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Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, Consejo Nacional de Investigaciones Cientificas y Técnicas, Universidad Nacional de General San Martín, Buenos Aires, Argentina
Received 4 October 2003/ Returned for modification 27 October 2003/ Accepted 15 December 2003
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ABSTRACT |
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INTRODUCTION |
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Brucella, Agrobacterium, and Rhizobium belong, according to 16S rRNA sequences, to the 
-2 subgroup of the Proteobacteria (28). Complete-genome sequencing reveals similarities in transport, metabolic capabilities, and genome structure with these soil- and plant-associated bacteria. Extensive gene synteny between chromosome 1 and the genome of the plant symbiont Mesorhizobium loti emphasizes the similarity between this animal pathogen with plant pathogens and symbionts. A limited repertoire of genes homologous to known bacterial virulence factors were identified (13, 31, 44). Several studies revealed common themes between animal and plant pathogens and endosymbionts. For example the Brucella two-component regulatory system BvrS-BvrR (39) is highly similar to the two-component regulatory systems ChvG-ChvI of Agrobacterium tumefaciens (9) and ExoS-ChvI of Sinorhizobium meliloti (10). These two-component regulatory systems are equivalent to the Salmonella PhoP-PhoQ (40) and the Bordetella bronchiseptica BvgA-BvgS systems (42). These two-component sensory systems are involved in controlling virulence or, in the case of Rhizobium, nodule invasion; in B. abortus bvrS-bvrR, mutants also display reduced invasiveness and virulence (32, 39).
The Brucella virB operon was recently identified (29, 38); it is highly homologous to the A. tumefaciens virB operon. The B. abortus virB10 mutant lost the ability to multiply in HeLa cells and was not recovered from the spleens of infected BALB/c mice (38). The same results were obtained with a Brucella suis virB10 mutant (29), thus demonstrating that in Brucella, as in Agrobacterium, the virB operon is involved in virulence.
A highly conserved B. abortus homologue of the S. meliloti bacA gene, which encodes a putative cytoplasmic membrane transport protein required for symbiosis, was identified (26). The B. abortus bacA mutant shows decreased survival in macrophages and reduced virulence in BALB/c mice (26).
Brucella, like Agrobacterium and Rhizobium, produces cyclic ß-1,2-glucans (45). chvB in A. tumefaciens and ndvB in S. meliloti are the genes coding for the cyclic ß-1,2-glucan synthetase (cgs) (50). The biosynthesis of cyclic ß-1,2-glucan proceeds in Brucella by the same mechanism as in Rhizobium and Agrobacterium (7). Cgs acts as an intermediate during the synthesis of the cyclic ß-1,2-glucan (21). Cyclic glucan is required for effective nodule invasion and symbiotic nitrogen fixation in S. meliloti and for crown gall tumor induction in A. tumefaciens (4). Agrobacterium cyclic ß-1,2-glucan mutants have several altered cell surface properties, including loss of motility due to a defective assembly of flagella and increased sensitivity to certain antibiotics and detergents (4). B. abortus cgs codes for the cyclic ß-1,2-glucan synthetase (21). B. abortus cgs mutants showed reduced survival in BALB/c mouse spleen tissues and impeded intracellular multiplication, indicating that, as in Rhizobium and Agrobacterium, cyclic glucan is required for effective host interaction (6). Moreover, Agrobacterium or Rhizobium cyclic ß-1,2-glucan mutants can be complemented by the Brucella cgs gene, indicating that their functions are highly conserved (6, 21).
The presence of cyclic ß-1,2-glucan in the periplasmic space is also required for effective S. meliloti nodule invasion (17, 41) and A. tumefaciens crown gall tumor induction (15, 22). Two chromosomal homologue loci, ndvA in S. meliloti and chvA in A. tumefaciens, code for cyclic ß-1,2-glucan transport genes (cgt). ndvA and chvA genes are interchangeable, and mutations in one gene can be complemented by the other, indicating that their functions are highly conserved (15). S. meliloti NdvA and A. tumefaciens ChvA are membrane proteins with homology to bacterial ATP-binding transporters of the ABC transporter superfamily (22, 41). ABC transporters utilize the energy of ATP hydrolysis to transport a wide variety of molecules across cellular membranes. These molecular pumps are found in all phyla and form a large protein family (37). The amino acid sequence of the ATPase domain contains the characteristic Walker A and B motifs involved in ATP binding (48). However, the intervening sequence between these two motifs is usually longer than in other ATP-binding enzymes, and the unique signature motif LSGGQ absent in other ATPases is always present.
Once cyclic ß-1,2-glucan is transported into the periplasm, a variety of nonglycosidic substituents (glycerol phosphate, succinate, and/or methyl malonate) are added, leading to the accumulation of periplasmic anionic cyclic ß-1,2-glucans (5, 8, 22). S. meliloti ndvA and A. tumefaciens chvA mutants have less than 15% of the wild-type levels of anionic periplasmic cyclic ß-1,2-glucan (41). The arrangement of ndvA and ndvB in the S. meliloti chromosome is similar to that of chvA and chvB in A. tumefaciens, with the two loci being adjacent to each other and transcribed in a convergent fashion (15, 17).
In this report we describe the isolation of the B. abortus cyclic ß-1,2-glucan transporter gene (cgt). B. abortus cgt complemented the phenotypes associated with A. tumefaciens chvA and S. meliloti ndvA mutations. On the other hand, B. abortus cgt mutants do not accumulate anionic cyclic ß-1,2-glucan and have reduced virulence in mice and defective intracellular multiplication in HeLa and J774 cells. These results suggest that, as in Agrobacterium and Rhizobium, B. abortus cyclic ß-1,2-glucan must be transported into the periplasm to exert its action.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cloning and DNA sequencing. The putative B. abortus cgt gene was amplified from B. abortus S2308 genomic DNA by PCR with primers cgt-fw (5'-CTCGCCCGCATCCACAATCT-3') and cgt-rev (5'-CCGCACCCAAGCCATTTTTC-3'), designed according to the sequence of a B. abortus gene highly homologue to A. tumefaciens chvA (complete genome of B. abortus strain 2308) (unpublished data).
The amplified products were ligated to pBluescript II KS(+) (Stratagene, La Jolla, Calif.) and digested with EcoRV by following manufacturer's instructions. The resulting plasmid, containing a 2.3-kb fragment, was named pBKcgt. DNA sequencing was carried out by the dideoxy method with an automated model 373 DNA sequencer (Perkin-Elmer Applied Biosystems Division, Foster City, Calif.) according to the manufacturer's instructions.
TLC of cyclic ß-1,2-glucan. Cells from cultures grown for 48 h were harvested by centrifugation at 10,000 x g for 10 min. Cyclic ß-1,2-glucans were extracted from cell pellets with ethanol (70% ethanol, 1 h at 37°C). Ethanolic extracts were centrifuged in an Eppendorf centrifuge, and supernatants were dried in a speed-vac centrifuge. Extracted glucans were dissolved in 70% ethanol and submitted to thin-layer chromatography (TLC) as described previously (7). TLC plates were developed by spraying with 5% sulfuric acid in ethanol and heating for 5 min at 120°C.
Nodulation and virulence test. Alfalfa seeds were surface sterilized with concentrated sulfuric acid for 30 s and washed several times with sterile distilled water until total removal of the acid. Seeds were germinated on wet filter paper in petri dishes. Two-day-old seedlings were planted in autoclaved modified Leonard jars filled with Jensen's N-free solution (47). Seedlings were dipped into 2-day-old cultures immediately before planting. After 4 weeks, plants were removed and strains were isolated from nodules as described previously (20). Virulence assays were carried out on Kalanchoe leaves as previously described (18).
Construction of B. abortus ß-1,2-glucan transporter mutant. An NcoI fragment (300 bp) of cgt was digested from pBKcgt. The deleted plasmid was blunt ended with T4 DNA polymerase (New England BioLabs) and ligated to a 1.3-kb HincII fragment containing a kanamycin resistance cassette (30). The recombinant plasmid was electroporated into B. abortus S19 and B. abortus S2308 strains, where it is incapable of autonomous replication. Transconjugants were selected in BB agar with kanamycin (50 µg/ml). Double crossover events were selected by streaking colonies in duplicate in BB agar with kanamycin (50 µg/ml) and BB agar with ampicillin (100 µg/ml). Kanamycin-resistant, ampicillin-sensitive clones were selected as possible double recombinants. Putative double recombinants were confirmed by colony PCR with primers 5'-TCAGCAATGTTTCGGTGG-3' and 5'-GAACGGCGGCTGACGGTG-3'. Genetic complementation of cgt mutants was carried out with plasmids pBB4cgt and pBB4522, containing a wild-type copy of the B. abortus and A. tumefaciens cgt genes, respectively. Plasmids pBB4cgt and pBB4522 were introduced in the mutants by biparental mating with E. coli S17.1 as the donor strain (6, 11).
Cell culture and infection assay. HeLa cells were cultured at 37°C in 5% CO2 atmosphere in minimal essential medium (Gibco, Paisley, Scotland), supplemented with 2 mM glutamine and 5% fetal calf serum without antibiotic (cell culture medium). Infection of cells with different Brucella strains was performed at a multiplicity of infection of 100 as previously described (32, 38). The murine macrophage-like J774 cell line was used to test phagocytic cells. Cells at 105/well were infected with a bacterial suspension prepared as described above for HeLa cells except for the following changes: the culture medium was RPMI 1640 (Gibco) supplemented with 5% fetal bovine serum (Gibco) and the multiplicity of infection was 50.
Virulence in mice. Nine-week-old female BALB/c mice were injected intraperitoneally with 0.2 ml of a suspension containing the appropriate number of viable Brucella organisms. Stock cultures were grown for 48 h on BB plates, and cells were suspended in sterile phosphate-buffered saline and adjusted turbidimetrically to the selected concentration. The exact bacterial concentration was calculated retrospectively by viable count. At selected postinfection times, groups of 5 mice were killed by cervical dislocation and spleens were homogenized in 1 ml of phosphate-buffered saline, serially diluted, and plated in triplicate on BB plates with the appropriate antibiotic (27).
Statistical analysis. Differences between the means of experimental groups were analyzed by using the Student t test. Differences were considered significant at P values of <0.05.
Nucleotide sequence accession number. The sequence of the B. abortus cyclic ß-1,2-glucan transporter gene (cgt) has been assigned GenBank accession number AY237159.
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RESULTS |
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Functional characterization of B. abortus cgt. S. meliloti ndvA and A. tumefaciens chvA mutants were used as heterologous backgrounds to study function and expression of B. abortus cgt. Two plasmids, pBB4cgt and pBB2cgt (see Materials and Methods and Table 2), were introduced by mating in S. meliloti LI1 ndvA (41) and A. tumefaciens ME104 chvA mutants (15, 22), respectively.
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A variety of pleiotropic phenotypes linked to the synthesis and secretion of cyclic ß-1,2-glucan were described previously (4) A. tumefaciens ME104 chvA and S. meliloti LI1 ndvA mutants are nonmotile due to defective flagellum assembly (4). Plasmids pBB2cgt and pBB4cgt restored to wild-type level the motility of chvA and ndvA mutants, respectively, thus indicating that B. abortus Cgt restored the correct assembly of flagella in these backgrounds.
Cyclic ß-1,2-glucan transport. It has been described that once cyclic ß-1,2-glucans are transported into the periplasm, they are replaced by nonglycosidic anionic substituents (4, 5). To study whether B. abortus cgt restores in A. tumefaciens and S. meliloti the transport of cyclic ß-1,2-glucan into the periplasmic space, cyclic ß-1,2-glucans were extracted from strains A. tumefaciens ME104(pBB2cgt) and S. meliloti LI1(pBB4cgt) and the presence of anionic glucans was analyzed by TLC as described in Materials and Methods. As shown in Fig. 3A and B and Table 2, the B. abortus cgt gene restored the accumulation of anionic glucan to wild-type levels in both mutants, thus indicating that Cgt is indeed a cyclic ß-1,2-glucan transporter. As shown in Fig. 3B, the A. tumefaciens chvA mutant has strongly reduced the amount of anionic glucans as well as the degree of polymerization of neutral glucans. Both effects where reverted to the wild-type phenotype by pBB2cgt and pCD522 plasmids.
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Glucan extraction and characterization by TLC. Localization of cyclic ß-1,2-glucan in the periplasm was studied indirectly by TLC determination of the presence of anionic glucans. As shown in Fig. 3C, B. abortus 2308 cgt::Km does not accumulate anionic glucans; the same result was obtained with B. abortus S19 cgt::Km (data not shown). It is shown that plasmid pBB4cgt, containing the B. abortus cgt, and plasmid pBB4522, containing A. tumefaciens chvA, restored the presence of anionic glucans to the wild-type level. These results demonstrate that cgt is the B. abortus cyclic glucan transporter gene and that the phenotype can also be complemented by the A. tumefaciens gene, thus cgt and chvA are fully interchangeable.
Intracellular multiplication of B. abortus cgt mutants. The importance of B. abortus cgt in invasion and intracellular survival was evaluated in HeLa and J774 cells as described in Materials and Methods. In HeLa cells, as shown in Fig. 4A and B, the number of intracellular bacteria recovered 4 h postinfection displayed no significant difference between the wild-type and cgt mutants, thus indicating that cgt does not have any apparent role during invasion. However, both B. abortus 2308 cgt::Km and B. abortus S19 cgt::Km mutants showed reduce intracellular multiplication at 24 and 48 h postinfection. J774 cells were infected with 2308 and the 2308 cgt mutant (Fig. 4C). Both strains invade the cells to the same extent and were equally recovered at 14 h postinfection. However, intracellular multiplication at 24 and 48 h were significantly reduced. Intracellular multiplication was restored to wild-type levels after complementation of the mutants with plasmid pBB4cgt. The intracellular multiplication of cgt mutants is similar to that described for cgs mutants (6), thus suggesting that cyclic glucan must be transported into the periplasm to exert its action.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ABC transporters are a major class of cellular translocation machinery in all bacterial species (37, 43). In this study, we have identified, sequenced, and disrupted a B. abortus ABC transporter that exhibits high homology to the cyclic ß-1,2-glucan transporters of A. tumefaciens ChvA (60% identity) (15) and S. meliloti NdvA (59% identity) (17). Accordingly, the gene was name B. abortus cgt, for cyclic glucan transporter.
Computer analysis of the predicted amino acid sequence revealed the presence of Walker and ABC signatures found in all ABC transporters in the C-terminal domain of Brucella Cgt.
Contrary to what happens in S. meliloti and A. tumefaciens, in which cyclic ß-1,2-glucan transporter (cgt) and cyclic ß-1,2-glucan synthase (cgs) are contiguous and convergently transcribed, in Brucella, cgs and cgt genes are separated by 857 kb. This suggests that Brucella cgs and cgt were either acquired independently or have suffered a severe genome rearrangement during evolution.
Cross-complementation studies revealed that this B. abortus ABC transporter gene restores cyclic ß-1,2-glucan transport of S. meliloti ndvA and A. tumefaciens chvA mutants. Conversely, the A. tumefaciens chvA gene complemented glucan transport of the B. abortus ABC transporter mutant. Thus, we concluded that this gene codes for the Brucella cyclic ß-1,2-glucan transporter.
B. abortus cgt mutants accumulate nonsubstituted neutral forms of cellular cyclic ß-1,2-glucan. The accumulation of nonsubstituted cyclic ß-1,2-glucan may be due either to a defect of the enzymes or substrates required for the modifying reaction or to a defect in the transport of cyclic ß-1,2-glucan to the periplasmic space. It was well established that cyclic ß-1,2-glucan modifying reactions take place in the periplasmatic space of the bacteria (3-5). Cross-complementation of Brucella cgt mutants with the A. tumefaciens cyclic ß-1,2-glucan transporter chvA gene demonstrated that the absence of anionic cyclic ß-1,2-glucan in B. abortus cgt mutants is due to the absence of cyclic ß-1,2-glucan transport into the periplasm.
A large number of genes with high homology to ABC transporters were identified in the genome of brucellae; however, this is the first report in which the role of a B. abortus ABC transporter was assigned. Recently, an ABC transporter homologue to S. meliloti ExsA was identified and characterized in B. abortus. exsA is critical for full B. abortus virulence (34); however, the substrate transported by ExsA was not identified. Mutants in the Brucella ATP-binding genes bapA and bapB (24) and in a B. abortus ABC transporter mapping in chromosome 1 have no effect on cyclic ß-1,2-glucan transport (M. Roset, unpublished data), thus indicating the high specificity of ABC transporters.
In a previous work, it was described that the absence of cyclic ß-1,2-glucan reduced the virulence of B. abortus in mice and impeded normal intracellular multiplication in HeLa cells (6). Cgs, the enzyme responsible for cyclic ß-1,2-glucan synthesis, is a 300-kDa inner membrane protein. All the cgs mutants studied so far were obtained by transposon insertion; thus, the lack of cyclic glucan was always accompanied by the lack of the 300-kDa inner membrane protein. Accordingly, the observed phenotype may be due to the lack of cyclic glucan, the lack of the 300-kDa inner membrane protein, or the lack of both.
In this study, we have obtained a B. abortus cyclic ß-1,2-glucan transport mutant. The mutant lacks the ABC-type transport protein Cgt but contains an intact Cgs and accumulates cytoplasmic nonsubstituted neutral cyclic ß-1,2-glucan. Interestingly, cgt mutants have reduced virulence in mice and defective intracellular multiplication in HeLa and J774 cells, a phenotype identical to that described for cgs null mutants. These results suggest that the presence of cyclic glucan in the periplasmic space is required in B. abortus for appropriate host interaction and full expression of virulence.
Further work is required to determine whether cyclic ß-1,2-glucan is an extracellular signal recognized by the host or whether the presence of the glucan in the periplasmic space stabilizes and/or promotes the correct folding of other membrane proteins required for successful bacterium-host interaction.
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ACKNOWLEDGMENTS |
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We thank J. J. Cazzulo for critical reading of the manuscript. We acknowledge Fabio Fraga and Pablo Briones, University of General San Martín, for technical assistance and L. Ielpi for providing the S. meliloti LI1 strain.
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FOOTNOTES |
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REFERENCES |
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