Brucella abortus VirB12 Is Expressed during Infection but Is Not an Essential Component of the Type IV Secretion System

Bacterial strains, media, and culture conditions.Bacterial strains used were Brucella abortus 2308, Brucella melitensis 16 M, and Brucella suis 1330, and their characteristics are listed in Table 1. Strains were routinely cultured on tryptic soy agar (TSA; Difco/Becton-Dickinson, Sparks, MD) or in tryptic soy broth (TSB) at 37°C on a rotary shaker. Bacterial inocula for infection of mice were cultured on TSA plus 5% defibrinated sheep blood. Antibiotics, when required, were added at the following concentrations: carbenicillin, 100 mg/liter; kanamycin (Km), 100 mg/liter; and chloramphenicol, 5 to 30 mg/liter. All work with live Brucella strains was performed at Biosafety Level 3.

Strain construction and recombinant DNA techniques.For construction of virB12 deletion mutants in B. abortus (AK/ORF12), B. melitensis (MK/ORF12), and B. suis (SK/ORF12), a pCR2.1 (Invitrogen)-based plasmid was generated using a three-step cloning strategy. This method takes advantage of the organization of the pCR2.1 TOPO vector. Plasmid pCR2.1 TOPO contains two PstI sites, and cleavage with PstI releases a fragment containing the 3′ end of the lacZ alpha fragment, the f1 origin, and the 5′ end of the kanamycin resistance gene (vector data are available from Invitrogen). Deletion of this 1.19-kb PstI fragment disrupts the kanamycin resistance gene but does not affect plasmid replication, and therefore pCR2.1 TOPO was used to generate a construct for the allelic exchange of virB12 with a deleted copy marked with a kanamycin resistance gene. In the first cloning step, a fragment in the 5′ region of virB12 (designated UP) was amplified from B. abortus genomic DNA using primers ORF12K1-F and ORF12K1-R (Fig. 1). A fragment in the 3′ region of virB12 (designated DN) was amplified using primers ORF12K2-F (with SmaI) and ORF12K2-R. The resulting fragments were TOPO cloned into pCR2.1 to yield the two plasmids pUP/ORF12 and pDN/ORF12.

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FIG. 1.

Construction of virB12 deletion mutants in Brucella. Top: Diagram showing overlap of a putative orf13 with the 5′ end of virB12. Bent arrows indicate the primers used for construction of virB12 deletion mutants. Middle: Map of pUKD/ORF12 showing KIXX flanked by upstream and downstream fragments cloned into the 2.7-kb pCR2.1 backbone (dotted line). The dotted arrow indicates the unique PstI site. Bottom: Replacement of an internal fragment of virB12 with KIXX does not disrupt the predicted orf13. The numbering of the virB12 deletion corresponds to that of the B. abortus virB operon in GenBank accession number AF226278 (22).

The orientations of the cloned inserts in pUP/ORF12 and pDN/ORF12 were determined by PCR and restriction analysis. Clones were identified that carry the SmaI site of the 5′ (UP)-virB12 fragment located at the end of the insert opposite PstI in pUP/ORF12. For pDN/ORF12, clones were identified that carry the SmaI site of the 3′-virB12 fragment located at the same end of the cloned insert as PstI. A 1,008-bp SmaI/PstI UP fragment of pUP/ORF12 was introduced into SmaI/PstI-cleaved pDN/ORF12 (the kanamycin resistance gene of pCR2.1 was truncated by this digestion) to link the UP and DN fragments of virB12 together. The resulting plasmid, named pUD/ORF12, was selected for ampicillin resistance. In the third cloning step, a 1.3-kb SmaI fragment of pUC4-KIXX (Pharmacia) containing the Tn5 kanamycin resistance gene was cloned into the SmaI site of pUD/ORF12 to generate pUKD/ORF12.

Plasmid pUKD/ORF12 was introduced into B. abortus 2308, B. melitensis 16 M, and B. suis 1330 by electroporation, and recombinants resistant to kanamycin and sensitive to carbenicillin (the resistance encoded on the backbone of pUKD/ORF12) were screened. The resulting strains were designated AK/ORF12 (B. abortus virB12::Km), MK/ORF12 (B. melitensis virB12::Km), and SK/ORF12 (B. suis virB12::Km). The plasmids constructed in this work are listed in Table 1, and the primers used in the construction of the plasmids are listed in Table 2. Plasmid DNA was isolated using ion exchange columns from QIAGEN, and the orientation of the cloned fragments in pUKD/ORF12 was confirmed by DNA sequencing. Standard methods were used for Southern blotting, PCR, restriction endonuclease analyses, and ligation and transformation of plasmid DNA into Escherichia coli (1). PCR products were cloned into pCR2.1-TOPO using a TOPO-TA cloning kit (Invitrogen).

Cell lines.The mouse macrophage-like cell line J774A.1 (18), obtained from ATCC, was cultured in Dulbecco's modified Eagle's medium (Gibco, Rockville, MD) supplemented with 10% heat-inactivated fetal bovine serum, 1% nonessential amino acids, and 1 mM glutamine (DMEMsup). For macrophage killing assays, 24-well microtiter plates were seeded with macrophages at concentrations of 1 × 10⁵ to 2 × 10⁵ cells/well in 0.5-ml portions of DMEMsup and incubated overnight at 37°C in 5% CO₂. The inocula were prepared by growing with shaking in TSB for 24 h and then subsequently diluting them in DMEMsup to concentrations of 4 × 10⁷ CFU/ml. Approximately 2 × 10⁷ bacteria in 0.5 ml of DMEMsup, containing a 1:1 mixture of the wild type and the isogenic mutant, were added to each well of macrophages. Microtiter plates were centrifuged at 250 × g for 5 min at room temperature in order to synchronize infection. Cells were incubated for 20 min at 37°C in 5% CO₂, free bacteria were removed by three washes with phosphate-buffered saline (PBS), and the zero time point was taken as described below. DMEMsup plus 50 μg gentamicin per ml was added to the wells, and the cells were incubated at 37°C in 5% CO₂. After 1 h, the DMEMsup plus 50 μg/ml gentamicin was replaced with medium containing 25 μg/ml gentamicin. Wells were sampled at 0 and 48 h after infection by aspirating the medium, lysing the macrophages with 0.5 ml of 0.5% Tween 20, and rinsing each well with 0.5 ml of PBS. Viable bacteria were quantified by dilution in sterile PBS and plating on TSA and TSA plus Km.

Infection of mice.Female BALB/c ByJ mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and used at ages of 6 to 10 weeks. For mixed-infection experiments, groups of four or five mice were inoculated intraperitoneally (i.p.) with 0.5-ml portions of PBS containing 2 × 10⁵ CFU of a 1:1 mixture of B. abortus, B. melitensis, or B. suis wild type and the isogenic virB12 mutant. Infected mice were held in microisolator cages in a Biosafety Level 3 facility. At 4 weeks postinfection, mice were euthanized by CO₂ asphyxiation, and the spleens were collected aseptically at necropsy. Spleens were homogenized in 3 ml of PBS and serial dilutions of the homogenate plated on TSA and TSA containing kanamycin for enumeration of mutant and wild-type CFU.

For assaying VirB12-specific antibody responses, mice were infected i.p. with 1 × 10⁵ CFU of B. abortus 2308 or of the B. abortus virB12 deletion mutant AK/ORF12. Blood samples were collected from the saphenous vein at various time points after infection. All animal experiments were approved by the Texas A&M University Laboratory Animal Care and Use Committee and were conducted in accordance with institutional guidelines.

Generation of polyclonal specific antisera.The gene encoding Bcsp31 (14) was PCR amplified from B. abortus using primers F1499-F and F1499-R (Table 2) and cloned into directional C-terminal His-tagged fusion protein expression vector pET101 (Invitrogen). For generation of VirB12-specific antiserum, a 504-bp fragment of the virB12 gene was PCR amplified from B. abortus using primers VirB11084F and VirB11563R (Table 2), cloned into pCR2.1 (Invitrogen), and subsequently cloned in pIVEX2.4bNdeI (Roche) with restriction enzymes NotI and PstI to generate an N-terminal fusion with the six-His tag. Both Bcsp31-6xHis and 6xHis-VirB12 were overexpressed and purified according to standard protocols (1) and used to raise polyclonal antisera in rabbits at the Texas A&M Comparative Medicine Program facility. To eliminate background reactivity to whole B. abortus, the VirB12 immune rabbit serum was affinity purified using 6xHis-VirB12 bound to a HiTrap column (Amersham Pharmacia) according to the manufacturer's instructions.

Western blotting. Brucella cultures inoculated in tryptic soy broth to a starting optical density at 600 nm of 0.01 were incubated at 37°C with shaking at 200 rpm. After 18 h, the cultures' optical densities at 600 nm were 1.2 to 1.5, and bacteria were pelleted and resuspended in 1× Laemmli sample buffer and heated at 100°C for 5 min, and the total protein equivalent to 1 × 10⁸ CFU per well was loaded for separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (13). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes by electroblotting and were detected using polyclonal rabbit serum and goat anti-rabbit immunoglobulin G (IgG) conjugated to horseradish peroxidase (HRP). HRP activity was detected with a chemiluminescent substrate (NEN). To determine protein expression levels, immunoblots were quantified by measuring the relative optical densities and areas of the corresponding bands with a computerized image analysis system (AlphaImager 2200; Alpha Innotech Corporation). Data were expressed as integrated density values and calculated as ratios of VirB12 to Bcsp31 or VirB5 to Bcsp31.

Detection of VirB12-specific IgG in mouse serum.The presence of antibody reactivities against the recombinant protein VirB12 of Brucella abortus in the serum samples from 10 BALB/c mice infected with B. abortus 2308 and 5 mice infected with B. abortus AK/ORF12 (virB12) was determined by indirect enzyme-linked immunosorbent assay (ELISA). Ni-nitrilotriacetic acid HisSorb plates from QIAGEN (Valencia, CA) were coated with 100 ng of 6xHis-VirB12 per well in PBS with 0.2% BSA (PBS-B), and plates were incubated at 4°C overnight. After washing with PBS and 0.5% Tween 20 (PBS-T), the pooled serum samples were diluted 1:100 in PBS-B and incubated overnight at 4°C. After washing with PBS-T, the reactivity was measured using HRP anti-mouse IgG (1:1,000; BD Pharmingen) by incubating the plates at 37°C for 1 h. The reaction was developed with Sigma Fast o-phenylenediamine dihydrochloride tablet sets. The resulting color was read at 410 nm with an ELISA microplate reader (Dynatech MR5000). Data points are the averages of duplicate dilutions, with each measurement being performed twice.

Statistical methods.For macrophage killing assays, all experiments were performed independently in triplicate at least three times, and data were expressed as the geometric mean of the logs of CFU/well ± standard deviation. For competitive infection of mice, the mutant and wild-type CFU data were expressed as the mean of log-transformed CFU/spleen ± standard deviation for each group of four or five mice. For both in vitro and in vivo infections, competitive indexes were calculated as log(CFU mutant/CFU wild type) and adjusted in each case to the ratio of mutant to wild type in the inoculum. The statistical significance of differences between mutants and wild types was determined by paired Student's t test. A P value of <0.05 was considered significant.

Construction and characterization of virB12 mutants.To determine whether virB12 contributes an essential function to the T4SS apparatus, we constructed mutants of B. abortus, B. melitensis, and B. suis, each carrying an internal 334-bp deletion marked by the Tn5 kanamycin resistance gene (see Materials and Methods for details on the construction). Since a predicted orf13 overlaps the first 99 bp of virB12 in B. abortus (22), we designed a deletion construct that left the first 143 bp of virB12 intact. Expression of this fragment would be expected to result in a 48-amino-acid N-terminal fragment containing a predicted lipoprotein signal sequence, which after cleavage would be predicted to yield a 33-amino-acid N-terminal fragment of a putative VirB12 protein (Fig. 1).

Plasmid pUKD/ORF12, carrying a copy of virB12 with an internal deletion of 334 bp replaced by the Tn5 kanamycin resistance gene, was constructed (see Materials and Methods) and introduced into B. abortus 2308, B. melitensis 16 M, and B. suis 1330 by electroporation. Recombinants resistant to kanamycin and sensitive to carbenicillin (the resistance encoded on pUKD/ORF12) were identified, and the resulting strains were designated AK/ORF12 (B. abortus virB12), MK/ORF12 (B. melitensis virB12), and SK/ORF12 (B. suis virB12) (Fig. 1). These strains were screened by PCR for the replacement of the 334-bp virB12 fragment by the Tn5 kanamycin resistance gene (1.4 kb) (Fig. 2A). This result was confirmed by a Southern blot of chromosomal DNA with a probe containing the deleted region of virB12 (Fig. 2B). All three wild-type Brucella strains and all three virB12 mutant strains hybridized with a virB2 probe, but the three virB12 deletion strains failed to hybridize with the virB12 probe, demonstrating that this part of the virB12 gene was deleted in all three virB12 mutants. As a further control, we generated and purified a 6xHis-VirB12 fusion protein (see Materials and Methods) and raised a polyclonal rabbit serum specific for VirB12. On Western blots, the antiserum reacted with a protein of approximately 17 kDa in lysates of B. abortus 2308, B. melitensis 16 M, and B. suis 1330 (Fig. 2C) grown to stationary phase in TSB. No proteins were detected in lysates any of these three virB12 mutants with this antiserum. Consistent with previous reports describing expression conditions for the virB genes, we found that virB12 was expressed at lower levels by B. suis than by B. abortus or B. melitensis under these growth conditions (3, 19). These results provide the first evidence that virB12 encodes a protein.

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FIG. 2.

Characterization of virB12 mutants. (A) Confirmation of virB12 deletion by PCR. Chromosomal DNAs of B. abortus 2308, B. melitensis 16 M, and B. suis 1330 and their respective virB12 mutants were amplified with primer pair ORF12C-F and ORF12C-R (Fig. 1 and Table 2). Wild-type strains yield an amplicon of 520 bp of virB12, while mutants yield one of 1.3 kb, since they contain the Km resistance determinant KIXX. (B) Confirmation of virB12 deletion by Southern blotting. EcoRI-digested genomic DNAs of wild-type Brucella strains and virB12 mutants were hybridized with either a virB2 probe (corresponding to the sequence from 1609 to 1926 of B. abortus virB) or an internal virB12 probe (corresponding to the sequence from 11181 to 11554 of B. abortus virB). The virB2 probe was generated by PCR using the primer pair virB2C-F and virB2C-R. The virB12 probe was generated using primers ORF12I-F and ORF12C-R. Primer sequences are shown in Table 2. (C) Detection of VirB12 protein in stationary-phase cultures of B. abortus 2308, B. melitensis 16 M, and B. suis 1330. Proteins from 1 × 10⁸ CFU of stationary-phase culture were separated on a 12% SDS-PAGE gel, transferred to a PVDF membrane, and probed with anti-VirB12 antiserum.

B. abortus mutants with polar insertions in the virB operon still express virB12.In order to determine whether B. abortus virB12 is coregulated with other genes in the virB operon, we assayed levels of VirB12 protein and compared them to levels of VirB5, whose expression was reduced by polar insertions in virB1 and virB2 (8). As a basis for comparison, we used the protein Bcsp31 (14). Bcsp31 is an immunogenic periplasmic protein that is not required for the growth of B. abortus in cultured epithelial cells or macrophages (10, 17). Our previous results (not shown) indicated that the abundance of Bcsp31 in B. abortus was not affected by mutations in the virB operon. Polar mutations in the virB1 to virB2 intergenic region (BA41) or in virB2 (ADH1) reduced the detection of VirB5 to a greater extent than they did that of VirB12. A transposon insertion in virB10 reduced the abundance of VirB12 but not of VirB5, suggesting that this insertion is polar on the virB12 expression. As expected, no VirB12 was detected in the virB12 mutant AK/ORF12, and neither VirB5 nor VirB12 was detected in the virB1 to virB12 operon deletion mutant, ADH4.2. These results suggest that polar mutations in the operon upstream of virB5 exert a greater effect on the expression of virB5 than they do on the expression of the downstream gene virB12.

B. abortus produces VirB12 protein during infection of mice.If virB12 is expressed during in vivo infection, then we would expect an infected host to develop an antibody response, since VirB12 was highly immunogenic in rabbits. To determine whether virB12 is expressed during infections of a model host with Brucella spp., we assayed for VirB12-specific IgG in sera from mice infected with B. abortus 2308 or virB12 mutant AK/ORF12. The results of ELISAs depicted in Fig. 3 show that the titer of IgG specific for 6xHis-VirB12 increases above the titer of naïve mice, starting at 21 days after infection. Titers of VirB12-specific IgG increased until 56 days postinfection, after which they remained at high levels through the end of the experiment at day 70. For mice infected with AK/ORF12, the IgG titers did not differ from the preinoculation titers over the course of the experiment, showing that VirB12-specific antibodies detected in mice infected with wild-type B. abortus are not elicited by other cross-reactive B. abortus proteins. These results indicate that VirB12 is synthesized and presented to the host immune system during infections of mice.

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FIG. 3.

VirB12-specific total IgG in serum samples of mice taken after infection of BALB/c mice with B. abortus 2308 (n = 10; filled bars) or its isogenic virB12 mutant, AK/ORF12 (n = 5; open bars). Samples taken at the indicated time points postinfection were diluted and measured by ELISA using His-VirB12 bound to HisSorb plates (QIAGEN). Bars indicate the averages ± standard deviations of duplicate measurements. The dotted line represents the background reactivity in mice before infection with B. abortus.

Brucella mutants lacking virB12 survive in murine macrophages like their wild-type parent strains.To determine whether VirB12 is required for intracellular survival of B. abortus, B. melitensis, or B. suis, we assayed the ability of virB12 mutant strains to survive in J774A.1 macrophages. For these experiments, we performed a coinfection of each virB12 mutant with its respective wild-type strain, as coinfection gives a sensitive measure of differences between the wild type and the mutant with regard to intracellular survival (Fig. 4). Inocula containing 1:1 mixtures of wild type and virB12 mutant were used to infect J774A.1 cultures. Coinfection of B. abortus 2308 and B. abortus BA41 (virB1-virB2::mTn5Km2) showed that at 48 h after infection, the CFU of BA41 recovered was 1/100 of that of B. abortus 2308, demonstrating that under these conditions the wild type is not able to rescue a mutant lacking a functional T4SS apparatus (Fig. 4). In contrast, the virB12 mutants AK/ORF12, MK/ORF12, and SK/ORF12 were recovered from J774A.1 cells in numbers indistinguishable from those of the wild type. These results show that VirB12 is not required for the survival of B. abortus, B. melitensis, or B. suis in this macrophage line.

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FIG. 4.

Recovery of B. abortus, B. suis, and B. melitensis virB12 mutants after coinfection of J774 macrophages with their respective wild-type strains (WTs). A mixed inoculum of the virB12 mutant and the wild type was used to infect J774 cells, and intracellular bacteria were recovered after 48 h as described in Materials and Methods. Data shown are the means of three independent assays done in triplicate ± standard deviations and plotted logarithmically. Top: Log-transformed CFU of the wild type and virB12 mutants recovered from J774 cells are given. Bottom: Competitive index (C.I.), calculated as log(CFU mutant/CFU wild type), is shown on the y axis. A significant difference (*) was seen only in the control experiment using B. abortus mutant BA41 (B. abortus virB1-virB2::mTn5) in comparison with the wild type (P = 0.04).

Brucella mutants lacking virB12 persist in murine spleens at wild-type levels.To test the ability of virB12 mutants to persist in an animal model of infection, we used the murine model to compare colonization levels of each virB12 mutant and its virulent parent strain. To this end, we performed coinfection studies with each mutant and its respective wild-type strain. For mice, coinfection gives a sensitive readout of attenuation with small experimental groups of animals. In addition, this experimental design allowed us to assess whether VirB12 is required for infection in the context of the host response to wild-type B. abortus. Groups of four to five mice were infected i.p. with inocula of 2 × 10⁵ CFU containing 1:1 mixtures of the mutant and the wild-type parent strain. While the B. abortus virB mutant BA41 was not detected in the spleen by 4 weeks, the numbers of CFU of virB12 mutants AK/ORF12, MK/ORF12, and SK/ORF12 recovered from murine spleens were not significantly different from those of the parent strains, showing that virB12 is not essential for persistence in this infection model (Fig. 5).

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FIG. 5.

Recovery of B. abortus, B. melitensis, and B. suis virB12 deletion mutants from mouse splenic tissue after mixed infection with their respective wild-type strains (WTs). Bacteria were recovered 4 weeks after i.p. infections with 2 × 10⁵ CFU of 1:1 mixtures of the wild type and the virB12 mutant. Data shown are the averages of groups of four or five mice ± standard deviations and are plotted logarithmically. Top panel: Log-transformed CFU recovered from mouse spleens are given for wild-type and virB12 mutant strains. The dashed line indicates the limit of detection in the spleen. Bottom: Competitive index (C.I.), calculated as log(CFU mutant/CFU wild type), is shown on the y axis. A significant difference (*) was seen in the experiment using B. abortus mutant BA41 (virB1-virB2::mTn5) in comparison with the wild type (P = 0.04).