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Previous Article | Next Article 
Infection and Immunity, December 2003, p. 7053-7060, Vol. 71, No. 12
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.12.7053-7060.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Unité de Recherche en Biologie Moléculaire (URBM), Laboratoire d'Immunologie et de Microbiologie, University of Namur, 5000 Namur, Belgium
Received 12 May 2003/ Returned for modification 17 June 2003/ Accepted 14 August 2003
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ABSTRACT |
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 Top Abstract IntroductionMaterials and
METHODSResultsDiscussionReferences |
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-subunit of the RNA polymerase. This disruption leaves a
partially functional protein, impaired for the activation of
virB transcription, as demonstrated by the absence of
induction of the virB promoter in the
Tn5::rpoA background. The results
presented here highlight the fact that the ability of Brucella
to induce pathogenesis shares similarities with the molecular
mechanisms used by both Rhizobium and Agrobacterium
to colonize their
hosts. |
INTRODUCTION |
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Brucella is able to survive in professional and nonprofessional phagocytes by subverting the intracellular trafficking of eukaryotic cells (3, 44, 45). Studies in epithelial cells have shown that the ability of Brucella to escape from the classical cellular trafficking pathway, which normally leads to the lysosome, needs at least the VirB system (homologous to a type IV secretion machinery) and the BvrR/BvrS two-component system (8, 10, 53). It has also been shown that Brucella recruits actin and activates small GTPases during its internalization in HeLa cells (20).
While genome analysis revealed some genes that could be related to virulence (e.g., adhesins, hemolysins, and invasins), it showed that Brucella lacks classical virulence-related sequences and genes, such as pathogenicity islands, type III secretion systems, toxins, pilus biogenesis genes, etc. (36). Therefore, to draw a complete map of the molecular basis of Brucella pathogenesis, unbiased approaches are still needed. Moreover, these approaches will help in the functional assignment of Brucella open reading frames (ORFs).
Recently, different methods have been used to detect genes which are induced or necessary in cellular models (16, 17, 27, 28), leading to the identification of several virulence genes. Although these models are relevant for Brucella virulence, they do not reflect the complexity of environments encountered by the bacterium in the host. In order to approach as much as possible the natural conditions, in vivo studies are required. One of the most powerful technologies for in vivo screening is signature-tagged mutagenesis (STM) (23), which allows screening for loss of virulence in an animal model of infection. The use of this technology led to the identification of new virulence genes in several pathogenic bacteria and fungi (for review, see reference 51). The method has been applied to Brucella in an acute infection model (29) as well as in a model of chronic persistence (25).
In this study, we present the results obtained by screening 1,152 new STM mutants during the acute phase of infection in a mouse model. We identified 36 attenuated mutants that were characterized regarding the ability to grow in different cellular models, sensitivity to oxidative and acidic stress, and lipopolysaccharide (LPS) phenotype. Molecular analysis of the mutants revealed that four of them are affected in genes that are only conserved in Rhizobiaceae. The presence of a fliF mutant allowed us to implicate a role for flagella in virulence. In this study, we focused on an unexpected mutant, which is disrupted in the essential gene rpoA. We showed that this mutant is impaired in the proper regulation of the virB operon and suggest that the C-terminal part of RpoA interacts with the virB transcriptional regulator.
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MATERIALS AND METHODS |
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pir (recA thi pro hsd (r-
m+)
RP4::2-Tc::Mu::Km
Tn7 lysogenized with pir phage)
(35), CC118 pir
[
(are-leu) araD
lacX74 galE galK phoA20 thi-1
rpsE rpoB argE recA1 lysogenized with pir phage]
(9), and TOP10
[F- mcrA
(mrr-hsdRMS-mcrBC)
80lacZM15 lacX14
recA1 deoR araD139
(ara-leu)7697 galU galK
rpsL (Strr) endA1 nupG]
(Invitrogen). E. coli strains were grown on Luria-Bertani (LB)
medium with appropriate antibiotics. Antibiotics were used at the
following concentrations for E. coli and B.
melitensis: ampicillin (Amp), 50 µg/ml; kanamycin (Kan),
50 µg/ml; nalidixic acid (Nal), 25 µg/ml. A modified
minimal medium was used (nitrogen and carbon sources were supplied with
glutamic acid, lactic acid, and glycerol)
(18,
29). The plasmids used in
this study were pUTmini-Tn5Km2
(23), pCR TOPO 2.1
(Invitrogen), and pBBR1-KGFP-virB
(7). Matings. Matings were performed by mixing equal volumes (20 µl) of liquid cultures of E. coli S17 donor cells (optical density at 600 nm of 0.6) and the B. melitensis 16M Nalr recipient strain (overnight culture) on a 0.22-µm-pore-size filter. The filter was left for 1 h on an LB plate without antibiotics and then transferred onto a 2YT plate containing Kan and Nal. After 3 days of incubation at 37°C, the exconjugates were replicated on 2YT Nal Kan and on 2YT Nal Amp. The Amp-resistant clones (about 4% of the clones) were discarded, and the Amp-sensitive clones were transferred to 96-well plates.
Amplification and labeling of DNA tags. For amplification and labeling of tags, we modified a previously described protocol (24). For both the input and the output pools, bacteria from plates containing approximately 104 colonies were resuspended in phosphate-buffered saline (PBS) and then centrifuged, and genomic DNA from the pelleted bacteria was recovered by the cetyltrimethylammonium bromide method (4). Tags were initially amplified by PCR from genomic DNA with the primers P2 (5'-TACCTACAACCTCAAGCT-3') and P4 (5'-TACCCATTCTAACCAAGC-3'). The amplicons were purified in Microbio spin columns (Bio-Rad), and a fraction was used as target for a second PCR including [32P]dCTP to radiolabel the tags. The cycling conditions for both PCRs were as follows: 5 min at 94°C followed by 20 cycles of 94°C for 30 s, 50°C for 45 s, and 72°C for 10 s. The reactions were performed with Taq polymerase from Biotools in a PTC-150 Minicycler (MJ Research).
Molecular techniques. DNA manipulation was performed according to standard techniques (4). Restriction enzymes were purchased from Roche and primers were from Amersham Pharmacia. For dot blots, the selected pUTmini-Tn5Km2 was amplified from a plasmid midi-preparation using primers P3 (5'-CATGGTACCCATTCTAAC-3') and P5 (5'-CTAGGTACCTACAACCTC-3'), with the following cycling conditions: 5 min at 94°C followed by 30 cycles of 94°C for 30 s, 50°C for 45 s, and 72°C for 10 s. One hundred microliters of a 1:10 dilution of the PCR products was transferred to a positively charged nylon membrane (Hybond N+; Amersham) with a dot blot apparatus (Bio-Rad). The blots and hybridization reactions were processed as described by Holden and Hensel (24).
Transposon
insertion sites were amplified by arbitrary PCR
(42). For the first
round, the primers were ARB1
[5'-GGCCACGCGTCGACTAGTAC(N)9GATAT-3'],
ARB3
[5'-GGCCACGCGTCGACTAGTAC(N)9CAGTC-3'],
ARB4
[5'-GGCCACGCGTCGACTAGTAC(N)9TACGT-3'],
and ARB5
[5'-GGCCACGCGTCGACTAGTAC(N)9GTTAC-3'],
used with transposon-specific primers. P4 was used to
amplify sequences from the I end and P7
(5'-GCACTTGTGTATAAGAGTCAG-3') was
used to amplify sequences from the O end. Ten microliters of the PCR
product was used in the second round with the primers ARB2
(5'-GGCCACGCGTCGACTAGTAC-3') and
P9 (5'-CGGCCGCACTTGTGTATAAG-3')
for the product obtained with P4. The primers ARB2
(5'-GGCCACGCGTCGACTAGTAC-3') and
P8 (5'-CGCAGGGCTTTATTGATTC-3')
were used in the second round for the fragment obtained with
P7. Arbitrary PCR was also performed with genomic DNA from the wild
type as a negative control. The PCR products were cloned into pCR TOPO
2.1 (Invitrogen). The inserts were sequenced by the dye terminator
method (Big Dye terminator kit; Perkin Elmer) with an ABI 377
sequencer. Sequences were analyzed by performing searches with the
Blastx program (2) against
the nonredundant peptide database
(http://www.ncbi.nlm.nih.gov/)
and against the B. melitensis database of the URBM
bioinformatic group
(http://serine.urbm.fundp.ac.be/
seqbruce/GENOMES/).
Screening of the STM Library. Mutants were grown at 37°C in 200 µl of 2YT medium in 96-well microtiter plates with appropriate antibiotics for 48 h. The bacteria were then pooled, centrifuged at 4,000 rpm for 10 min in a Jouan centrifuge, and resuspended in 2 ml of 0.9% NaCl. The bacterial suspension was then diluted to a final concentration of 5 x 105 CFU in 100 µl of 0.9% NaCl. The number of bacteria was confirmed by plating dilutions on 2YT plates. The bacterial suspension (100 µl) was injected intraperitoneally into 5-week-old female BALB/c mice (n = 2). The remaining part of the suspension was plated onto medium for DNA isolation. Five days after the infection, animals were sacrificed and the spleens were removed aseptically. For recovery of bacteria, the spleens were homogenized in PBS-0.1% Triton X-100 (Roche) and dilutions were plated on 2YT medium. Plates containing approximately 104 clones were used for DNA extraction.
CI. In competition experiments, mutant (Nalr Kanr) and wild-type (Nalr) bacteria were grown for 48 h in 2YT medium, and then equal amounts of bacteria (about 2.5 x 105 each in 100 µl of 0.9% NaCl) were mixed and injected intraperitoneally into mice (n = 2). Dilutions of the infecting doses were plated on 2YT and 2YT Kan media to estimate the ratio of mutant-to-wild-type bacteria in the inoculum. Mice were sacrificed after 5 days, and the spleens were removed and homogenized. To determine the proportion of mutant-to-wild-type bacteria, dilutions of spleen homogenates were plated on 2YT and 2YT Kan media. The competitive index (CI) was calculated as the proportion of mutant-to-wild-type bacteria recovered from the animals divided by the proportion of mutant-to-wild-type bacteria in the inoculum.
Phenotypical characterization of mutants. The crystal violet method (58) was used to stain B. melitensis 16M for the rough phenotype. Smooth colonies do not take up the dye, whereas rough colonies become colored. H2O2 sensitivity tests were performed as described previously (15).
For the pH sensitivity test, bacteria were grown on solid media buffered with acetate and citrate phosphate buffers (pHs 5, 5.4, and 7).
Infection of macrophages and HeLa cells. Subconfluent monolayers (2 x 104) of bovine macrophages (54) or human HeLa cells were inoculated with bacteria diluted to 6 x 106 CFU ml-1 in cell culture medium. Plates were centrifuged for 10 min at 1,000 rpm at room temperature in a Jouan centrifuge and placed in a 5% CO2 atmosphere at 37°C. After 1 h, wells were washed three times and incubated for 48 h with cell culture medium supplemented with 50 µg of gentamicin per ml. At the end of the infection time, the monolayers were washed three times with cell culture medium and treated for 20 min with 200 µl of 0.1% Triton X-100 (Roche) in PBS. Serial dilutions of the lysates were plated onto 2YT plates for determination of CFU. Each infection was performed in triplicate.
Immunofluorescence assays. Labeling of intracellular bacteria was performed as described previously (44). Briefly, a goat polyclonal anti-B. melitensis antibody was used to detect the bacterium and a Texas red-conjugated anti-goat immunoglobulin G was used as secondary antibody (Jackson ImmunoResearch Laboratories, Immunotech, Marseille, France). Glass coverslips (13-mm diameter) in 24-well plates were seeded with HeLa cells and macrophages suspended in culture medium (105 cells/well). The cells were inoculated with bacteria at a multiplicity of infection of 200 to 300 and washings were performed as described above. The infected cells were fixed for 20 min in 3% paraformaldehyde, pH 7.4, at room temperature at different times postinfection for immunofluorescence staining. Splenocytes from infected mice were collected at 5 days postinfection from spleens homogenized with a Potter-Elvehjem homogenizer in an NH4Cl solution. Cells were washed two times in PBS, pH 7.4, and resuspended in 2 ml of PBS. Five hundred microliters was deposed on glass coverslips saturated with poly-D-lysine and was left for 10 min at room temperature before fixation.
Fluorescence-activated cell sorting (FACS) analysis. Flow cytometric analysis of bacterial virB expression was performed with a FACScalibur machine. About 10,000 individual events were excited with a 488-nm argon ion laser, and emission light was detected through a 530-nm bandpass filter. Flowjo software (Treestar) was used for the quantitation of fluorescence.
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RESULTS |
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-Proteobacteria (Table
1). The 20 remaining mutants had insertions in genes for which the function assignment (based on homology) was obvious. These 20 genes can be classified into seven different functional classes (Table 1).
Of the four mutants affected in transport function, one had a transposon insertion in a macrolide efflux system, one had an insertion in a trigger factor that helps protein folding and secretion, and two had insertions in homologous genes coding for a UgpA-related protein.
Among the mutants classified by function as related to amino acid and sugar metabolism, two were related to amino acid metabolism, one was related to vitamin B-12 synthesis, and two were related to sugar metabolism. One of particular interest was mutated in a gene similar to mosA. In Rhizobium, mosA encodes a methylase involved in the synthesis of rhizopine, a compound synthesized in the plant nodule (39).
We found four mutants affected in regulation systems, three of them in classical transcriptional regulators and one in feuQ, encoding the sensor of a two-component system, FeuP/FeuQ. A feuP mutant has been described and was shown to be fully virulent in mice and macrophages (14). Our finding that a feuQ mutant of B. melitensis is attenuated in mice as well as in cellular models might suggest that feuP is not the sole response regulator interacting with feuQ or that this two-component system is involved in a different role in B. melitensis and B. suis.
The DNA-RNA metabolism class contains two mutants, one
affected in xseA, a gene coding for an exodeoxyribonuclease,
and one (9A3) in which the transposon is inserted at the 3' end
of the rpoA gene. rpoA codes for the
-subunit of the RNA polymerase, which is essential for
transcription. Since the transposon is located close to the end of the
gene, it is likely that the bacterium expresses a C-terminally
truncated protein, allowing growth in laboratory medium.
The exopolysaccharide class contains one mutant, which has an insertion in the gene for phosphomannomutase, a protein involved in LPS biosynthesis (1).
We also found a mutant (9C6) in the fliF gene coding for the MS ring of the flagellar apparatus. It has been known for a long time that Brucella contains flagellar genes (22), and the genome sequence has revealed that it contains all of the genes necessary to build a flagellum but that it is deprived of any chemotactic system (30). It has been suggested that these genes are cryptic, because flagellated Brucella has never been observed. This is the first indication that the flagellar genes are not cryptic and may be involved in Brucella pathogenesis.
Three strains were mutated in sequences involved in oxidoreduction, including norE, which is involved in nitric oxide reduction.
One of the attenuated strains has a mutation in an intergenic region. In this mutant, the transposon is integrated at nucleotide 340509 of chr I and is therefore located 217 bp downstream of a gene that in Rhodobacter sphaeroides codes for an enzyme involved in succinylation of osmoregulated periplasmic glucans (7a) and 271 bp upstream of a gene coding for a protein of unknown function. The insertion of the transposon in such a place could have an effect on transcriptional termination of the upstream gene, or more likely, it might disrupt the promoter region of the downstream gene.
In vitro characterization of the attenuated mutants. To further analyze the phenotypes of the mutants, we performed in vitro assays. Expression of the complete LPS molecule is known to be required for full virulence of Brucella (1, 19, 34, 46). Therefore, all mutants were tested for expression of a full LPS molecule by crystal violet colony staining. The sole rough mutant detected was strain 17F3, in which the gene coding for phosphomannomutase was disrupted. During its intracellular travel, Brucella is thought to resist oxidative stress as well as acidic pH (47), and to mimic these conditions, H2O2 and pH sensitivity of all mutants was studied. The mutants 8B9, 9A4, 13G7, and 16A11 showed a significant increase in sensitivity to H2O2, as tested by a disk sensitivity assay (diameters of 4.61 cm [P < 0.01], 4.15 cm [P < 0.05], 4.2 cm [P < 0.05], and 4.16 cm [P < 0.05], respectively, compared to 3.5 cm for the wild type). Interestingly, mutant 9A3 (Tn5::rpoA) was shown to be more resistant to oxidative stress. Mutants 8B9, 9A4, 14D3, and 16H1 were unable to grow at pH 5.4, unlike the wild type. Mutant 9A3 was the only strain that was able to grow at pH 5, whereas the wild type could not.
Intracellular growth of the mutants. The pathogenicity of Brucella is critically dependent on its ability to infect and multiply in professional and nonprofessional phagocytes. We therefore established whether the attenuated mutants were also affected in the ability to invade into and survive within bovine macrophages and epithelial cells. The results showed that most mutants were attenuated in both bovine macrophages and HeLa cells (Table 1). However, mutant 9A3 (Tn5::rpoA) is attenuated only in HeLa cells.
RpoA is involved in
virB regulation in vitro.
The transposon in the mutant 9A3
(Tn5::rpoA) is integrated at the
3' end of the gene (at nucleotide 1007 of the 1,014-bp ORF).
The mutant is predicted to express a protein in which the last residues
of its C-terminal part are replaced by residues encoded by the I end of
the transposon. It is known that the C-terminal part of the
-subunit is involved in the regulation of transcription
through contact with cis or trans elements while the
N-terminal domain is involved in the assembly of the polymerase (for
review, see reference
48). Since it has been
shown that RpoA is involved in the regulation of virB operon
transcription in Agrobacterium tumefaciens
(32,
33), we compared
virB regulation in the Brucella
rpoA::Tn5 mutant and wild-type
strains. The plasmid pBBR1-KGFP-virB
(7), containing the
putative virB promoter fused with promoterless gfp,
was shown to be suitable for virB expression studies
(7). This plasmid was
electroporated into B. melitensis 16M, into the rpoA
transpositional mutant, and as a negative control into a characterized
mutant (vjbR) known to be unable to activate transcription of
the virB operon (R.-M. Delrue, personal communication). Green
fluorescent protein production was monitored and measured by
fluorescence-activated cell sorting (FACS). With the wild-type strain,
fluorescence was detected during exponential growth in rich medium and
after a 4-h shock in minimal medium, whereas no fluorescence was
detected for the rpoA mutant nor for the negative control
(Fig.
1).
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We found a mutant in norE, a gene coding for a component of the anaerobic respiratory chain that transforms nitrate to dinitrogen. This finding suggests two different possibilities: (i) the bacterium replicates in an environment deprived of oxygen and switches to anaerobic growth by using nitrate as an electron acceptor, as has already been suggested by the narG mutant described by Kohler et al. (27), with the absence of functional NorE leading to a toxic accumulation of NO in the bacterium, or (ii) the bacterium detoxifies nitric oxide produced by the cells as proposed by Wang et al. (57).
Genomic sequences from B. melitensis and B. suis revealed strong similarities with bacteria from the Rhizobium/Agrobacterium group (43). It has also been shown that the intracellular lifestyle of Brucella spp. requires genes that are also involved in the virulence of A. tumefaciens and in Rhizobium symbiosis (21, 26, 31, 53). The data presented here highlight the similarities between the two groups, as explained below.
The pathological and symbiotic processes developed by Agrobacterium and Rhizobium share many features, one of them being the ability to synthesize or induce synthesis of specific compounds known as opines for Agrobacterium and rhizopine for Rhizobium. Rhizopine (L-3-O-methyl-scyllo-inosamine [3-O-MSI]) is specifically synthesized by Rhizobium in the plant nodule (38, 39). The mos operon, composed of four genes (orf1, mosA, mosB, and mosC), encodes the enzymes of the 3-O-MSI synthesis pathway (38). It has been shown that MosA, MosB, and MosC are required for the synthesis of 3-O-MSI, whereas the function of orf1 still needs to be investigated (40). Rhizopine is a specific growth substrate for strains of Rhizobium carrying the moc operon and is thought to give a specific advantage to moc+ strains in the rhizosphere (49). In this study, we show that Brucella contains in its genome a gene that is highly homologous to mosA and that a mutation of this gene causes attenuation in vivo and in two cellular models, suggesting that Brucella might also produce an inositol derivative important for the colonization of its host. This result is reinforced by the finding that a gene homologous to mocC is specifically induced in macrophages (16). Moreover, analysis of the B. melitensis genome allowed us to find homologues of mocC, mocA, and mosC, and the implication of these genes in rhizopine metabolism and Brucella pathogenesis is under investigation in our lab. Preliminary results suggest that rhizopine might be a signal molecule affecting the growth of Brucella. However, we still do not know if Brucella does effectively synthesize rhizopine.
Bacteria from the Rhizobium/Agrobacterium group are usually motile and their flagella are involved in host colonization, whereas Brucella spp. are classically described as nonmotile. The presence of flagellar genes within the B. abortus genome has been described (22, 30, 50), but to date, no relation between these genes and virulence and/or motility has been reported. In this study, we have isolated an STM mutant in which the fliF gene is disrupted. This gene encodes a protein similar to the MS ring monomer, a basal component of the flagellum. This mutant is outcompeted by a wild-type strain in mice and is unable to replicate in macrophages and HeLa cells, suggesting an important role of the Brucella flagellar genes in virulence. Given the structural similarity between flagellar export machinery and the type III secretion system, Brucella flagellar genes might be involved in secretion rather than in motility. For example, for Yersinia (sensu stricto) it has been shown that flagellar components may act as a secretory apparatus involved in the export of phospholipase C (59).
The type four
secretion system encoded by the Ti plasmid from the virB
operon is an essential component of Agrobacterium virulence.
In Agrobacterium, the transcription of the virB
operon requires the interaction of the RpoA C-terminal domain with the
transcriptional activator VirG
(32,
33). In our study, we
isolated a mutant of rpoA. Analysis of the transposon
insertion site showed that the last 7 nucleotides of the gene are
replaced by an unrelated sequence from the I end of the transposon. Our
results show that this mutant is unable to induce transcription of the
virB operon under all conditions tested. This led us to
suggest that the change in the C-terminal domain of the protein
impaired its interaction with different transcriptional regulators,
among them the virB regulator. The VirB apparatus is known to
be important for intracellular replication in both macrophages and HeLa
cells; however, our mutant is attenuated in HeLa cells but not in
macrophages and showed a rather puzzling phenotype, as it is resistant
to acidic and oxidative stress. This complex phenotype may be due to a
deregulation of transcription at some locus, as it is known, for
example, that in E. coli the C-terminal part of the
-subunit interacts with OxyR, a transcriptional regulator for
hydrogen peroxide-inducible genes
(55). The persistence in
macrophages is in contradiction with the absence of virB
expression. However, in cellular models, we used inactivated
macrophages, and infection study cultures stand for 
48 h, a
lapse of time during which its higher stress resistance might allow the
mutant to persist in a place that is different from the natural
Brucella replication vacuole. In mice, the infection lasts for
a longer period of time and therefore macrophages may be activated.
This is also illustrated by the observation that even with the wild
type, it is rare to find a splenocyte full of Brucella,
whereas in ex vivo models, HeLa cells and macrophages contain large
numbers of bacteria after 48 h of infection. This may reflect
less efficient replication in macrophages in vivo and emphasize the
importance of using in vivo models of infection.
Finally, we also
found six unknown virulence genes which, to date, are only shared among
members of the -proteobacteria. Studies of these genes might
allow a better understanding of the phylogeny of the
-proteobacteria and of the story of their interaction with
eukaryotic cells.
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ACKNOWLEDGMENTS |
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This work was supported by the Commission of the European Communities, contract no. QLK2-CT-1999-00014.
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FOOTNOTES |
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