- Research article
- Open Access
The CO2-dependence of Brucella ovis and Brucella abortus biovars is caused by defective carbonic anhydrases
© The Author(s) 2018
- Received: 3 July 2018
- Accepted: 3 August 2018
- Published: 5 September 2018
Brucella bacteria cause brucellosis, a major zoonosis whose control requires efficient diagnosis and vaccines. Identification of classical Brucella spp. has traditionally relied on phenotypic characterization, including surface antigens and 5–10% CO2 necessity for growth (CO2-dependence), a trait of Brucella ovis and most Brucella abortus biovars 1–4 strains. Although molecular tests are replacing phenotypic methods, CO2-dependence remains of interest as it conditions isolation and propagation and reflects Brucella metabolism, an area of active research. Here, we investigated the connection of CO2-dependence and carbonic anhydrases (CA), the enzymes catalyzing the hydration of CO2 to the bicarbonate used by anaplerotic and biosynthetic carboxylases. Based on the previous demonstration that B. suis carries two functional CAs (CAI and CAII), we analyzed the CA sequences of CO2-dependent and -independent brucellae and spontaneous mutants. The comparisons strongly suggested that CAII is not functional in CO2-dependent B. abortus and B. ovis, and that a modified CAII sequence explains the CO2-independent phenotype of spontaneous mutants. Then, by mutagenesis and heterologous plasmid complementation and chromosomal insertion we proved that CAI alone is enough to support CO2-independent growth of B. suis in rich media but not of B. abortus in rich media or B. suis in minimal media. Finally, we also found that insertion of a heterologous active CAII into B. ovis reverted the CO2-dependence but did not alter its virulence in the mouse model. These results allow a better understanding of central aspects of Brucella metabolism and, in the case of B. ovis, provide tools for large-scale production of diagnostic antigens and vaccines.
Brucella is a genus of gram-negative bacteria of the α-2 subdivision of the class Proteobacteria  that includes the causal agents of brucellosis, a zoonosis producing important economical loses and human suffering in many developing countries . Currently, the genus contains twelve nominal species often showing host preference. Those spp. that were identified early (frequently referred to as the classical Brucella spp.) are Brucella abortus, preferentially infecting cattle, B. melitensis, usually infecting sheep and goats, B. suis, infecting pigs, hares, reindeer ad several wild rodents, B. canis, found in dogs, B. neotomae, isolated from desert wood rats, and Brucella ovis, a non-zoonotic species that is restricted to sheep and causes a condition known as B. ovis ovine epididymitis . More recently, brucellae have been isolated from marine mammals, voles and other wild life vertebrates, and new species proposed [4–8].
Conventional identification of the classical Brucella spp. and biovars has traditionally relied on dye and phage sensitivity, H2S production, urease activity, requirement of 5–10% CO2 atmospheres (0.04% in normal air) for growth (CO2-dependence) and surface antigens . Even though these methods are being rapidly replaced by molecular tests, antigenic structure and CO2-dependence remain of immediate practical interest as these properties affect the conditions for primary isolation and propagation in vitro and the implementation of diagnostic tests. Antigenically, the classical Brucella spp. are divided in two groups: the rough (R) species (B. canis and B. ovis), which carry R-type lipopolysaccharides (LPS), and the smooth (S) brucellae, which carry S-LPS [3, 10]. Concerning CO2-dependence, this is a trait of B. ovis and most strains of B. abortus biovars 1–4 [3, 10]. In both cases, CO2-independent variants may appear with low frequency  and, for obvious practical reasons, some of these variants have been used for B. ovis and B. abortus antigen production [3, 10]. Similarly, B. abortus vaccines have been developed on CO2-independent backgrounds .
The classical Brucella spp. are facultative intracellular parasites able to circumvent early proinflammatory responses and endowed with a type IV secretion system involved in the control of intracellular trafficking [12–15]. Moreover, it is postulated that these bacteria have progressively adapted their metabolism to the nutrients encountered within cells as an essential part of their intracellular strategy [16–21]. In this regard, despite being a notorious phenotype of practical importance, Brucella CO2-dependence has deserved no attention since the demonstration over 60 years ago that CO2-rich atmospheres are not required to reduce oxygen tension and that CO2 is used as a nutrient per se (reviewed in ). Indeed, CO2 assimilation requires carbonic anhydrases (CAs), a group of critically important ubiquitous enzymes distributed into six evolutionary distinct classes named α to η, with the β class present in bacteria . In heterotrophs, CAs are involved in C acquisition via assimilatory and anaplerotic reactions linked to several biosynthetic processes , and CO2-dependence has been related to defects in CA function in several microorganisms [24–30]. However, to the best of our knowledge, the role of CAs in CO2-dependence has not been investigated in Brucella spp. where the information is limited to recent investigations in search for targets for new drugs [31–34]. These investigations have described that B. suis (thus CO2-independent) strain 1330 has two ORFs (BRA0788 and BR1829) that code for β CAs (henceforth Bs1330CAI and Bs1330CAII). Both CAs are predicted to contain all the amino acid residues involved in the catalytic site and, more important, their activity was verified upon purification and found to be better for Bs1330CAII [31–33]. The demonstration that these B. suis ORFs actually code for enzymes with the predicted activity, together with the availability of the genome sequences of both CO2-independent and -dependent Brucella spp. and biovars, open the way to investigate the mechanisms underlying CO2-dependence in Brucella. The aim of the work described here was twofold: to investigate the genetic background behind the Brucella CO2-independent and -dependent phenotypes and, for B. ovis, a species that shows constant CO2-dependence, to construct a CO2-independent strain suitable for vaccine and antigen production.
Bacterial strains and growth conditions
Characteristics of the Brucella strains used in CO 2 -dependence studies
Other relevant characteristics
B. suis 1330 (1)
Virulent; reference strain of biovar 1; ATCC 23444
B. suis 513 (5)
Virulent; reference strain of biovar 5; NCTC 11996
B. abortus 2308W (1)
Virulent; Wisconsin replicate of USDA challenge strain 2308
B. abortus 544 (1)
Virulent; reference strain of biovar 1; ATCC 23448
B. abortus 292 (4)
Virulent; reference strain of biovar 4; ATCC 23451
B. ovis PA (n.a.)a
Virulent; challenge strain used in B. ovis vaccine studies.
B. ovis REO198 (n.a.)a
Attenuated; genome not sequenced; used for R antigen production for serodiagnosis of ovine epididymitis
B. abortus AB0339 (1)
B. abortus AB0127 (3)
B. abortus AB0130 (3)
BoPA-CO 2 mut (n.a.)a
B. ovis PA mutant isolated during routine work at CITA
This work (Ov-2357)
AB0339-CO 2 mut (1)
B. abortus mutant isolated during routine work at University of Navarra
This work (AZB250)
AB0127-CO 2 mut (3)
B. abortus mutant isolated during routine work at University of Navarra
This work (AZB251)
AB0130-CO 2 mut (3)
B. abortus mutant isolated during routine work at University of Navarra
This work (AZB252)
Genomic sequences of B. suis 1330, B. suis 513 (not annotated), B. abortus 544 (not annotated) and B. abortus 292 were obtained from the databases at National Center for Biotechnology Information (NCBI), Kyoto Encyclopedia of Genes and Genomes (KEGG) or The Broad Institute. The genomic sequence of B. abortus 2308W was obtained from the European Nucleotide Archive (ENA) and compared with its sibling 2308 sequence in KEGG. When genomic sequences were not available (B. ovis PA, B. ovis REO198, B. abortus AB0339, AB0339-CO 2 mut , B. abortus AB0127, AB0127-CO 2 mut , B. abortus AB0130, AB0130-CO 2 mut and BoPA-CO 2 mut ) ORFs were PCR amplified and then sequenced. DNA sequencing was carried out by “Servicio de Secuenciación de CIMA (Centro de Investigación Médica Aplicada, Pamplona, Spain)”. Sequence alignments were performed with Clustal Omega.
Plasmid and chromosomal DNA were extracted with QIAprep Spin Miniprep (Qiagen) and Ultraclean Microbial DNA Isolation kit (Mo Bio Laboratories), respectively. When needed, DNA was purified from agarose gels using QIAquick Gel Extraction Kit (Qiagen). Primers (Additional file 3) were synthesized by Sigma (Haverhill, United Kingdom). Restriction modification enzymes were used under the conditions recommended by the manufacturer.
Construction of B. abortus 2308W and B. suis mutants by gene disruption
For the construction of the CAI mutants, an internal region of 323 bp was amplified with oligonucleotides CAI-F1-ins (5′-GAATTTCTATGGATCGGCTGTT-3′) and CAI-R2-ins (5′-CGGTCCTGCGTGTTTTCTAT-3′). The resulting fragment containing an internal region of the ORF was cloned into pCR2.1-TOPO® vector (Invitrogen) to generate plasmid pCR2.1Ba2308WCAI (Additional file 2) and then, sequenced to verify the insertion. After sequencing, this fragment was cloned into the BamHI and XbaI sites of the suicide vector pJQKm . The resulting plasmid pJQKmBa2308WCAI (Additional file 2) was transformed into competent E. coli S17 λpir [38, 39] and transferred into B. abortus 2308W, B. suis 1330 and B. suis 513 by conjugation, where a single crossover led to disruption of the wild type locus. Integrative mutants were selected on a medium containing kanamycin and nalidixic acid or polymyxin and called B. abortus 2308W::pJQKm-CAI, B. suis 513::pJQKm-CAI and B. suis 1330::pJQKm-CAI (Additional file 1). Since the orientation of the insert in the pJQKm vector was known after sequencing, gene disruption was confirmed by detecting PCR products with primers CAI-Fw and M13Fw and primers CAI-Rv and M13Rv.
The CAII mutants were constructed in a similar way. A 302 bp internal fragment of CAII was amplified with oligonucleotides CAII-F1-Ins (5′-CAATGTGGCCAATCTCATTC-3′) and CAII-R2-ins (5′-GCGAATAGCGGATCGAAATA-3′). The resulting fragment was cloned into pCR2.1-TOPO® vector (Invitrogen) to generate plasmid pCR2.1Ba2308W CAII (Additional file 2), sequenced to verify the insertion and subsequently cloned into the BamHI and XbaI sites of the suicide vector pJQKm . The mutants were named B. suis 513::pJQKm-CAII and B. suis 1330::pJQKm-CAII (Additional file 1). Since the orientation of the insert in the pJQKm vector was known after sequencing, the site of the insertion was confirmed by independent PCR rounds with primers CAII-Fw and M13Fw, and primers CAII-Rv and M13Rv. After several attempts, no mutant in B. abortus 2308W CAII was obtained either under normal or CO2-enriched conditions.
Selection of CO2-independent spontaneous mutants
To obtain CO2-independent spontaneous mutants from CO2-dependent bacteria, B. ovis PA and three B. abortus isolates (one biovar 1 and two biovar 3) were plated on TYSA or TSA and incubated at 37 °C without CO2. After 5 days, one colony was picked and the genes encoding CAI and CAII were PCR amplified using primers CAI-Fw and CAI-Rv, and CAII-Fw and CAII-Rv (see Additional file 3). DNA sequencing with these primers and CAI-F1-Sec and CAII-F1-Sec primers (Additional file 3) allowed identification of mutations by comparison with the nucleotide sequence of the parental CO2-dependent strains.
Construction of the plasmid carrying CAIIba2308W and introduction into B. abortus 292 and 544
For the construction of the expression plasmid encoding Ba2308WCAII oligonucleotides CAII-Fw-Gw (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCGCTGCCGTGTTTGAAATCA-3′) and CAII-Rv-Gw (5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAAAGTTCAGGGCGTTTGAA-3′) that contain sequences attB (underlined) were used to amplify CAII and the promoter from B. abortus 2308W. The resulting PCR product was cloned into pDONR223 to generate plasmid pDONOR223Ba2308WCAII (Additional file 2). After sequence verification, the ORF encoding CAII was transferred from pDONOR223Ba2308WCAII to pRH001 . The resulting plasmid, pRH001Ba2308WCAII (Additional file 2) was transformed into competent E. coli S17 λpir and introduced into Brucella strains by conjugation. The clones that had acquired the plasmid were selected by kanamycin resistance and confirmed by PCR using primers CAII-Fw-Gw and CAII-Rv-Gw, and M13F-M13R. The strains were called B. abortus 292 pRH001Ba2308WCAII and B. abortus 544 pRH001Ba2308WCAII (Additional file 1).
Construction of miniTn7T-KmR plasmids carrying CAI or CAII and introduction into Brucella strains
Using DNA from B. abortus 2308W, oligonucleotides CAII-IF-F1 (5′-CCGGGCTGCAGGAATTCGCTGCCGTGTTTGAAATCA-3′) and CAII-IF-R2 (5′-AGCTTCTCGAGGAATTTCAAAGTTCAGGGCGTTTGAA-3′) amplified a 966 bp region containing CAII and the promoter region. This fragment was cloned into the linearized vector (EcoRI) pUC18R6KT-miniTn7T-KmR  using the In-Fusion HD Enzyme Premix (Clontech). The resulting plasmid was called pUC18R6KT-miniTn7T-KmRBa2308WCAII (Additional file 2) and transformed into E. coli PIR1 and subsequently to E. coli S17 λpir. Then, it was transferred into Brucella by a tetraparental conjugation . The resulting constructs (B. abortus 292 Tn7Ba2308WCAII, B. abortus 544 Tn7Ba2308WCAII and B. ovis PA Tn7Ba2308WCAII; Additional file 1) were confirmed by PCR for the correct insertion and orientation of the mini-Tn7 between genes glmS and recG. Primers GlmS_B (5′-GTCCTTATGGGAACGGACGT-3′) and Ptn7-R (5′-CACAGCATAACTGGACTGATT-3′) were used to confirm insertion downstream glmS; Ptn7-L (5′-ATTAGCTTACGACGCTACACCC-3′) and RecG (5′-TATATTCTGGCGAGCGATCC-3′) insertion upstream recG and GlmS_B and RecG presence of transposon.
Oligonucleotides CAI-IF-F1 (5′-CCGGGCTGCAGGAATTTGTGGAATTGCACCGACAC-3′) and CAI-IF-R2 (5′-AGCTTCTCGAGGAATTCAATTATTCTGCCGGTTGG-3′) amplified a 987 bp fragment from B. suis 513 DNA containing CAI and the promoter. This fragment was subsequently cloned into the linearized vector (EcoRI) pUC18R6KT-miniTn7T-KmR using the In-Fusion HD Enzyme Premix (Clontech). The resulting plasmid was called pUC18R6KT-miniTn7T-KmRBs513CAI (Additional file 2) and was transformed into E. coli PIR1 and then to E. coli S17 λpir. After, the plasmid was introduced into the different Brucella strains by a tetraparental conjugation . The strains were called B. abortus 2308W Tn7Bs513CAI, B. abortus 292 Tn7Bs513CAI and B. abortus 544 Tn7 Bs513CAI. The insertion of the transposon was confirmed by PCR (see above and Additional file 3).
When necessary, constructs without kanamycin resistance cassette were obtained following the protocol set up by Martínez-Gómez et al. .
The strains were inoculated into 10 mL of TSB or TYSB in a 50 mL flask and incubated at 37 °C for 18 h with or without orbital shaking, in an atmosphere with 5% CO2 in the case of CO2-dependent strains. Then, these bacteria were harvested by centrifugation, resuspended in 10 mL of the test medium at an optical density at 600 nm (OD600nm) of 0.1, and incubated under the same conditions for 18 h. These exponentially growing bacteria were harvested by centrifugation, resuspended at an OD600nm of 0.1 (equivalent to 0.05 readings in the Bioscreen apparatus) in the test medium in appropriate multiwell plates (200 μL/well) and cultivated in a Bioscreen C (Lab Systems) apparatus with continuous shaking at 37 °C. Absorbance values at 420–580 nm were automatically recorded at 30 min-intervals. All experiments were performed in triplicate. Controls with medium and no bacteria were included in all experiments.
Studies in mice
Seven-week-old female BALB/c mice (Harlan Laboratories; Bicester, United Kingdom) were accommodated in the facilities of “Centro de Investigación y Tecnología Agroalimentaria de Aragón” (CITA; Registration code ES502970012025) for 2 weeks before and during the experiments, with water and food ad libitum under P3 biosafety containment conditions. The animal handling and other procedures were in accordance with the current European (directive 86/609/EEC) and Spanish (RD 53/2013) legislations, supervised by the Animal Welfare Committee of the CITA (2014-20).
To prepare inocula, BABS-grown bacteria were harvested, adjusted spectrophotometrically (OD600nm = 0.170) in sterile buffered saline (BSS; 0.85% NaCl, 0.1% KH2PO4, 0.2% K2HPO4; pH 6.85) and diluted in the same diluent up to approximately 5 × 107 CFU/mL. For each bacterial strain, five mice were intraperitoneally inoculated with 0.1 mL/mouse, the exact doses assessed retrospectively by plating dilutions of the inocula. The number of CFU in spleen was determined at 3 and 8 weeks post-inoculation. For this, the spleens were aseptically removed and individually weighed and homogenized in 9 volumes of BSS. Serial tenfold dilutions of each homogenate were performed, and each dilution was plated by triplicate. Plates were incubated at 37 °C, without CO2, for 5 days. The identity of the spleen isolates was confirmed by PCR. The individual number of CFU/spleen was normalized by logarithmic transformation, and the mean log CFU/spleen values and the standard deviations (n = 5) were calculated. Statistical comparisons were performed by Student’s t-test.
ORF sequences suggest a critical role of CAII in CO2-independence
We first analyzed whether the sequences of Bs1330CAI and Bs1330CAII, respectively encoded by BRA0788 and BR1829 of B. suis 1330 and with proved CA activity, had orthologues in reference and collection strains representative of the CO2-independent and -dependent Brucella phenotypes (Table 1). This analysis showed that all these brucellae carry Bs1330CAI and Bs1330CAII orthologues, with the peculiarities summarized below (for further details, see Additional files 4 and 5).
B. suis 513 Bs1330CAI orthologue differed from Bs1330CAI only at position 40 (serine instead of leucine) and carried a CAII identical to Bs1330CAII. The B. abortus 2308W Bs1330CAI orthologue differed from Bs1330CAI in that the serine at position 40 and valine in position 76 were both substituted by glycine. Similarly, the Bs1330CAII orthologue had an extra amino acid (alanine) at position 114. B. abortus 292 and 544, both CO2-dependent, contained a Bs1330CAI orthologue with the same serine and valine substitutions as strain 2308W, and a cytosine insertion at position 338 of the Bs1330CAII orthologue leading to a frameshift affecting almost 50% of the protein. In the B. ovis PA Bs1330CAI orthologue, a deletion of 24 nucleotides at positions 217–240 results in a protein lacking amino acids 74–81, and the insertion of a guanine at the Bs1330CAII orthologue originates a frameshift and a protein defective in the last 40 amino acids. B. ovis REO198, which is CO2-independent, is identical to B. ovis PA with respect to the Bs1330CAI orthologue. However, the lack of a guanine in the Bs1330CAII orthologue three positions after the 521 position guanine of its B. ovis PA counterpart restores the reading frame and should allow synthesis of a protein identical to that of B. suis 1330 (Additional files 4 and 5). Altogether, the observations strongly suggest that the CO2-dependence of B. ovis PA, B. abortus 292 and 544 is caused by the lack of an active CAII (activity defined empirically as that allowing growth in a normal atmosphere) and, conversely, that mutations in B. ovis CAII could account for the spontaneous emergence of CO2-independent strains in at least B. ovis. On the other hand, these analyses did not allow inferring the relevance of CAI, which was apparently complete in B. abortus and B. suis.
CAII is mutated in spontaneous CO2-independent mutants
We examined first the validity of the hypothesis on the relevance of CAII for growth in normal air by comparing the putative CA genes of several spontaneous CO2-independent mutants that appeared during routine laboratory manipulations with their parental counterparts (Table 1). For the B. ovis PA CO2-independent mutant (BoPA-CO 2 mut ), we observed that while the guanine in the CAII gene causing the above-described frameshift was absent, the CAI gene had not undergone any changes. The CO2-independent mutants of three recent B. abortus isolates (one biovar 1 and two biovar 3)  lacked a guanine at position 340 of the CAII gene that was however present in the CO2-dependent parental isolates. Altogether, these results support the starting hypothesis that CAII mutations are involved in the emergence of CO2-independent mutants and indirectly suggest that CAI is less relevant in the uptake of CO2.
An active CAI is enough by itself to support CO2-independent growth of B. suis but not of B. abortus
The CO2-independence mediated by CAI is conditioned by nutrient availability
An active CAII reverts the CO2-dependence of B. ovis PA and does not alter its multiplication in the mouse model
The metalloenzymes generically designated as CAs catalyze the reversible hydration of CO2 into bicarbonate, the substrate of key anaplerotic and biosynthetic enzymes . Many aerobic microorganisms can obtain enough bicarbonate from ambient air (about 0.04% CO2) and not surprisingly CA mutants of at least Ralstonia eutropha, Streptococcus pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Corynebacterium glutamicum are unable to grow under these conditions [25–30]. All these observations made very likely the existence of a relationship between CA deficiencies and the CO2-dependence that is characteristic of some Brucella biovars and species, a hypothesis proved in this work. If we define CA inactivity as that not high enough to make the bacteria able to grow in a normal atmosphere, we have demonstrated that while B. suis 1330 and 513 genomes encode two active CAs (CAI and CAII) only CAII is active in B. abortus 2308W and none in representative strains of the CO2-dependent B. abortus biovars or in B. ovis PA. Indeed, the fact that the gene encoding CAII is conserved in B. abortus 2308W is in agreement with previous in vitro enzymatic analysis that show that B. suis 1330 CAII is a better catalyst for the conversion of CO2 to bicarbonate (with an enzymatic activity 1.85 times higher) than Bs1330CAI .
It has to be stressed that the functional definition of activity used here pertains to the particular physiology of each strain. Although the number of strains tested was necessarily limited, we found evidence supporting the hypothesis that CAI is insufficient to support growth of Brucella when the medium is limited to simple C substrates, or even in rich media for those brucellae that display comparatively reduced biosynthetic abilities. Indeed, whereas growth on the minimal media used here requires bicarbonate being incorporated by the reactions catalyzed by enzymes such as phosphoenolpyruvate carboxylase, pyruvate carboxylase, carbamoyl phosphate synthetase, 5-aminoimidazole ribotide carboxylase and enoyl-CoA carboxylases/reductases, growth on rich media does not entail an intense biosynthesis of amino acids and nucleic acid precursors and, therefore, most if not all of the linked pathways should pose no stringent demands for bicarbonate. In the context of this hypothesis, B. suis, the fast-growing B. suis biovar 5 (strain 513) in particular, on one hand, and B. ovis, on the other, would respectively represent two opposite situations. The existence of Brucella strains carrying inactivated CAs strongly suggests that this enzymatic activity is not necessary for the persistency in nature of at least B. abortus and B. ovis. Indeed, the presence of mutations inactivating the metabolic genes may result from the absence of a positive selective pressure, reflecting an adaptation during which the cognate functions become dispensable because of the nutritional environment. Such a Brucella adaptation would not be a novelty in intracellular parasites because, while a majority of the genome-sequenced Proteobacteria retain a CA gene, intracellular genera such as Buchnera and Rickettsia contain CA-defective representatives . It remains to be investigated whether such a CA dispensability represents a high CO2 tension in their niche, as described for Symbiobacterium thermophilum , the exploitation of host CAs or the presence of nutrients bypassing metabolic steps connected to CA activity.
It is important to highlight the practical implications of this work. Unraveling the genetic background of Brucella CO2-dependence allowed us to construct a B. ovis CO2-independent mutant with practical implications on the diagnosis and control of B. ovis infection. Because of the non-zoonotic nature of B. ovis, this disease may not always deserve the attention of official programs and it is often overlooked. Control and eventual eradication of B. melitensis brucellosis of small ruminants is based on the use of diagnostic tests detecting antibodies to the S-LPS  and vaccination with B. melitensis Rev 1, a vaccine that also protects against B. ovis. However, Rev 1 may interfere in serological diagnosis  and it is virulent for humans  and resistant to streptomycin (an antibiotic of choice to treat human brucellosis). Owing to these drawbacks, Rev 1 vaccination is discontinued and finally banned in those regions or countries where B. melitensis prevalence is considered low enough to implement an exclusively test and slaughter strategy. Withdrawal of Rev 1 vaccination leaves animals unprotected against B. ovis, thus favoring the emergence of the disease in areas where B. melitensis is almost or totally eradicated. Moreover, B. ovis has remained endemic in many areas where B. melitensis is not present and Rev 1 vaccination was never implemented [51, 52]. Accordingly, research on B. ovis-specific vaccines is an area of increasing interest as these vaccines would neither pose risk of zoonotic infection nor interfere in those B. melitensis serological tests detecting S-LPS O-polysaccharide antibodies (i.e., rose bengal and complement fixation tests) [53–56]. The CO2 requirement represents a significant obstacle in the development of a B. ovis live attenuated vaccine for large-scale production. We have demonstrated that the B. ovis PA Tn7Ba2308WCAII described here not only grows under normal atmospheric conditions but also retains the virulence in at least the accepted laboratory model, thus representing an appropriate tool for the development of such CO2-independent attenuated vaccines. For instance, B. ovis PA Tn7Ba2308WCAII can be used as the background to apply the strategy proposed by Conde-Álvarez et al.  based on the deletion of LPS core glycosyltransferases that results in a truncated structure that by uncovering innate immunity targets triggers a potent protective Th1 response. In fact, Soler-Lloréns et al.  recently demonstrated that deletion of two of such glycosyltransferases in B. ovis PA results in attenuation and suitable vaccine properties in the mouse model. Similarly, the B. ovis PA Tn7Ba2308WCAII construct could be used to produce the R-specific antigen currently used in B. ovis serological tests. This antigen is made of vesicles rich in outer membrane proteins and R-LPS, and both types of components have been shown to be important for optimal sensitivity [58, 59]. Currently, this R antigen is obtained from B. ovis REO198 taking advantage of the unusual CO2-independence of this strain (Table 1). Yet, B. ovis REO198 LPS carries a core oligosaccharide defect that damages the diagnostic epitopes of the R LPS and it is thus likely to yield suboptimal results in serodiagnosis . If this is confirmed, it could be advantageously replaced by B. ovis PA Tn7Ba2308WCAII. Research is in progress to evaluate the attenuation and protection against B. ovis of B. ovis PA Tn7Ba2308WCAII core glycosyltransferase mutants as well as the diagnostic properties of R antigens obtained from this strain. In summary, the evidence presented in this work not only clarifies the biochemical basis of an important Brucella phenotype but also provides a tool for large-scale production of B. ovis diagnostic antigens and vaccines.
The authors declare that they have no competing interests.
IM, MI and AZ-R conceived the study. LP-E, AZ-R, RC-Á, MK, MJM and PMM carried out the experimental work. IM, LP-E and AZ-R wrote the paper. All authors read and approved the final manuscript.
The authors are grateful to F. J. Sangari and J. M. Blasco for helpful discussions and suggestions. The authors thank Sara Serrano and María Uriarte for excellent technical assistance.
This research was supported by MINECO grant AGL2014-58795-C4-1-R (UNAV) and AGL2014-58795-C4-3-R (CITA), as well as by the Institute for Tropical Health funders (Obra Social La Caixa, Fundaciones Caja Navarra and Roviralta, PROFAND, Ubesol, ACUNSA and Artai). LP-E was supported by a grant from the Institute for Tropical Health. Work at CITA was sustained also by Aragon Government (Grupo de Investigación en Desarrollo A13-17D).
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