j and Pc715j-72, respectively. Purified PSCP was applied in the second dimension of the SDS-PAGE. (c, d) Western immunoblots of the extracellular protein profiles of B. cepacia Pc715j and Pc715j-72, respectively. PSCP is only present in the parent strain and is indicated by an arrow. ‘*’ indicates the 40 kDa protein.

 
The proteolytic activities of the mutants were compared to their parent strains using skim milk agar and hide blue azure as substrates. On skim milk agar Pc715j(pUCP28Tc) produced 4·3±0·3 mm zones of clearing compared to 3·3±0·3 mm in the zmpA mutant, Pc715j-72(pUCP28Tc) and 5·0±0 mm in Pc715j-72(pCC14). The difference in zone size between the three strains was significant (P<0·05; ANOVA). There was a more dramatic difference in the zone sizes of K56-2 and its zmpA mutant, K56-2-9, which produced little or no zones of clearing on skim milk agar after 24 h. K56-2(pUCP26) produced 3·2±0·6 mm zones of clearing compared to 0·5±0·0 mm by K56-2-9(pUCP26) and 2·7±0·3 mm by K56-2-9(pCC13). When hide blue azure was used as a substrate, the cell-free supernatant of Pc715j-72(pUCP28Tc) contained 53 % less proteolytic activity than Pc715j(pUCP28Tc), whereas Pc715j-72(pCC14) demonstrated a 3·6-fold increase in the amount of proteolytic activity compared to the wild-type Pc715j(pUCP28Tc) (data not shown). As previously reported K56-2 cell-free supernatants did not contain sufficient proteolytic activity to be quantified in this assay (Lewenza et al., 1999).

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.


View this table:
[in this window]
[in a new window]
 
Table 2. Comparison of the virulence of B. cepacia parent and zmpA mutants using a chronic respiratory infection model

 
Rats infected with K56-2-9 had significantly less lung pathological changes than rats infected with K56-2 on both days 7 and 14 p.i. (P<0·01; Student's unpaired t test). In contrast, there were no significant differences in the degree of lung pathology observed between B. cepacia Pc715j and Pc715j-72 on either day 7 or day 14 (Table 2). These data suggest that PSCP may be an important virulence factor in some strains of B. cepacia, but production of additional proteases may compensate for a loss of PSCP in strains such as Pc715j.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study we identified genes from B. cepacia and B. pseudomallei that encode zinc metalloproteases. The deduced amino acid sequences of the B. cepacia and B. pseudomallei extracellular zinc metalloproteases have a preproenzyme structure similar to members of the thermolysin-like metalloprotease family (Hase & Finkelstein, 1991; Kessler & Safrin, 1988; McIver et al., 1991). The preproenzyme structure dictates secretion through the general secretory pathway (Pugsley, 1993). The signal peptide directs secretion through the periplasmic membrane, followed by cleavage of the signal peptide on the outer side of the periplasmic membrane (Pugsley, 1993). The pro-sequences of P. aeruginosa elastase (LasB) and Bacillus thermoproteolyticus thermolysin act as molecular chaperones that mediate folding of the mature enzymes within the periplasm (Braun et al., 1996; Marie-Claire et al., 1999; McIver et al., 1995; O'Donohue & Beaumont, 1996). The propeptides remain non-covalently associated with the mature enzymes to inhibit their proteolytic activity until liberation from the cell (Braun et al., 1998; Kessler & Safrin, 1994; Kessler et al., 1998; O'Donohue & Beaumont, 1996).

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.


   ACKNOWLEDGEMENTS
 
This study was supported by the Canadian Institutes for Health Research and the Canadian Bacterial Diseases Networks Center for Excellence. M. N. B. was supported by a studentship award from the Alberta Heritage Foundation for Medical Research.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Abe, M. & Nakazawa, T. (1996). The dsbB gene product is required for protease production by Burkholderia cepacia. Infect Immun 64, 4378–4380.[Abstract]

Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.[Abstract/Free Full Text]

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (editors) (1989). Current Protocols in Molecular Biology. New York: Wiley.

Banbula, A., Potempa, J., Travis, J., Fernandez-Catalan, C., Mann, K., Huber, R., Bode, W. & Medrano, F. (1998). Amino-acid sequence and three-dimensional structure of the Staphylococcus aureus metalloproteinase at 1·72 Å resolution. Structure 6, 1185–1193.[Medline]

Bever, R. A. & Iglewski, B. H. (1988). Molecular characterization and nucleotide sequence of the Pseudomonas aeruginosa elastase structural gene. J Bacteriol 170, 4309–4314.[Medline]

Braun, P., Tommassen, J. & Filloux, A. (1996). Role of the propeptide in folding and secretion of elastase of Pseudomonas aeruginosa. Mol Microbiol 19, 297–306.[Medline]

Braun, P., de Groot, A., Bitter, W. & Tommassen, J. (1998). Secretion of elastinolytic enzymes and their propeptides by Pseudomonas aeruginosa. J Bacteriol 180, 3467–3469.[Abstract/Free Full Text]

Cash, H. A., Woods, D. E., McCullough, B., Johanson, W. G., Jr & Bass, J. A. (1979). A rat model of chronic respiratory infection with Pseudomonas aeruginosa. Am Rev Respir Dis 119, 453–459.[Medline]

Chaowagul, W., White, N. J., Dance, D. A., Wattanagoon, Y., Naigowit, P., Davis, T. M., Looareesuwan, S. & Pitakwatchara, N. (1989). Melioidosis: a major cause of community-acquired septicemia in northeastern Thailand. J Infect Dis 159, 890–899.[Medline]

Colman, P. M., Jansonius, J. N. & Matthews, B. W. (1972). The structure of thermolysin: an electron density map at 2–3 Å resolution. J Mol Biol 70, 701–724.[Medline]

Dance, D. A. (1991). Melioidosis: the tip of the iceberg? Clin Microbiol Rev 4, 52–60.[Medline]

Darling, P., Chan, M., Cox, A. D. & Sokol, P. A. (1998). Siderophore production by cystic fibrosis isolates of Burkholderia cepacia. Infect Immun 66, 874–877.[Abstract/Free Full Text]

Dennis, J. J. & Sokol, P. A. (1995). Electrotransformation of Pseudomonas. Methods Mol Biol 47, 125–133.[Medline]

Dennis, J. J. & Zylstra, G. J. (1998). Plasposons: modular self-cloning minitransposon derivatives for rapid genetic analysis of gram-negative bacterial genomes. Appl Environ Microbiol 64, 2710–2715.[Abstract/Free Full Text]

DeShazer, D. & Woods, D. E. (1996). Broad-host-range cloning and cassette vectors based on the R388 trimethoprim resistance gene. Biotechniques 20, 762–764.[Medline]

Dunnil, M. S. (1962). Quantitative methods in the study of pulmonary pathology. Thorax 17, 320–328.

Figurski, D. H. & Helinski, D. R. (1979). Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76, 1648–1652.[Abstract]

Gessner, A. R. & Mortensen, J. E. (1990). Pathogenic factors of Pseudomonas cepacia isolates from patients with cystic fibrosis. J Med Microbiol 33, 115–120.[Abstract]

Gilligan, P. H. (1991). Microbiology of airway disease in patients with cystic fibrosis. Clin Microbiol Rev 4, 35–51.[Medline]

Gotschlich, A., Huber, B., Geisenberger, O. & 11 other authors (2001). Synthesis of multiple N-acylhomoserine lactones is widespread among the members of the Burkholderia cepacia complex. Syst Appl Microbiol 24, 1–14.[Medline]

Hase, C. C. & Finkelstein, R. A. (1991). Cloning and nucleotide sequence of the Vibrio cholerae hemagglutinin/protease (HA/protease) gene and construction of an HA/protease-negative strain. J Bacteriol 173, 3311–3317.[Medline]

Henry, D. A., Campbell, M. E., LiPuma, J. J. & Speert, D. P. (1997). Identification of Burkholderia cepacia isolates from patients with cystic fibrosis and use of a simple new selective medium. J Clin Microbiol 35, 614–619.[Abstract]

Hoang, T. T., Karkhoff-Schweizer, R. R., Kutchma, A. J. & Schweizer, H. P. (1998). A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212, 77–86.[CrossRef][Medline]

Kessler, E. & Safrin, M. (1988). Synthesis, processing, and transport of Pseudomonas aeruginosa elastase. J Bacteriol 170, 5241–5247.[Medline]

Kessler, E. & Safrin, M. (1994). The propeptide of Pseudomonas aeruginosa elastase acts an elastase inhibitor. J Biol Chem 269, 22726–22731.[Abstract/Free Full Text]

Kessler, E., Safrin, M., Gustin, J. K. & Ohman, D. E. (1998). Elastase and the LasA protease of Pseudomonas aeruginosa are secreted with their propeptides. J Biol Chem 273, 30225–30231.[Abstract/Free Full Text]

Kooi, C., Cox, A., Darling, P. & Sokol, P. A. (1994). Neutralizing monoclonal antibodies to an extracellular Pseudomonas cepacia protease. Infect Immun 62, 2811–2817.[Abstract]

Kooi, C., Hodges, R. S. & Sokol, P. A. (1997). Identification of neutralizing epitopes on Pseudomonas aeruginosa elastase and effects of cross-reactions on other thermolysin-like proteases. Infect Immun 65, 472–477.[Abstract]

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[Medline]

Lewenza, S., Conway, B., Greenberg, E. P. & Sokol, P. A. (1999). Quorum sensing in Burkholderia cepacia: identification of the LuxRI homologs CepRI. J Bacteriol 181, 748–756.[Abstract/Free Full Text]

LiPuma, J. J., Spilker, T., Gill, L. H., Campbell, P. W., 3rd, Liu, L. & Mahenthiralingam, E. (2001). Disproportionate distribution of Burkholderia cepacia complex species and transmissibility markers in cystic fibrosis. Am J Respir Crit Care Med 164, 92–96.[Abstract/Free Full Text]

Madden, T. L., Tatusov, R. L. & Zhang, J. (1996). Applications of network BLAST server. Methods Enzymol 266, 131–141.[Medline]

Mahenthiralingam, E., Coenye, T., Chung, J. W., Speert, D. P., Govan, J. R., Taylor, P. & Vandamme, P. (2000). Diagnostically and experimentally useful panel of strains from the Burkholderia cepacia complex. J Clin Microbiol 38, 910–913.[Abstract/Free Full Text]

Marie-Claire, C., Ruffet, E., Beaumont, A. & Roques, B. P. (1999). The prosequence of thermolysin acts as an intramolecular chaperone when expressed in trans with the mature sequence in Escherichia coli. J Mol Biol 285, 1911–1915.[CrossRef][Medline]

McIver, K., Kessler, E. & Ohman, D. E. (1991). Substitution of active-site His-223 in Pseudomonas aeruginosa elastase and expression of the mutated lasB alleles in Escherichia coli show evidence for autoproteolytic processing of proelastase. J Bacteriol 173, 7781–7789.[Medline]

McIver, K. S., Kessler, E., Olson, J. C. & Ohman, D. E. (1995). The elastase propeptide functions as an intramolecular chaperone required for elastase activity and secretion in Pseudomonas aeruginosa. Mol Microbiol 18, 877–889.[CrossRef][Medline]

McKevitt, A. I. & Woods, D. E. (1984). Characterization of Pseudomonas cepacia isolates from patients with cystic fibrosis. J Clin Microbiol 19, 291–293.[Medline]

McKevitt, A. I., Bajaksouzian, S., Klinger, J. D. & Woods, D. E. (1989). Purification and characterization of an extracellular protease from Pseudomonas cepacia. Infect Immun 57, 771–778.[Medline]

Mohr, C. D., Tomich, M. & Herfst, C. A. (2001). Cellular aspects of Burkholderia cepacia infection. Microbes Infect 3, 425–435.[CrossRef][Medline]

Morihara, K. (1995). Pseudolysin and other pathogen endopeptidases of thermolysin family. Methods Enzymol 248, 242–253.[Medline]

Nakazawa, T. & Abe, M. (1996). Pathogenesis of Burkholderia cepacia and export of protease by the general secretory pathway involving disulfide bond formation in the periplasm. In Molecular Biology of Pseudomonads. Edited by T. Nakazawa. Washington, DC: American Society for Microbiology.

Nakazawa, T., Yamada, Y. & Ishibashi, M. (1987). Characterization of hemolysin in extracellular products of Pseudomonas cepacia. J Clin Microbiol 25, 195–198.[Medline]

Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10, 1–6.[Abstract]

O'Donohue, M. J. & Beaumont, A. (1996). The roles of the prosequence of thermolysin in enzyme inhibition and folding in vitro. J Biol Chem 271, 26477–26481.[Abstract/Free Full Text]

O'Donohue, M. J., Roques, B. P. & Beaumont, A. (1994). Cloning and expression in Bacillus subtilis of the npr gene from Bacillus thermoproteolyticus Rokko coding for the thermostable metalloprotease thermolysin. Biochem J 300, 599–603.[Medline]

Ohman, D. E., Sadoff, J. C. & Iglewski, B. H. (1980). Toxin A-deficient mutants of Pseudomonas aeruginosa PA103: isolation and characterization. Infect Immun 28, 899–908.[Medline]

Pugsley, A. P. (1993). The complete general secretory pathway in gram-negative bacteria. Microbiol Rev 57, 50–108.[Medline]

Rawlings, N. D. & Barrett, A. J. (1995). Evolutionary families of metallopeptidases. Methods Enzymol 248, 183–228.[Medline]

Rinderknecht, H., Geokas, M. C., Silverman, P. & Haverback, B. J. (1968). A new ultrasensitive method for the determination of proteolytic activity. Clin Chim Acta 21, 197–203.[CrossRef][Medline]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Schweizer, H. P., Klassen, T. & Hoang, T. (1996). Improved methods for gene analysis and expression in Pseudomonas spp. In Molecular Biology of Pseudomonads, pp. 229–237. Edited by K. F. T. Nakazawa, D. Haas & S. Silver. Washington, DC: American Society for Microbiology.

Sokol, P. A., Ohman, D. E. & Iglewski, B. H. (1979). A more sensitive plate assay for detection of protease production by Pseudomonas aeruginosa. J Clin Microbiol 9, 538–540.[Medline]

Sokol, P. A., Kooi, C., Hodges, R. S., Cachia, P. & Woods, D. E. (2000). Immunization with a Pseudomonas aeruginosa elastase peptide reduces severity of experimental lung infections due to P. aeruginosa or Burkholderia cepacia. J Infect Dis 181, 1682–1692.[CrossRef][Medline]

Speert, D. P., Henry, D., Vandamme, P., Corey, M. & Mahenthiralingam, E. (2002). Epidemiology of Burkholderia cepacia complex in patients with cystic fibrosis, Canada. Emerg Infect Dis 8, 181–187.[Medline]

Teufel, P. & Gotz, F. (1993). Characterization of an extracellular metalloprotease with elastase activity from Staphylococcus epidermidis. J Bacteriol 175, 4218–4224.[Abstract]

Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76, 4350–4354.[Abstract]

Vandamme, P., Holmes, B., Vancanneyt, M. & 8 other authors (1997). Occurrence of multiple genomovars of Burkholderia cepacia in cystic fibrosis patients and proposal of Burkholderia multivorans sp. nov. Int J Syst Bacteriol 47, 1188–1200.[Abstract/Free Full Text]

Vermis, K., Coenye, T., Mahenthiralingam, E., Nelis, H. J. & Vandamme, P. (2002). Evaluation of species-specific recA-based PCR tests for genomovar level identification within the Burkholderia cepacia complex. J Med Microbiol 51, 937–940.[Abstract/Free Full Text]

West, S. E., Schweizer, H. P., Dall, C., Sample, A. K. & Runyen-Janecky, L. J. (1994). Construction of improved EscherichiaPseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 148, 81–86.[CrossRef][Medline]

Woods, D. (1984). Oligonucleotide screening of cDNA libraries. Focus 6, 1–2.

Yabuuchi, E., Kosako, Y., Oyaizu, H., Yano, I., Hotta, H., Hashimoto, Y., Ezaki, T. & Arakawa, M. (1992). Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov. Microbiol Immunol 36, 1251–1275.[Medline]

Received 17 January 2003; revised 14 April 2003; accepted 25 April 2003.