Characterization and Protective Property of Brucella abortus cydC and looP Mutants

ABSTRACT Brucella abortus readily multiplies in professional or nonprofessional phagocytes in vitro and is highly virulent in mice. Isogenic mutants of B. abortus biovar 1 strain IVKB9007 lacking the ATP/GDP-binding protein motif A (P-loop) (named looP; designated here the IVKB9007 looP::Tn5 mutant) and the ATP-binding/permease protein (cydC; designated here the IVKB9007 cydC::Tn5 mutant) were identified and characterized by transposon mutagenesis using the mini-Tn5Km2 transposon. Both mutants were found to be virtually incapable of intracellular replication in both murine macrophages (RAW264.7) and the HeLa cell line, and their virulence was significantly impaired in BALB/c mice. Respective complementation of the IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants restored their ability to survive in vitro and in vivo to a level comparable with that of the wild type. These findings indicate that the cydC and looP genes play important roles in the virulence of B. abortus. In addition, intraperitoneal immunization of mice with a dose of the live IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants provided a high degree of protection against challenge with pathogenic B. abortus strain 544. Both mutants should be evaluated further as a live attenuated vaccine against bovine brucellosis for their ability to stimulate a protective immune response.

B rucella abortus, the causative agent of bovine brucellosis, causes abortion and reduced fertility in cattle and undulant fever in humans (1). Unlike other pathogenic bacteria, brucellae do not have classical virulence factors. A key aspect of the virulence of B. abortus is its ability to invade, survive, and proliferate within host phagocytic cells; it successfully bypasses the bactericidal activities of phagocytes and thereby establishes long-lasting chronic infections (2,3). The B. abortus vaccine strains S19 and RB51 have been used worldwide for the prevention of brucellosis in cattle but have several drawbacks, including interference with diagnosis, residual virulence, and some pathogenicity for humans as well as cattle (4,5). Therefore, understanding the virulence of B. abortus on a genetic level and the host response against B. abortus will provide important information for the development of a new vaccine for controlling brucellosis.
Transposon mutagenesis is an extensively used approach for the identification of genes involved in the virulence of bacterial pathogens (6)(7)(8). To understand the sustained intracellular residence of brucellae in host cells, a transposon-based approach has been employed to identify genes in brucellae that are critically required for lipopolysaccharide biosynthesis, metabolic processes, stress responses, nutrient deprivation, and invasion of and survival in host cells. Mutations in these genes led to the attenuation of Brucella compared with the virulence of the parental strain; attenuation was recognized by the reduced intracellular survival and replication or rapid clearance of the mutants from both macrophages and mice (6)(7)(8)(9)(10)(11). However, these attenuated mutants were considered either not safe or not sufficiently studied for use as possible vaccines in animals. Therefore, identification of additional vaccine targets is required.
In the present study, in an effort to identify additional genes that control the intracellular fate of B. abortus, transposon-generated mutants were selected based on their decreased survival in HeLa cells. We describe here the identification of transposon ATP-binding/permease protein (CydC) and ATP/GDP-binding protein motif A (P-loop) B. abortus mutants. The cydC gene in Escherichia coli has been well described and was originally identified by its requirement for the assembly of the cytochrome bd-type terminal oxidase (12)(13)(14). Mutants defective in cydC have a deficiency of cytochrome bd oxidase and an inability to exit from stationary phase and are hypersensitive to zinc and oxidative stress. However, the looP gene has not been fully characterized (15,16). The roles of the looP and cydC genes in the virulence of B. abortus are not well established. In the present study, both looP and cydC mutants of B. abortus were characterized and evaluated for intracellular replication in cell models, their virulence in mice, and their ability to induce protection against a wild-type B. abortus challenge in mice.

MATERIALS AND METHODS
Bacterial culture and media. The virulent wild-type B. abortus biovar 1 strain IVKB9007 was isolated from an aborted bovine fetus in South Korea, passaged twice on tryptic soy agar (TSA; Difco, Sparks, MD, USA), and stored frozen at Ϫ70°C in 25% glycerol. The B. abortus vaccine strain RB51, the virulent B. abortus challenge strain 544, the B. abortus IVKB9007 strain, and the isogenic IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants were routinely grown on TSA or tryptic soy broth (TSB; Difco) containing 5% bovine serum at 37°C in 5% CO 2 . E. coli DH5␣ and E. coli S17-1 pir pUT mini-Tn5Km2 were used for transformation and as the donor strain for conjugation, respectively (6). Each E. coli strain was cultivated in Luria-Bertani (LB) broth or agar (Difco, Sparks, MD, USA). If necessary, the medium was supplemented with an appropriate reagent and antibiotics, namely, 0.5% glycerol, 50 g/ml rifampin (RP), 50 g/ml kanamycin (KM), 30 g/ml nalidixic acid (NA), and 100 g/ml ampicillin (AMP). Bacterial strains and plasmids used in this study are described in Table 1.
Construction of mini-Tn5Km2 mutants. Wild-type B. abortus IVKB9007 was subjected to random transposon mutagenesis using a mini-Tn5Km2 transposon that was delivered on the pUT suicide vector as described previously (6). The transposon mutant was constructed by conjugal transfer of pUT mini-Tn5Km2 from the E. coli S17-1 pir donor strain into the wild-type B. abortus IVKB9007 strain. Transconjugants were isolated on TSA containing 50 g/ml KM and 30 g/ml NA to select against both B. abortus and E. coli donor strains, and mutants were screened for defects in intracellular survival within HeLa cells as described below. All mutants were kept in 25% glycerol in TSB at Ϫ70°C until the screening.
Screening for an intracellular-survival defect of B. abortus mutants. Intracellular-survival assays were performed using the modified method described previously (6). Briefly, HeLa cells were grown and maintained at 37°C in 5% CO 2 in complete medium (CM; Dulbecco's modified Eagle's medium [DMEM; GIBCO, Carlsbad, CA, USA] containing 10% fetal bovine serum [GenDEPOT, USA]) with an antibiotic or antimycotic (Life Technologies, Gaithersburg, MD, USA). The HeLa cells were dispensed into 96-well tissue culture plates 24 h prior to bacterial infection. The mutants were deposited onto the cells grown in 96-well microplates, and the plates were centrifuged at 150 ϫ g for 10 min at room temperature. Subsequently, the cells were incubated at 37°C in 5% CO 2 for 1 h, washed twice with sterile phosphate-buffered saline (PBS; pH 7.0), and incubated with CM plus 30 g/ml gentamicin (GM; Sigma, St. Louis, MO, USA) for 48 h. The infected cells were then washed and lysed with 0.1% Triton X-100 in sterile distilled deionized water (sDDW), and 30 l of the cell lysate was inoculated onto TSA and incubated at 37°C in 5% CO 2 for 72 h. The mutants showing no growth on TSA were considered intracellularsurvival-defective mutants and were selected for further assays.
Cloning and sequencing of transposon insertion sites. The DNA sequences adjacent to the transposon insertion sites were determined as described previously (6). Briefly, chromosomal DNA was isolated from transposon mutants and digested with one of the several restriction enzymes (EcoRI or KpnI; New England BioLabs, Hitchin, United Kingdom) that cut at either end of the transposon, leaving the KM resistance gene intact. The digested fragments were ligated into similarly cleaved pBluescript II KS (ϩ) (Stratagene, La Jolla, CA, USA) and transformed into competent E. coli DH5␣ cells (Invitrogen, Carlsbad, CA, USA). The transformants were selected on LB agar plates containing 50 g/ml KM and 100 g/ml AMP. The plasmid DNA from each transformant, containing both transposon and flanking DNA, was purified using a HiYield plasmid minikit (RBC Bioscience, Taipei, Taiwan), and automated sequence analysis was performed (Bionic, South Korea) using the mini-Tn5Km2 transposon outside-end (O=-end) primer (5=-CCTCTAGAGTCGACCTGCAG-3=). The DNA sequences flanking the transposon insertion were assembled, and homology searches were carried out using the public BLASTn and BLASTx databases at http://www.ncbi.nlm.nih.gov.
Determination of the intracellular growth efficiency of mutants. The intracellular bacterial growth assays were performed as described previously (19,20). Briefly, RAW264.7 and HeLa cells were seeded at 10 5 cells/well into a 24-well tissue culture plate and infected with wild-type IVKB9007 and its isogenic mutants at a multiplicity of infection (MOI) of 20. The plates were centrifuged at 150 ϫ g for 10 min at room temperature and then incubated at 37°C in 5% CO 2 for 1 h. The nonadherent bacteria were removed by washing the plates three times with CM without antibiotics, and then cells were incubated with CM containing 50 g/ml GM to kill extracellular bacteria. The infected cells were washed twice with CM, the medium was replaced with CM containing 10 g/ml GM, and the plate was incubated at 37°C for 0, 4, 24, and 48 h. At different times , the infected cells were washed three times with sterile PBS and lysed with 0.1% Triton X-100 in sDDW. Serial dilutions of the lysates were plated on TSA agar to enumerate the CFU. These assays were performed at least three times, and the means Ϯ standard deviations (SD) were calculated. Measurement of sensitivity of mutants to environmental stresses. The parental IVKB9007 strain and mutants were evaluated for sensitivity to stress conditions and for acid and oxidative-stress tolerance. To measure sensitivity to hydrogen peroxide (H 2 O 2 ), a hydrogen peroxide agar diffusion assay was conducted (9). Briefly, bacterial cultures (optical density at 600 nm [OD 600 ] of 0.8) were pelleted by centrifugation, and the pellets were washed twice in PBS and resuspended in PBS at a concentration of approximately 10 8 CFU/ml. One hundred microliters of each culture was spread on TSA plates in triplicate. Five microliters of 30% H 2 O 2 was spotted onto 6-mm-diameter sterile blank paper (BD Bioscience, NJ, USA), and the paper was placed at the center of each plate. After incubation for 3 days at 37°C, the diameter of the zone of bacterial clearance around each disk was measured.
To determine the sensitivity to acid, bacteria were grown to early stationary phase (OD 600 , 0.8) at 37°C. The cultures were collected and then pelleted by centrifugation at 12,000 ϫ g for 5 min at room temperature, and the pellets were resuspended in TSB adjusted to pH 7.0, 4.5, or 3.5 with concentrated HCl. After 3 h of incubation with agitation at 37°C, CFU were counted by plating of serial dilutions on TSA plates. The assay was repeated at least three times independently, and the means Ϯ SD were calculated.
Assessment of sensitivity to heavy metals and respiratory chain inhibitors was performed as described previously (9). Early-stationary-phase bacteria were serially diluted and plated in triplicate on TSA or TSA supplemented with zinc sulfate (ZnSO 4 ; Sigma) and sodium azide (NaN 3 ; Sigma). Bacterial survival was determined after 5 days of incubation by enumerating the number of bacteria grown on plates of TSA and TSA containing ZnSO 4 /NaN 3 . Experiments were performed at least three times, and the means Ϯ SD were calculated.
Persistence of B. abortus mutants in mice. The virulence in vivo of the wild-type B. abortus IVKB9007 strain and mutants was determined by measuring the persistence of the strains in the spleens of mice after 1, 2, 3, and 7 weeks p.i. All mice were kept under controlled conditions in a micro ventilation cage system (MVCS; Threeshine, Daejeon, South Korea). The animal experiments were approved by the Animal Research Committee of Kangwon National University. Female 8-week-old BALB/c mice were inoculated intraperitoneally (i.p.) with 2.0 ϫ 10 5 CFU/mouse of the wildtype IVKB9007 strain, the IVKB9007 looP::Tn5 mutant, the IVKB9007 cydC::Tn5 mutant, or the complemented IVKB9007 C-looP::Tn5 or C-cydC::Tn5 mutant or with 2.5 ϫ 10 8 CFU/mouse of rough vaccine strain RB51 in 0.2 ml of PBS. As a control, one group (n ϭ 5) of mice was inoculated with sterile saline. At 1, 2, 3, and 7 weeks p.i., mice from each group (n ϭ 5) were euthanized by cervical dislocation, and their spleens were removed aseptically and weighed. The spleens were homogenized in 1 ml sterile PBS using a TissueLyser system (Qiagen, Valencia, CA, USA), and spleen homogenates were serially diluted in PBS and plated in duplicate on TSA agar. Plates were then incubated for 3 to 5 days at 37°C with 5% CO 2 to assess spleen colonization.
Protective efficacy of the mutants against challenge with strain 544. At 5 weeks postvaccination, groups of five mice were challenged i.p. with 1.5 ϫ 10 5 CFU/mouse of virulent B. abortus strain 544. At 2 weeks postchallenge, the mice were euthanized by cervical dislocation, and the spleens were aseptically removed, weighed, and homogenized in 1 ml PBS. Serial dilutions of homogenized spleens were plated in duplicate onto TSA plates or TSA plates supplemented with 50 g/ml KM or 50 g/ml RP. After incubation for 3 to 5 days at 37°C in 5% CO 2 , bacterial colonies were enumerated, and the number of CFU per spleen was calculated. To differentiate between the mutants or RB51 vaccine strain and the challenge strain, different antibiotics were added to TSA according to the antibiotic resistance phenotype of the mutants (Km r ) or the RB51 vaccine strain (Rp r ), and the number of CFU of the challenge strain was determined by subtracting the number of CFU on antibiotic-containing agar from the number of CFU on agar without antibiotics. The degrees of protection were expressed as the mean number of CFU Ϯ SD of B. abortus strain 544 for each mouse group obtained after challenge. Log 10 units of protection (U) were obtained by subtracting the mean log 10 number of CFU for the vaccinated group from the mean log 10 number of CFU for the PBS control group.
Statistical analysis. The bacterial CFU, survival, and morphometric analysis data were analyzed using GraphPad Prism (version 5; GraphPad Software, La Jolla, CA, USA). Analyses of variance (ANOVAs) and Student t tests were performed to compare individual mutant groups with groups infected with complementation strains or the wild-type control and for comparing the vaccine group with unvaccinated controls. P values of less than 0.05 were considered significant.

Isolation of intracellular-survival-defective B. abortus mutants.
To identify B. abortus genes required for virulence and intracellular survival, transposon mutagenesis of a wild-type B. abortus IVKB9007 strain was performed as described previously (6). Nine hundred ninety mutants were screened to select mutants defective in intracellular survival within HeLa cells in at least two independent assays. Finally, B. abortus IVKB9007 looP::Tn5 and cydC::Tn5 mutants were selected because, while they retained a smooth phenotype (data not shown), they displayed the most severe defects in intracellular survival in HeLa cells and were used for further analysis.
Determination of the transposon insertion site in B. abortus mutants. To determine the location of the transposon insertion site in the chromosomal DNA of the mutants, the genomic DNA of the mutants was extracted, digested, and cloned into the pBluescript II KS (ϩ) plasmid. The resulting plasmid DNA was isolated and subjected to DNA sequencing using the O=-end primer of the mini-Tn5Km2 transposon (6). The nucleotide and the deduced amino acid sequences flanking the transposon insertions in each mutant were compared with the Brucella sequence database available at the NCBI (BLAST). The genes of the mutants disrupted by transposon insertion were identified by their homology and demonstrated coding sequences which showed that a mini-Tn5Km2 transposon was inserted in the looP and cydC genes, encoding ATP/GDP-binding protein motif A (P-loop) and the ATP-binding/permease protein, respectively.  Fig. 1, the numbers of intracellular bacteria recovered 0 h p.i. did not differ significantly between the IVKB9007 strain and mutants, indicating that there was no defect in the mutants in internalization and invasion of either cells. However, we found almost a 1-log reduction in the number of intracellular bacteria of the mutant types (B. abortus IVKB9007 looP::Tn5 and cydC and looP Are Involved in Virulence of B. abortus cydC::Tn5 mutants) at 4 h p.i. compared with the number of bacteria of the B. abortus IVKB9007 strain (P Ͻ 0.05). At 24 h p.i., the numbers of CFU of IVKB9007 looP::Tn5 and cydC::Tn5 were dramatically decreased by nearly 1.5 and 2.5 logs, respectively, compared with that of the wild-type IVKB9007 strain (Fig. 1A) (P Ͻ 0.001). At 48 h p.i., the numbers of CFU of the B. abortus IVKB9007 looP::Tn5 and cydC::Tn5 mutants continued to decrease, with approximately 3.4-and 4.5-log reductions, respectively, relative to the number for the wild-type strain (P Ͻ 0.001). Infection with the complemented C-looP::Tn5 and C-cydC::Tn5 mutants indicated that the introduction of intact copies of the looP and cydC genes in the looP::Tn5 and cydC::Tn5 mutants restored their intracellular-survival capability to near the level of the wildtype B. abortus IVKB9007 strain. This result confirmed that the intracellular growth defects of the looP::Tn5 and cydC::Tn5 mutants were caused by disruption of the looP and cydC genes.
To determine whether sensitivity to respiratory inhibitors and intracellular-stress conditions play a role in the attenuation of the IVKB9007 looP::Tn5 and cydC::Tn5 mutants, the survival ability of all strains under stress conditions was determined. As shown in Fig. 2A, the looP::Tn5 and cydC::Tn5 mutants showed increased sensitivity to respiratory inhibitors (P Ͻ 0.001). In particular, the cydC::Tn5 mutant was highly sensitive compared with the parental IVKB9007 strain to the combination of ZnSO 4 and NaN 3 (P Ͻ 0.001). Our results suggest, similarly to the results obtained with E. coli cydDC and B. abortus cydB strains (9,12), that the cydC gene is required for a functional cytochrome bd oxidase of B. abortus.
Oxidative killing was determined by measuring the diameter of the clearance zone around a disk containing 30% hydrogen peroxide. The zone of inhibition for the looP::Tn5 and cydC::Tn5 mutants was significantly larger than the zone of inhibition for the wild-type IVKB9007 strain, indicating that the mutants are more susceptible to oxidative stress (P Ͻ 0.001) (Fig. 2B). Figure 2C shows that the levels of resistance of the B. abortus IVKB9007 wild type and the mutants to pH 7.0 were similar. However, there were remarkable differences between the wild type and the mutants in their sensitivities to low pH. The numbers of CFU of the looP::Tn5 and cydC::Tn5 mutants were significantly reduced by 2.5 and 5 logs at pHs 4.4 and 3.5, respectively (P Ͻ 0.001). The survival abilities of the complemented strains (B. abortus IVKB9007 C-looP::Tn5 and C-cydC::Tn5 strains) of the looP::Tn5 and cydC:: Tn5 mutants with intact cydC and looP genes were restored to nearly the wild-type level at a low pH (Fig. 2). Taken together, these findings indicated that the looP and cydC genes are involved in Brucella stress adaptation, and the intracellular growth defects of the looP::Tn5 and cydC::Tn5 mutants may be caused by increased sensitivity to a harsh intracellular environment.
The B. abortus IVKB9007 looP::Tn5 and cydC::Tn5 mutants are attenuated in BALB/c mice. To assess the persistence and level of splenic colonization of tested strains in mice, the same dose (10 5 CFU) of the wild type, looP::Tn5 mutant, cydC::Tn5 mutant, and complemented C-looP::Tn5 and C-cydC::Tn5 mutants were inoculated i.p. into BALB/c mice. At 1, 2, 3, and 7 weeks p.i., five mice from each group were sacrificed, their spleens were removed, and CFU were counted. As shown in Fig. 3A, a significant difference in CFU numbers between the wild type and the mutants was observed at all times tested. While the wild type colonized and replicated rapidly in the spleen, without dramatic decreases in numbers, the colonization of the looP::Tn5 and cydC::Tn5 mutants in the spleen was significantly reduced at 1 and 2 weeks p.i. (P Ͻ 0.001). The numbers of CFU in the spleen dropped sharply thereafter; in particular, there were no detectable CFU in the spleens of mice inoculated with the cydC::Tn5 mutant at 3 and 7 weeks p.i., whereas nearly 2.6 log CFU of the looP::Tn5 mutant were still detected at 7 weeks p.i. (P Ͻ 0.001).
Spleen weights were compared between the groups. The weights of the spleens in mice that received the looP::Tn5 and cydC::Tn5 mutants were significantly lower than those in wildtype-infected mice during the experimental period (P Ͻ 0.001), as was evident by the lack of splenomegaly (Fig. 3B). Additionally, the decreased virulence of the looP::Tn5 and cydC::Tn5 mutants could be fully restored to wild-type levels by complementation, as shown by the C-looP::Tn5 and C-cydC::Tn5 strains (Fig. 3). From these results, we can conclude that mutations in the looP and cydC genes of B. abortus IVKB9007 conferred the attenuated phenotype and resulted in a major reduction in virulence.
Immunization with the B. abortus mutants conferred protection to mice. To determine whether IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants are able to induce protection against challenge, mice were immunized with B. abortus IVKB9007 looP::Tn5 and cydC::Tn5 mutants and the commercial RB51 vaccine strain. At 5 weeks p.i., these mice were challenged with the virulent B. abortus 544 strain, and the level of protection against challenge provided by the mutants was assessed (Table 2). At 2 weeks postchallenge, BALB/c mice immunized with the looP:: Tn5 and cydC::Tn5 mutants and the RB51 strain had significantly fewer splenic bacteria than nonimmunized mice (P Ͻ 0.001). Mice vaccinated with the RB51 strain and looP::Tn5 mutant exhibited a significant degree of protection (1.51 U and 2.19 U, respectively) against challenge (P Ͻ 0.001). Interestingly, the level of protection following challenge in mice immunized with the cydC:: Tn5 mutant (2.31 U) was higher than that in mice immunized with the looP::Tn5 mutant and RB51 strain, even though it was cleared more quickly from the spleen. Splenomegaly postchallenge was significantly reduced following vaccination with either the looP::Tn5 or the cydC::Tn5 mutant compared with that of RB51-vaccinated (P Ͻ 0.001) and nonvaccinated (P Ͻ 0.001) mice, resulting in significantly lower levels of 544 infection. Taken together, these results suggest that both mutants, especially the cydC::Tn5 mutant, are very safe and show protective ability against virulent B. abortus challenge.

DISCUSSION
A virulence factor is defined as any factor that contributes to the ability of the pathogen to colonize, persist, propagate, and cause disease in hosts. Therefore, to better understand the interplay between Brucella and its host that leads to the development and persistence of disease, it is crucial to identify genes encoding putative virulence determinants. The primary objective of this study was to identify B. abortus mutants that are well attenuated, capable of inducing protection against virulent challenge, and able to be used for a live attenuated vaccine. We produced transposon mutations in the B. abortus IVKB9007 field isolate obtained from a cow and compared the resulting mutants with the parental strain for virulence in vitro and in vivo. In addition, we assessed their ability to provide protection against challenge with a virulent strain. We identified among the mutants the looP::Tn5 and cydC:: Tn5 mutants, which have a transposon insertion in the looP and cydC genes, respectively, as meeting the desired criteria.
The P-loop motif is present in ATP/GDP-binding protein Walker A, a member of the ATP-binding cassette (ABC) superfamily of transporters, which are known to be involved in the uptake or secretion of diverse molecules across the bacterial membrane and to be important for survival in the host (21,22). The looP gene has been identified previously in E. coli, Saccharomyces cerevisiae, and Porphyromonas gingivalis, and the motif is commonly found in many nucleotide-binding proteins, including ATPases, ATP synthases, kinases, elongation factors, and myosin (15,16,23). In fact, a mutation within the P-loop ATP-binding motif abolished most of the activity of the ribosome-activated (C) Sensitivity to acidic stress was determined after all strains were exposed for 3 h to TSB adjusted to pH 7.0, pH 4.4, or pH 3.5. The CFU were enumerated on TSA plates. Data points and error bars represent the means of results from three independent trials and their standard deviations. The asterisks indicate a significant difference in mean values from those for the IVKB9007 strain (**, P Ͻ 0.001; *, P Ͻ 0.05).
cydC and looP Are Involved in Virulence of B. abortus ATPase and reduced its intrinsic ATPase activity, leading to increased oxidative stress and increased sensitivity to respiratory inhibitors (15,16,(23)(24)(25)(26). However, the role of ATP/GDP-binding protein motif A (P-loop) in the virulence of brucellae is still unknown.
The B. abortus cydDC genes are located upstream of cydAB, a gene cluster encoding a high-oxygen-affinity cytochrome bd terminal oxidase (9). This experimental finding is consistent with observations for the E. coli cydDC genes, which encode a heterodimeric, membrane-bound ABC type of transporter that is required for the cydAB-encoded cytochrome bd terminal-oxidase assembly (13,27). This transporter functions in energy conservation under microaerobiosis and protects the cell from oxidative stress and respiratory inhibitors (28)(29)(30). The cytochrome bd terminal oxidase is used by Brucella species to survive inside host cells, where it contributes to intracellular replication (9,31). It has been previously demonstrated by Kohler et al. that mutation of Brucella suis cydD results in defective intracellular growth in cultured macrophages, but its characterization and attenuation in vivo have yet to be reported (8). Moreover, the cytochrome bd oxidase-deficient cydB mutants of B. abortus and B. suis display very different phenotypes in vitro and in vivo (9,31): while the B. abortus cydB mutant was attenuated, the B. suis cydB mutant was hypervirulent. These results suggested a differential use of terminal oxidases in these two species. Therefore, the roles of the cydDC genes in the virulence of B. suis and B. abortus may differ and should be further evaluated.
In this study, we found that the looP::Tn5 and cydC::Tn5 mutants have a reduced ability compared with that of the parental IVKB9007 strain to survive and proliferate within RAW264.7 and HeLa cells. The mutants also showed heightened sensitivity to respiratory inhibitors, acidic pH, and highly reactive oxygen species. These results suggest that disruption of the looP and cydC genes impaired the ability of the IVKB9007 strain to cope with different environmental stresses and to reach its intracellular replicative niche within the host cell. Similarly to a previously published B. abortus cydB mutant (9), our B. abortus mutant lacking cydC displayed a sensitivity to environmental stressors that was significantly greater than that of the parental IVKB9007 strain. In addition, we found that the virulences of the looP::Tn5 and cydC:: Tn5 mutants were significantly attenuated in mice. The decrease in intracellular numbers of CFU of mutants in vivo is consistent with the intracellular growth defects observed in cell culture. To address the exact role of the looP and cydC genes in B. abortus virulence, complementation strains of the looP::Tn5 and cydC:: Tn5 mutants were constructed. The complemented strains restored the intracellular survival and replication, persistence, and splenomegaly in mice to the level observed in the parental IVKB9007 strain. Therefore, the data indicated that the looP and cydC genes are involved in the virulence of B. abortus.
Of note, the hyperattenuation observed in the cydC::Tn5 mutant was possibly because of a deficiency in cytochrome bd terminal oxidase. Consistently with this, the cydC::Tn5 mutant may be attenuated because of insufficient oxygen exposure and heightened sensitivity to environmental stresses, which is essential for the proliferation and metabolism of Brucella inside the host. Remarkably, while the B. abortus cydB mutant remained viable until 8 weeks p.i. in mice (9), the IVKB9007 cydC::Tn5 mutant was rapidly cleared after 3 weeks p.i.; even the inoculum dose was nearly 10-fold higher than that of the B. abortus cydB mutant.  These observations suggest that cydD, a component of the ABC cassette-type transporter, is required for cytochrome bd oxidase activity and that mutation of cydC has a more deleterious effect on B. abortus virulence than that of the absence of cydB-encoded cytochrome bd oxidase. In fact, however, the exact mechanisms responsible for the rapid clearance of the IVKB9007 cydC::Tn5 mutant are still not known. Since we have not measured either cydAB expression or accumulation of the defective cytochrome terminal oxidases in the mutant, we cannot explain the precise mechanism by which cydC contributes to the cytochrome bd oxidase expression. Notably, a recent study on cydX of B. abortus demonstrated that the promoter controlling the expression of cydAB and cydX is located between the cydDC and cydAB genes (32). Therefore, it is less likely that mutation of cydC will affect the regulation of cytochrome bd expression. In E. coli and other bacterial species where cydDC genes, an ATP-binding cassette type, have clearly been identified, these genes are adjacent but separate from cydAB genes (29,(33)(34)(35). Mutation of either cydD or cydC displayed complex phenotypes in addition to being required for cytochrome bd oxidase assembly or activity. These include the loss of periplasmic c-type cytochrome and transport of glutathione from the cytoplasm to the periplasm (14,29,33). Therefore, it is reasonable to propose that a more severe colonization defect of the IVKB9007 cydC::Tn5 mutant possibly resulted from one of the other phenotypes associated with the lack of glutathione uptake or cytochrome c oxidase assembly.
The differences in survival exhibited by the IVKB9007 looP:: Tn5 and cydC::Tn5 mutants might prove to be potential advantages for vaccine strains. It is possible that the rapid clearance of the IVKB9007 cydC::Tn5 mutant may enhance its safety, but this may provide only modest protection, because it may not produce in vivo some key antigens necessary for the induction of a protective immunity. Therefore, the protective efficacy of the IVKB9007 cydC::Tn5 mutant should be investigated. It is of interest that the IVKB9007 cydC::Tn5 mutant showed better protection than the RB51 vaccine strain, as did the IVKB9007 looP::Tn5 mutant. The reason why both mutants showed better protection than RB51 seems to be related to lipopolysaccharide (LPS) biosynthesis. Both mutants retained the smooth phenotype of the parental IVKB9007 strain, while the RB51 strain has a rough phenotype because it does not have the O side chain of LPS (O-LPS) (36). B. abortus LPS O antigen is known to be an essential virulence factor, and this antigen plays an important role in eliciting protective immunity against challenge infection with virulent B. abortus (36,37). Therefore, it might be explained that the mice vaccinated with either mutant showed higher protection than the mice vaccinated with RB51 because the mice vaccinated with the mutants may have had protective immunity to smooth LPS. The high level of protection provided by both the IVKB9007 looP::Tn5 and cydC:: Tn5 mutants suggests that the mutants retained their immunogenic properties enough to confer protection against wild-type B. abortus infection. Further study should be needed for characterizing the cydDC operon and particularly the cydC mutant as a live vaccine.
In present study, a higher level of protection conferred by the IVKB9007 cydC::Tn5 mutant was surprising, although a precise explanation for this observation is not available at this time. We hypothesize that the IVKB9007 cydC::Tn5 mutant retained all the known virulence factors that may contribute to protective immu-nity, which is similar to the phenotypes observed in Brucella cydBA mutants (38,39). A possible explanation for a slightly lower level of protection observed in mice immunized with the IVKB9007 looP::Tn5 mutant is that disruption of the looP gene may produce unidentified effects on the induction of protective immunity. Future research will be need to elucidate the exact mechanisms of induction of immunity by the mutants.
In summary, we describe two B. abortus transposon mutants, looP and cydC mutants, which exhibit attenuated phenotypes both in cell infection models and in mice and afford better protection than the RB51 vaccine strain. This indicates that both mutants, especially the cydC mutant, might provide useful information for the construction of a new live vaccine candidate against bovine brucellosis.