| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Correspondence |
Division of Medical Microbiology and Genitourinary Medicine, University of Liverpool, Daulby Street, Liverpool L69 3GA, UK
Correspondence
Craig Winstanley
(C.Winstanley{at}liv.ac.uk)
The Burkholderia cepacia complex (BCC) consists of a group of related genospecies that include important opportunistic pathogens, especially in relation to cystic fibrosis (CF) (Mahenthiralingam et al., 2005). The most common and pathogenically important member of the BCC is Burkholderia cenocepacia, which along with Burkholderia multivorans accounts for the vast majority of BCC isolates from CF patients.
Type III secretion (TTS) systems are implicated in the pathogenicity of a number of Gram-negative bacterial pathogens (Winstanley & Hart, 2001). Previously we cloned and sequenced the TTS gene cluster from B. cenocepacia ET12 lineage strain J2315 (GenBank AY028431). Furthermore we identified and sequenced TTS gene regions for strains representing genomovar I (B. cepacia) and B. multivorans (strain E243). Whilst genomovar I lacks TTS structural genes, it contains flanking genes, suggesting that a deletion has occurred. B. multivorans contains a gene cluster equivalent to that of B. cenocepacia J2315 (Glendinning et al., 2004). It has been demonstrated that a TTS mutant of B. cenocepacia J2315 is attenuated for virulence in a murine model of infection (Tomich et al., 2003), suggesting a role for TTS in pathogenicity. However, the precise role of TTS remains unclear, and the secreted effectors themselves have yet to be identified.
The Caenorhabditis elegans infection model has been used extensively to study bacterial virulence and host–pathogen interactions (Sifri et al., 2005; Alegado et al., 2003). The model has been tested with various members of the BCC (Cardona et al., 2005), but has been used most effectively with B. cenocepacia strain H111 to demonstrate the importance of quorum-sensing (QS) regulation in virulence (Huber et al., 2004). As an alternative to mammalian animal models, we investigated the potential use of a C. elegans killing assay to study the role of TTS in B. cenocepacia. Using the pKNOCK vector system (Alexeyev, 1999), TTS knock-out mutants were constructed in two different strains of B. cenocepacia. A bscsN mutant of B. cenocepacia H111, analogous to the mutant used in a previous study (Tomich et al., 2003), was constructed by cloning an internal region of the bcscN gene (encoding ATPase) into pKNOCK-Km. The region was PCR-amplified from B. cenocepacia using the oligonucleotide primers 5'-ACCTCTAGAAAGATGATCGACACGCCG-3' (forward primer containing an XbaI restriction site) and 5'-ATCCTGCAGCTGGTACTTCGCGATCAAC-3' (reverse primer containing a PstI restriction site), and cloned into pKNOCK-Km plasmid digested with XbaI and PstI. In addition, a bcscQR-virB1 knock-out mutant (bcscQ mutant) of B. cenocepacia C5424 (ET12 lineage) was constructed using the pKNOCK-Cm vector (Alexeyev, 1999). A 2.5 kb SacII–XhoI fragment of the B. cenocepacia J2315 genome, comprising part of bcscQ, all of bcscR and part of virB1 (Glendinning et al., 2004), was cloned into pKNOCK-Cm. The pKNOCK-Km-bcscN and pKNOCK-Cm-bcscQ constructs were subsequently introduced into B. cenocepacia H111 and B. cenocepacia C5424, respectively, by electroporation. Cells from 5 ml overnight broth cultures were harvested, washed twice in 10 % (v/v) glycerol and subsequently resuspended in 0.5 ml of the same solution. Bacterial suspension (100 µl) was mixed with DNA, transferred to 0.2 cm electrocuvettes, and subjected to a pulse of 2.5 kV (25 µF capacitance and 400 
resistance) using a GenePulser II apparatus (Bio-Rad). After electroporation and the addition of 0.9 ml L broth, cells were incubated at 37 °C for 2 h prior to plating onto media containing kanamycin (50 µg ml–1; for pKNOCK-Km-bcscN) or chloramphenicol (50 µg ml–1; for pKNOCK-Cm-bcscQ). Genomic DNA was isolated from putative mutants using a Wizard Genomic DNA Purification kit (Promega). The position of the insertion was determined by cloning out the pKNOCK-Km/Cm regions into the KpnI site of pUC19 and sequencing using vector and internal primers. Sequencing confirmed that the pKNOCK construct had inserted into bcscN and bcscQ respectively. TTS mutant and wild-type strains were phenotypically similar with respect to growth rate in Luria broth and a series of 20 biochemical tests using the API ZYM kit (bioMérieux).
B. cenocepacia wild-type and two TTS mutant strains were tested in small-scale agar-plate assays for their ability to kill C. elegans, essentially using the method described by Köthe et al. (2003). Bacterial lawns of the test strains and an E. coli control strain were prepared on nematode growth (NG) media to test for infective (slow-killing) effects. Small-scale assays were used in multi-segmented Petri dishes, in which each test was performed in an area of 1.5 cm2. After bacterial growth for 24 h at 37 °C, L4 larvae or adult worms (
50 and 10 for the bcscQ and bcscN mutants, respectively) were placed on the bacterial lawn. The plates were incubated at 23 °C and scored for live worms at intervals using a microscope set at x50 magnification. Results were obtained from three independent experiments (or two for the bcscN mutant), each experiment testing the ability of the wild-type strain, mutant strain and E. coli OP50 to kill C. elegans.
A comparison of the bcscN TTS mutant with B. cenocepacia H111 wild-type in the C. elegans slow-killing model of infection at time intervals of 24, 48 and 72 h post-infection indicated a significant difference (P <0.05) at the 48 and 72 h time-points (Fig. 1a
). When the bcscQ mutant was compared with wild-type B. cenocepacia C5424, at time intervals of 6, 18, 24 and 42 h, a significant difference (P <0.05) was only observed at the 18 h time-point (Fig. 1b). At the 24 and 72 h time-points, all worms had been killed by both wild-type and mutant strains, whereas at the 6 h time-point, less than 10 % of worms had been killed by either wild-type or mutant. Our findings demonstrate that TTS mutants kill C. elegans more slowly than wild-type strains, but that the effect can only be observed at particular time-points that can vary between B. cenocepacia strains. Cardona et al. (2005) have reported significant variation between the C. elegans-killing activities of members of the BCC. Interestingly, strain C5424 was included in the study, but scored as non-pathogenic to C. elegans. Both in our study and the study of Cardona et al. (2005), B. cenocepacia C5424 was obtained as part of an experimental strain panel deemed representative of the BCC (Mahenthiralingam et al., 2000). In our hands, B. cenocepacia C5424 was effective at killing C. elegans. The discrepancy may indicate that one or other version of strain C5424 has mutated.
|
We would like to acknowledge funding from the UK Cystic Fibrosis Trust. In addition, we are grateful to Dr Mikhail Alexeyev (University of South Alabama) for providing the pKNOCK vectors.
REFERENCES
Alegado, R. A., Campbell, M. C., Chen, W. C., Slutz, S. S. & Tan, M. W. (2003). Characterization of mediators of microbial virulence and innate immunity using the Caenorhabditis elegans host–pathogen model. Cell Microbiol 5, 435–444.[CrossRef][Medline]
Alexeyev, M. F. (1999). The pKNOCK series of broad-host-range mobilizable suicide vectors for gene knockout and targeted DNA insertion into the chromosome of Gram-negative bacteria. Biotechniques 26, 824–828.[Medline]
Cardona, S. T., Wopperer, J., Eberl, L. & Valvano, M. A. (2005). Diverse pathogenicity of Burkholderia cepacia complex strains in the Caenorhabditis elegans host model. FEMS Microbiol Lett 250, 97–104.[Medline]
Glendinning, K. J., Parsons, Y. N., Duangsonk, K., Hales, B. A., Humphreys, D., Hart, C. A. & Winstanley, C. (2004). Sequence divergence in type III secretion gene clusters of the Burkholderia cepacia complex. FEMS Microbiol Lett 235, 229–235.[Medline]
Gravato-Nobre, M. J. & Hodgkin, J. (2005). Caenorhabditis elegans as a model for innate immunity to pathogens. Cell Microbiol 7, 741–751.[CrossRef][Medline]
Huber, B., Feldmann, F., Köthe, M., Vandamme, P., Wopperer, J., Riedel, K. & Eberl, L. (2004). Identification of a novel virulence factor in Burkholderia cenocepacia H111 required for efficient slow killing of Caenorhabditis elegans. Infect Immun 72, 7220–7230.
Köthe, M., Antl, M., Huber, B., Stoecker, K., Ebrecht, D., Steinmetz, I. & Eberl, L. (2003). Killing of Caenorhabditis elegans by Burkholderia cepacia is controlled by the cep quorum-sensing system. Cell Microbiol 5, 343–351.[CrossRef][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.
Mahenthiralingam, E., Urban, T. A. & Goldberg, J. B. (2005). The multifarious, multireplicon Burkholderia cepacia complex. Nat Rev Microbiol 3, 144–156.[CrossRef][Medline]
Sifri, C. D., Begun, J. & Ausubel, F. M. (2005). The worm has turned – microbial virulence modeled in Caenorhabditis elegans. Trends Microbiol 13, 119–127.[CrossRef][Medline]
Tomich, M., Griffith, A., Herfst, C. A., Burns, J. L. & Mohr, C. D. (2003). Attenuated virulence of a Burkholderia cepacia type III secretion mutant in a murine model of infection. Infect Immun 71, 1405–1415.
Winstanley, C. & Hart, C. A. (2001). Type III secretion systems and pathogenicity islands. J Med Microbiol 50, 116–126.
This article has been cited by other articles:
|
|
 |
|
  S. Uehlinger, S. Schwager, S. P. Bernier, K. Riedel, D. T. Nguyen, P. A. Sokol, and L. Eberl Identification of Specific and Universal Virulence Factors in Burkholderia cenocepacia Strains by Using Multiple Infection Hosts Infect. Immun., September 1, 2009; 77(9): 4102 - 4110. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. McClean and M. Callaghan Burkholderia cepacia complex: epithelial cell-pathogen confrontations and potential for therapeutic intervention J. Med. Microbiol., January 1, 2009; 58(1): 1 - 12. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. T. G. Holden, H. M. B. Seth-Smith, L. C. Crossman, M. Sebaihia, S. D. Bentley, A. M. Cerdeno-Tarraga, N. R. Thomson, N. Bason, M. A. Quail, S. Sharp, et al. The Genome of Burkholderia cenocepacia J2315, an Epidemic Pathogen of Cystic Fibrosis Patients J. Bacteriol., January 1, 2009; 191(1): 261 - 277. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. D. Seed and J. J. Dennis Development of Galleria mellonella as an Alternative Infection Model for the Burkholderia cepacia Complex Infect. Immun., March 1, 2008; 76(3): 1267 - 1275. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | J MED MICROBIOL | MICROBIOLOGY | J GEN VIROL | ALL SGM JOURNALS |
