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Previous Article | Next Article 
Infection and Immunity, March 2003, p. 1405-1415, Vol. 71, No. 3
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.3.1405-1415.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455,1 Division of Pediatric Infectious Disease, Children's Hospital and Regional Medical Center, Seattle, Washington 981052
Received 10 July 2002/ Returned for modification 23 September 2002/ Accepted 3 December 2002
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B. cepacia is actually a complex of bacteria belonging to at least seven genomic species, or genomovars (57). Four of these genomovars were recently designated distinct species: Burkholderia multivorans (genomovar II), Burkholderia stabilis (genomovar IV), Burkholderia vietnamiensis (genomovar V), and Burkholderia ambifaria (genomovar VI). Although strains representing many of the known genomovars of the B. cepacia complex have been associated with opportunistic infections in humans, strains belonging to genomovar III have been most commonly associated with severe and chronic infections in CF patients (32, 57).
Investigation into the pathogenesis of B. cepacia has provided growing evidence that the organism possesses mechanisms for adherence (42, 46), invasion (3, 33, 56), intracellular survival (33, 41), and modulation of the host immune response (22, 36, 37, 52). B. cepacia has been shown to adhere to human mucins and respiratory epithelial cells, and this adherence may promote colonization of the lungs (46, 47). Invasion and intracellular survival have been demonstrated by use of cultured respiratory epithelial cells and macrophages (3, 33, 41), and the recent examination of lung explants from infected CF patients revealed the presence of B. cepacia intracellularly in surface airway epithelial cells and luminal macrophages (43). The ability of B. cepacia exoproducts, including lipopolysaccharide, to elicit cytokine release from lung epithelial cells and human monocytes may also contribute to infection, through both the induction and the modulation of the host proinflammatory response (22, 37, 51, 52). Despite these new insights, the specific virulence determinants and corresponding genetic elements required for B. cepacia infection are largely unknown.
A wide range of gram-negative bacterial pathogens utilize a conserved secretory system, termed type III, to deliver virulence proteins directly into host cells (12). The type III system-dependent delivery of virulence proteins, referred to as effector proteins, into host cells has been associated with (i) invasion by Salmonella enterica serovar Typhimurium and Shigella flexneri (9, 34), (ii) lesion formation by enteropathogenic Escherichia coli (24), (iii) cytotoxicity caused by P. aeruginosa (16, 19), (iv) evasion of phagocytosis and the host immune response by Yersinia spp. and Bordetella bronchiseptica (11, 61), and (v) intracellular survival in S. enterica serovar Typhimurium (6, 20). Type III secretion systems are also utilized by phytopathogens to cause disease on susceptible plants, as well as to elicit the so-called hypersensitive response in resistant plants (1). Delivery of the bacterial type III effector proteins into host cells requires a secretory apparatus comprised of approximately 10 highly conserved proteins (21). This core secretory apparatus is also found in the flagellar type III secretion system, utilized for the export and assembly of the structural components of the bacterial flagellum (31).
In this study, we identified genes encoding core components of a type III secretion system in B. cepacia strain J2315, a clinical isolate associated with epidemic outbreaks and mortality in CF patients. One of these genes, designated bscN (Burkholderia secretion protein N), encodes a new member of a family of ATP-binding proteins that likely function in the generation of energy driving effector protein secretion. To examine the role of B. cepacia type III secretion in virulence, a defined null mutation was generated in bscN, and the null strain and wild-type parent strain were compared by use of a murine model of B. cepacia infection.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]dCTP (Amersham Pharmacia Biotech) by the random-primer method. Recombinant plasmids were introduced into E. coli and B. cepacia strain J2315 by electroporation with a Gene Pulser II (Bio-Rad). DNA sequencing. Nucleotide sequencing was performed at the Advanced Genetic Analysis Center at the University of Minnesota by using the dideoxy chain termination method and an ABI 1371A DNA sequencer (Applied Biosystems). The oligonucleotide primers used for sequencing were standard forward and reverse (T3 and T7) pBluescript primers or custom oligonucleotides synthesized at Integrated DNA Technologies. The nucleotide sequence was determined on both strands. Double-stranded sequences were aligned and assembled by using the EditSeq and SeqMan components of a demonstration version of the Lasergene sequence analysis software package (DNASTAR Inc.). Nucleotide and amino acid sequence searches and analyses were done with the BLASTX and BLASTP programs from the National Center for Biotechnology Information.
Identification of type III secretion genes in B. cepacia strain J2315. Degenerate primers for regions conserved in the Yersinia enterocolitica YscN protein (59) and its homologs in other bacteria were used in PCRs with genomic DNA from B. cepacia strain J2315. The degenerate primers used were Ysc1 (5'-GARMGNGGNMGNGARGT-3') and Ysc2 (5'-TRTGNCCRTCNARDAT-3'), where R is A or G; M is A or C; N is A, C, G, or T; and D is A, G, or T. After 35 cycles consisting of incubations at 95°C for 1 min, 40°C for 1 min, and 72°C for 1 min, the amplified products were gel purified, cloned into TA cloning vector pCRII (Invitrogen), and sequenced. One of the cloned PCR products (PCR1A) contained a partial open reading frame (ORF) that encoded a product with predicted amino acid sequence homology to YscN and its homologs. PCR1A was used to probe restriction digests of J2315 genomic DNA, and a cross-hybridizing 1.7-kb PstI fragment was identified and subcloned into pBluescript SK II(+), generating plasmid pCM20. The 1.7-kb fragment contains the entire bscN gene. To clone the regions flanking bscN, the 1.7-kb fragment in plasmid pCM21 was used to probe a cosmid library of B. cepacia strain J2315 (56). Subclones of cross-hybridizing cosmids were generated, and the DNA sequence of the B. cepacia type III secretion locus shown in Fig. 1 was determined on both strands.
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Quantitative invasion and adherence assays. Invasion and adherence assays with cultured A549 respiratory epithelial cells were performed as described previously (56). All quantitative assays were performed by using triplicate wells with two samplings per well.
Statistical analysis. The statistical significance of the observed differences in the mean log CFUs recovered from the animal infection experiments as well as the mean adherence and invasion frequencies in the A549 cell invasion and adherence assays was determined by calculating P values with the two-tailed Student t test for unpaired data sets.
Nucleotide sequence accession number. The sequence of the B. cepacia type III secretion locus determined in this study has been deposited in GenBank under accession number AY166598.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PCR1A, with translated amino acid sequence homology to YscN and its homologs, was used to probe restriction enzyme-digested chromosomal DNA of B. cepacia strain J2315. A 1.7-kb cross-hybridizing PstI fragment was identified and cloned. The PstI fragment carries a single ORF whose product has deduced amino acid sequence homology across its entire length to YscN (56% amino acid identity). A portion of the cloned region was identical in DNA sequence to the entire amplified product PCR1A, indicating that the corresponding gene had been cloned. We designated the gene bscN (Burkholderia secretion protein N), in keeping with the currently accepted nomenclature and the homology of its predicted product to known type III secretion ATP-binding proteins. The bscN gene encodes a predicted 52-kDa protein containing two conserved nucleotide-binding motifs, designated Walker boxes (58), suggesting that it can bind and hydrolyze ATP.
To further characterize the bscN locus, the 1.7-kb PstI fragment was used to probe a cosmid library of J2315 genomic DNA (56), and cross-hybridizing cosmids were identified. Subclones of these cosmids were generated and sequenced, revealing a locus containing multiple ORFs encoding products with predicted amino acid sequence homology to known type III secretion proteins. Homologs of the B. cepacia bscJ, bscT, and bscU genes shown in Fig. 1 encode both inner and outer membrane proteins involved in the formation of the channel through which type III effector proteins are secreted (39). Homologs of the bscK and bscL genes encode cytoplasmic components of type III secretion systems (39). The putative products of at least two ORFs (orf1 and orf2 in Fig. 1) within the B. cepacia type III secretion gene cluster have no significant homology to known type III secretion components, suggesting that they may encode functions specific to the type III secretion system in B. cepacia. No ORFs with homology to known type III secretion genes have been identified in the region immediately downstream of bscU (Fig. 1), suggesting that bscU may mark the end of the type III secretion gene cluster.
Generation of a bscN null strain. To characterize further the role of type III secretion in B. cepacia, we generated a defined null mutation in bscN, encoding the putative energy-generating component of the secretory system. In order to insertionally inactivate bscN, an internal fragment of the cloned bscN gene (including both of the putative Walker boxes) was deleted and replaced with a chloramphenicol resistance cassette (Fig. 2A). The disrupted copy of bscN was delivered to B. cepacia strain J2315 via conjugation, and inactivation of the chromosomal copy of bscN was generated by allelic exchange. Southern blot hybridization analysis confirmed that the chromosomal copy of bscN had been disrupted via a double-crossover event (Fig. 2B). The bscN null strain was designated CM56.
We first tested whether the bscN disruption affected motility, since homologs of bscN are known to be involved in flagellar export and assembly. The bscN null strain was not defective in motility, as determined by a swarm agar plate assay (56) or by light microscopy, indicating that the bscN gene product is not involved in flagellar biogenesis in B. cepacia (data not shown). There was also no significant difference in the growth rates of the bscN null strain and the wild-type parent strain in either minimal (M9 broth) or rich (LB) medium.
Attenuated virulence of the bscN mutant. To examine the role of bscN in B. cepacia virulence, wild-type strain J2315 and type III secretion mutant strain CM56 were examined in a mouse agar bead model of infection. In the initial experiment with groups of three mice, a significant difference was seen between the number of CFU recovered from animals infected with wild-type strain J2315 and the number of CFU recovered from animals infected with strain CM56. An average of 3 x 105 CFU were recovered from the lungs of mice infected with wild-type B. cepacia, compared to an average of less than 5 x 102 CFU recovered from the lungs of mice infected with the bscN null strain (Fig. 3). A comparison of bacterial counts in the spleens of infected animals revealed a similar trend, with significantly fewer CFU recovered from mice infected with the type III secretion mutant strain CM56 than from mice infected with wild-type strain J2315 (Fig. 3).
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To confirm the findings of the mouse infection studies, we repeated the experiments by using groups of five animals and obtained similar results (data not shown). We also examined the lungs and spleens of infected animals for bacteria at 7 days postinfection. All of the mice infected with the bscN null strain had cleared the bacteria from both the lungs and the spleen. In contrast, none of the mice infected with the wild-type strain had completely cleared the bacteria from either the lungs or the spleen. Bacteria were also recoverable at 7 days from the lungs of all of the mice infected with the complemented strain, and only one mouse had cleared the complemented strain from the spleen. Recovery at 7 days postinfection, however, was variable and significantly lower than that at 72 h, suggesting that the bacteria were being cleared by the animals. Together, the data from the mouse infection studies support a role for type III secretion in the virulence of B. cepacia.
Histopathological analysis. In order to determine the degree of pathologic changes and inflammatory responses associated with B. cepacia infection, lung sections from mice infected with either wild-type strain J2315 or the bscN null strain were examined for histopathological changes. The examination of lung sections from mice infected with wild-type strain J2315 revealed an extensive inflammatory cell infiltrate, comprised predominantly of macrophages and neutrophils (Fig. 4A and B). Other changes, including desquamated airway epithelium and interstitial inflammation, were also observed. In contrast, the pathologic changes in lung sections from mice infected with bscN mutant strain CM56 appeared less pronounced, with reduced parynchymal involvement and inflammatory cell infiltrate, as well as a more regular appearance of ciliated airway epithelial cells (Fig. 4C and D). The histopathological changes in lung sections from mice infected with complemented strain CM62 were similar to those in lung sections from mice infected with wild-type strain J2315 (Fig. 4E and F).
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DISCUSSION |
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At 3 days postinfection, significantly fewer bacteria were recovered from the lungs and spleens of mice infected with the bscN mutant strain than from those of mice infected with the parent strain. These findings suggest that bacteria lacking bscN are either less likely to establish infection or more likely to be cleared from infected organs. One hypothesis to explain the attenuated virulence of the bscN null strain is that type III secretion plays a role in modulation of the host immune response, allowing B. cepacia to evade host defense mechanisms and establish persistent infection. Histopathological changes in the lungs of mice infected with wild-type B. cepacia strain J2315 were characterized by an extensive inflammatory cell infiltrate, comprised predominantly of macrophages and neutrophils. Similar findings have been reported for B. cepacia in other animal models of infection (4, 45, 54). Using a mouse model of CF, Sajjan et al. (45) recently reported that B. cepacia infection was accompanied by infiltration of a mixed inflammatory cell population of macrophages and neutrophils into the lungs. Interestingly, however, the infiltrating immune cells appeared to be largely inactive, as determined by the reduced oxidant production and cell surface expression of the ß2 integrin CD11b (45). It is conceivable that the inactivation of host macrophages and neutrophils through the secretion of type III effector molecules impairs the normal clearance of bacteria, resulting in the establishment of persistent B. cepacia infection. The significant reduction in the recovery of bacteria from mice infected with the type III secretion mutant may reflect improved clearance by fully activated immune cells in the lungs.
Invasion assays with cultured A549 respiratory epithelial cells did not reveal a significant difference between the wild-type and type III secretion mutant strains in their abilities to invade cultured respiratory epithelial cells. We cannot, however, completely discount a possible role for type III secretion in B. cepacia invasion of host tissues in vivo. It is possible that B. cepacia entry into cultured respiratory epithelial cells involves mechanisms distinct from those required for invasion in vivo or that cell culture conditions may not adequately mimic the lung environment, where additional host-specific signals may be required for full induction of the type III secretion system. An invasion defect due to disruption of the type III secretion system could explain the reduced extrapulmonary spread of B. cepacia to the spleens of mice infected with the bscN null strain. Primary cell cultures were recently used to investigate the invasion mechanisms of B. cepacia (42, 50) and may allow further dissection of the role of type III secretion in invasion under conditions more closely mimicking the in vivo environment.
Based on the known role of bscN homologs in the secretion of effector proteins by other bacterial pathogens, it is reasonable to suggest that the reduced virulence of the bscN null strain is due to a disruption in the delivery of B. cepacia virulence determinants into host cells. While the results of the present study clearly implicate type III secretion in the virulence of B. cepacia, the identity of the putative secreted effector(s) is unknown at this time. Unlike other components of the secretory apparatus, the effector proteins of bacterial type III secretion systems often share little or no homology, likely reflecting the diversity of their host cell targets (12). At least two ORFs that were identified within the type III secretion gene cluster appeared to be specific to B. cepacia, since they have no known homologs in other systems. These ORFs might encode effector proteins exported by the B. cepacia type III translocon. Alternatively, the genes encoding the type III effector proteins might not be linked to the genes encoding other components of the secretory apparatus, as has been reported for several type III effector genes in S. enterica serovar Typhimurium and B. bronchiseptica (35, 61).
We have compared the extracellular protein profiles of the B. cepacia wild-type and bscN mutant strains grown under several conditions known to stimulate type III secretion in other bacteria; these conditions include growth in minimal and tissue culture media, growth in the presence of Congo red, and growth under calcium limitation. Thus far, we have not detected differences between the two strains (data not shown). It is possible that the laboratory conditions tested do not adequately reproduce the environment in the animal model and human host. Similar difficulties have been encountered in the identification of substrates for the type III secretion systems of the respiratory pathogen Bordetella pertussis and the plant pathogen Xanthomonas campestris (15, 27). In the latter, identification of the effector proteins required specific inducing conditions as well as mutagenesis of a key regulatory gene, allowing for constitutive expression of the type III secretion genes (40). It is likely that similar approaches will be necessary to identify the substrate(s) for the B. cepacia type III secretion system.
We have identified a locus of B. cepacia type III secretion genes encoding predicted structural as well as energy-generating components of the secretory system. The entire arrangement of the B. cepacia type III secretion genes is not found in any other known bacterial type III secretion system, although the gene order bscJKLN is conserved in the type III secretion systems of the closely related respiratory pathogens B. bronchiseptica and B. pertussis (15, 60). Notably missing from the B. cepacia type III secretion gene cluster are genes encoding homologs of the Yersinia YscQ and YscR proteins. Members of these protein families are conserved in all known type III secretion systems, and their corresponding genes are typically found within clusters encoding homologs of the bsc genes described in this report. Parsons and coworkers (38) recently identified genes encoding predicted proteins with homology to the archetypal YscQ and YscR proteins in B. cepacia strain J2315. The corresponding genes, designated bcscQ and bcscR in reference 38, appear to be located outside of the gene cluster described in our study, suggesting a genetic organization distinct from those of all other known type III secretion systems. Although the B. cepacia bcscQ and bcscR genes have not been characterized, the possible presence of a YscQ homolog in B. cepacia is of particular interest, since SpaO and Spa33, the YscQ homologs in Salmonella spp. and Shigella spp., respectively, have been shown to be secreted via their respective type III secretion systems (10, 49).
It is worth noting that the B. cepacia type III secretion genes that we have identified are homologous not only to type III secretion components in other human pathogens but also to type III secretion components in several plant pathogens, including X. campestris and Ralstonia solanacearum. Recent studies have provided evidence that type III secretion genes may be widely distributed in the B. cepacia complex, including clinical as well as environmental isolates (38). Given the known role of B. cepacia as a phytopathogen (2), it is tempting to speculate that type III secretion can play a role in the establishment of commensal or pathogenic relationships between B. cepacia and plants and is also utilized during the course of infection in humans.
In summary, we have identified components of a type III secretion system in B. cepacia and have provided evidence that type III secretion is an important virulence determinant for this organism. Future studies will focus on further defining the role of B. cepacia type III secretion both in vitro and in vivo, including the identification of type III secretion molecules and characterization of their specific effects on host cells.
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ACKNOWLEDGMENTS |
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This work was supported by Minnesota Medical Foundation grant 13700, by grant MOHR0010 from the Cystic Fibrosis Foundation to C.D.M., and by NIH grant RO1 HL65898 to J.L.B.
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FOOTNOTES |
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