Departments of Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, Ontario, Canada1
Department of Pediatrics and Communicable Diseases, University of Michigan, 8323 MSRB III, Box 0646, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, USA2
Author for correspondence: John J. LiPuma. Tel: +1 734 936 9767. Fax: +1 734 764 4279. e-mail: jlipuma{at}umich.edu
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ABSTRACT |
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Keywords: cblA gene, bacterial adherence, cystic fibrosis
Abbreviations: CF, cystic fibrosis; CK13, cytokeratin 13
a The GenBank accession numbers for the complete cblA nucleotide sequences for the isolates listed in Table 1 are AF455151AF455162.
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INTRODUCTION |
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Certain B. cepacia complex strains have attracted considerable attention because of their potential commercial use as biopesticidal and bioremedial agents (LiPuma & Mahenthiralingam, 1999 ; Parke & Gurian-Sherman, 2001
). At the same time, there has been a growing appreciation of the role B. cepacia complex species play as opportunistic human pathogens. Although generally not pathogenic for healthy humans, these species are capable of causing severe infection in certain vulnerable populations, particularly persons with cystic fibrosis (CF), the most common lethal genetic disease among whites (LiPuma, 1998
). In CF, respiratory tract infection with B. cepacia complex is associated with increased rates of morbidity and mortality. In fact, following infection, a significant proportion of CF patients will die, with rapidly progressive necrotizing pneumonia and sepsis.
The bacterial virulence factors and pathogenic mechanisms involved in human infection due to B. cepacia complex remain to be elucidated. Nevertheless, it is clear that some specific strains are more frequently recovered from CF patients than are others. One such epidemic strain, the ET12 lineage, is a genomovar III strain that predominates among patients in Ontario, Canada, and is found in about one-third of B. cepacia-infected patients in the United Kingdom (Pitt et al., 1996 ; Sun et al., 1995
). This strain expresses a distinctive pilus (the cblA-encoded cable pilus) and an associated adhesin that mediate bacterial binding to CF respiratory epithelium (Sajjan et al., 1995
, 2000a
, b
). The cognate receptor for cable-pili expressing B. cepacia, cytokeratin 13 (CK13), is a 55 kDa protein that is enriched in CF epithelia (Sajjan et al., 2000a
). Because the expression of cable pili may provide an important pathogenic mechanism contributing to inter-patient spread of B. cepacia complex, differential infection control measures based on the presence (or absence) of cblA have been proposed (Clode et al., 2000
).
In a recent study only one of 606 B. cepacia complex-infected US CF patients harboured a cblA-containing ET12 strain (LiPuma et al., 2001 ). Analyses employing cblA-specific dot-blot and PCR assays, and genotyping by PFGE indicated, however, that nine additional patients were infected with non-ET12 strains also positive for cblA sequences. An additional cblA-positive isolate was identified from stream sediment. Interestingly, all 10 of these cblA-positive, non-ET12 isolates were B. cepacia genomovar I, a species not frequently found in CF sputum culture (LiPuma et al., 2001
). In this study we investigated the expression of cable pili by these strains and characterized the cblA gene variants found therein.
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METHODS |
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ATCC 25416 (American Type Culture Collection, Manassas, VA, USA) is a cblA-negative genomovar I strain and was used as a negative control.
Bacterial growth conditions.
For DNA sequence determination and PFGE analyses, bacteria from frozen stock were recovered on MuellerHinton agar (Difco) after incubation at 35 °C for 2448 h. For Southern and Western blot analyses, and for electron microscopy, bacteria from frozen stock were grown on brain-heart infusion agar (Becton Dickinson). For CK13-binding assay, bacteria were grown in tryptic soy broth (Difco), as described by Sajjan et al. (2000b ).
cblA and upstream DNA sequence analysis.
By using cblA-specific PCR primers and reaction conditions previously described (Sajjan et al., 1995 ) DNA fragments of the predicted size were amplified from the 11 study isolates (Table 1
) as well as from the positive control strains BC7 and J2315. A second PCR assay employing forward primer 5'-GAGCTCGAATTCGATATCGAGTGG-3' and reverse primer 5'-CTTGTCGTTCGTGAAGATCTTCGTG-3' was designed to amplify DNA sequences immediately upstream of the cblA start codon. This assay was carried out in a 50 µl final volume that included 1·5 mM MgCl2, 0·8 mM dNTPs, 0·4 mM of each primer, 100 ng template DNA and 1 U Taq DNA polymerase. PCR conditions were the same as for the cblA PCR except that an annealing temperature of 62 °C was used. Nucleotide sequences of cblA and the upstream segments were determined by using an ABI PRISM model 3700 DNA sequencer (Perkin-Elmer Applied Biosystems). Sequence analyses were performed using EditSeq and MegAlign software (DNAStar). The GenBank accession numbers for the complete cblA nucleotide sequences for the isolates listed in Table 1
are AF455151AF455162.
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Southern blot analysis.
Chromosomal DNA (10 µg) from each isolate was digested with EcoRI and subjected to electrophoresis in 0·8% agarose. The resulting DNA fragments were transferred to a nylon membrane, fixed by UV cross-linking and hybridized with a digoxigenin-labelled cblA probe as described by Sajjan et al. (1995) . The bound probe was detected by using anti-digoxigenin antibody and a chemiluminescent substrate (CPD*, Roche Molecular Biochemicals).
Production of antibody to cable pilin protein.
Cable pilin subunit protein was prepared from B. cepacia strain BC7 as described previously (Sajjan et al., 1995 ). Two New Zealand White rabbits were injected subcutaneously with this preparation (10 µg), mixed with TiterMax classic adjuvant (Sigma-Aldrich), on days 1, 15, 30 and 45. Polyclonal antiserum was obtained 2 weeks after the last injection.
Reactivity to anti-cable pilus antibodies.
Whole-cell extracts of B. cepacia isolates were prepared as described previously (Sajjan & Forstner, 1992 ). Bacterial surface proteins were isolated after incubating bacteria at 60 °C for 15 min as described by Cravioto et al. (1982)
. Whole-cell extracts or surface proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 3% BSA for 2 h at room temperature, incubated with anti-cable pilin antiserum (1:1000 diluted) overnight at 4 °C and washed. Bound antibody was detected using anti-rabbit IgG conjugated with alkaline phosphatase (Bio-Rad), and a colour substrate (NBT-BCIP, Roche Molecular Biochemicals).
CK13 binding assay.
Binding of B. cepacia to CK13 was determined as described previously (Sajjan et al., 2000a ). In brief, a cytokeratin-rich fraction, isolated from buccal epithelial cells as described by Franke et al. (1981)
, was subjected to electrophoresis and the proteins were transferred to a nitrocellulose membrane. Membranes were blocked with 1% gelatin for 1 h at room temperature, incubated with 35S-labelled B. cepacia (1x109 c.f.u. ml-1) for 90 min at 37 °C, washed to remove unbound bacteria and exposed to X-ray film.
Electron microscopy.
Bacteria were grown on brain-heart infusion agar, lifted onto a Formvar-coated grid, and stained with 1% phosphotungstic acid for 30 s. Excess stain was removed by blotting with moist filter paper and the grid was allowed to air dry. Samples were examined by using a JEOL 1230 electron microscope equipped with a CCD camera.
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RESULTS |
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Although DNA fragments of the predicted size were amplified from all 11 isolates by cblA-specific PCR, only the product from the genomovar III isolate (AU0007) had 100% nucleotide sequence identity with the cblA amplified from the ET12 strains J2315 and BC7. The cblA nucleotide sequences from the 10 genomovar I isolates showed high identity (range 98·2100%) to each other, but lower identity (range 87·888·4%) to the ET12 cblA sequence. cblA sequences from three genomovar I isolates (AU1544, AU1555 and AU1693) shared 100% identity with each other.
To investigate whether differences in cable pili expression (see below) may result from upstream nucleotide sequence variations we amplified a 145 bp segment immediately upstream of the cblA start codon from all isolates. Again, the sequences from the ET12 isolates AU0007, J2315 and BC7 were identical to each other. The sequences from AU1544, AU1555 and AU1693 were also identical to each other and this sequence differed from the remaining seven genomovar I strains by a single nucleotide. The identity between the ET12 sequence and that of isolates AU1544, AU1555 and AU1693 was 96·6%; identity between the ET12 sequence and that of the remaining seven isolates was 95·9%. Although the putative -10 and -35 sequences are identical among all ET12 and genomovar I isolates, the 16 bp intervening region contains 3 bp that differ between the ET12 and the genomovar I isolates (Fig. 1).
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DISCUSSION |
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The factors that account for the frequency of ET12 and other so-called epidemic strains among CF patients are not clear. Few such strains have been characterized in detail (Mahenthiralingam et al., 1997 ) and factors critical to inter-patient spread have yet to be defined. The B. cepacia epidemic strain marker (BCESM), a 1·4 kb sequence encoding an ORF with homology to transcriptional regulatory genes, is found in ET12 and other strains that infect multiple CF patients (Mahenthiralingam et al., 1997
). This sequence is generally absent from strains found only in single patients (i.e. for which there is no evidence of patient-to-patient spread). The role this element may have in contributing to inter-patient transmission is unknown.
Cable pili, another feature of the ET12 lineage, have been better characterized (Sajjan et al., 1995 ; Sun et al., 1995
). These large intertwined peritrichous fibres, together with an associated adhesin, mediate bacterial adherence to respiratory epithelia via binding to CK13, a 55 kDa protein that has increased expression in CF (Sajjan et al., 2000a
). Recent work indicates that adherence mediated by cable pili also contributes to epithelial cell invasion and cytotoxicity during B. cepacia infection in CF (Sajjan et al., 2002
). Thus, expression of cable pili is of critical importance in the pathology of ET12, a strain accounting for significant infection in CF.
The identification of cable pili in ET12 initially raised hopes that screening of B. cepacia isolates for this phenotype (or, more specifically, for the presence of cblA) may allow identification of highly transmissible lineages (Hearst & Elliott, 1995 ). Such screening could, in turn, enable CF centres to selectively apply stringent infection control measures, isolating only those patients harbouring an epidemic strain. Similar recommendations for stratification of infection control based on the presence or absence of cblA have been made more recently (Clode et al., 2000
).
However, such strategies are limited. Although cable pili mediate events important in the pathogenesis of infection by ET12, it is not yet clear whether expression of cable pili confers an enhanced capacity for inter-patient transmission, per se. Other B. cepacia complex lineages implicated in inter-patient spread do not contain cblA sequences (Mahenthiralingam et al., 1997 ). Indeed, PHDC, the genomovar III strain that predominates among CF patients in the mid-Atlantic region of the US, contains neither BCESM nor cblA (Chen et al., 2001
). The B. multivorans strain responsible for a hospital-associated outbreak among CF patients in the UK (Whiteford et al. 1995
) similarly lacks BCESM and cblA (Mahenthiralingam, 2000b
). Thus, neither cblA nor cable pili expression is a sensitive marker of B. cepacia strains that seem to possess an enhanced ability for spread in CF.
The results of the present study also indicate that cblA sequences are not specific for ET12. We identified several non-ET12 isolates positive for cblA by both dot-blot and PCR assays. Variant cblA genes with varying degrees of identity to the ET12 cblA gene were obtained from these isolates by PCR amplification using cblA-directed primers. Although we demonstrated pili expression by the ET12 isolates included in the study, none of the 10 cblA-containing non-ET12 isolates expressed pili under the conditions examined based on functional assays; this was confirmed by electron microscopy. The non-ET12 isolates represented several distinct strains by PFGE analysis, but interestingly all belonged to genomovar I, a species not commonly found in CF (LiPuma et al., 2001 ). Furthermore, in all of these isolates the cblA sequences resided on an approximately 4 kbp EcoRI fragment, suggesting a common chromosomal location.
Others have similarly found cblA sequences among non-ET12 isolates, albeit at a low frequency. Sun et al. (1995) identified a single cblA-positive strain from among non-ET12 isolates recovered from over 100 CF patients. Similar to the isolates described in the present study, the cblA gene nucleotide sequence from this isolate had 88% identity with the ET12 cblA sequence. Although neither microscopic studies nor epithelial cell binding assays were performed, antibody against purified ET12 cable pili did not react with this isolate. Because description of this isolate preceded current knowledge of the taxonomy of the B. cepacia complex, the genomovar of this isolate is not known.
In a larger survey of 627 B. cepacia isolates representing 132 distinct strain types (defined by using randomly amplified polymorphic DNA typing) recovered from both CF and non-CF sources, Mahenthiralingam et al. (1997) detected DNA homologous to cblA in five non-ET12 isolates. Only one of these was from a CF patient; the remaining four were recovered from the environment. cblA sequence analysis and cable pili expression studies were not performed and again, genomovar analysis was not yet available.
More recently, Richardson et al. (2001) identified two non-ET12 strains, of unclear genomovar, containing cblA sequences from among 75 isolates of Burkholderia spp. Although functional assays of pili expression were not performed, the predicted amino acid sequences of these two strains had only 68% and 78% identity with that of the ET12 pilin subunit protein. In contrast, a survey of 117 isolates from CF patients receiving care in 40 hospitals in the UK by Clode et al. (2000)
demonstrated cblA sequences exclusively among ET12 lineage isolates; none of 76 non-ET12 isolates was cblA positive by PCR assay.
The frequency of cblA sequences in B. cepacia residing in the natural environment (or more specifically, among environmental genomovar I strains) is unknown. A more comprehensive survey of such isolates is needed to address this. Nevertheless, our findings suggest the possibility of inter-species transfer of potential virulence elements among B. cepacia complex species. Work is currently under way to assess this.
The reasons for the lack of cable pili expression in the cblA-positive genomovar I isolates identified in this study are not entirely clear. We found minor differences in the DNA upstream of the cblA start codon (specifically in the spacer region between the putative -10 and -35 sequences), and it is possible that this variation may affect transcription initiation. For example, substitution of AT by GC in this spacer region significantly increases the Ka of E. coli RNA polymerase (Auble et al., 1987 ). Perhaps the replacement of C and G at positions -18 and -25, respectively, in the ET12 sequence by T in genomovar I isolates decreases the Ka of RNA polymerase, resulting in a reduced rate of transcription. It is also entirely possible that the differences noted in this promoter region do not account for the lack of pili expression by these isolates. Several other genes are probably involved in cable pili expression. Perhaps nucleotide differences in one or more of these, particularly those residing upstream of cblA, are responsible for the lack of pili expression in these isolates. Ongoing studies are assessing this possibility.
The reasons for the failure of AU0007 to express typical cable pili and the associated adhesin also require elucidation. This genomovar III CF isolate is clearly of the ET12 clonal lineage based on PFGE analysis. It also shares 100% nucleotide identity with the other ET12 isolates analysed (BC7 and J2315) in cblA as well as in the 145 bp segment immediately upstream of the cblA start codon. Nevertheless, it is hyperpiliated with rigid-appearing pili and fails to express cable-pili-associated adhesin based on its lack of binding to CK13 (Sajjan et al., 2000b ); this is markedly different from other ET12 isolates examined to date. It is possible that the reasons for this lie elsewhere in the B. cepacia cbl pil operon; this is the subject of ongoing investigation.
In summary, although efforts to lessen the burden of strict infection control in CF are urgently needed, data from this study and those of others indicate that cblA lacks both the sensitivity and specificity to identify transmissible strains. Until the factors contributing to the apparent proclivity of select B. cepacia complex strains to cause infection in CF are better defined, stratification of infection control measures will be problematic. In the interim, assays to assess cable pili expression (rather than the presence of cblA sequences) may provide better specificity for identification of ET12 isolates. This will be particularly helpful in regions where tracking of ET12 is important in infection control surveillance.
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ACKNOWLEDGEMENTS |
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Received 18 March 2002;
revised 19 June 2002;
accepted 11 July 2002.