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The CO₂-dependence of Brucella ovis and Brucella abortus biovars is caused by defective carbonic anhydrases.

ABSTRACT: 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.

SUBMITTER: Perez-Etayo L

PROVIDER: S-EPMC6126018 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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The CO2-dependence of Brucella ovis and Brucella abortus biovars is caused by defective carbonic anhydrases.

Pérez-Etayo Lara L de Miguel María Jesús MJ Conde-Álvarez Raquel R Muñoz Pilar M PM Khames Mammar M Iriarte Maite M Moriyón Ignacio I Zúñiga-Ripa Amaia A

Veterinary research 20180905 1

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 condi ...[more]

PMID: 30185220

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Project description:Some Brucella isolates are known to require an increased concentration of CO2 for growth, especially in the case of primary cultures obtained directly from infected animals. Moreover, the different Brucella species and biovars show a characteristic pattern of CO2 requirement, and this trait has been included among the routine typing tests used for species and biovar differentiation. By comparing the differences in gene content among different CO2-dependent and CO2-independent Brucella strains, we have confirmed that carbonic anhydrase (CA) II is the enzyme responsible for this phenotype in all the Brucella strains tested. Brucella species contain two CAs of the ? family, CA I and CA II; genetic polymorphisms exist for both of them in different isolates, but only those putatively affecting the activity of CA II correlate with the CO2 requirement of the corresponding isolate. Analysis of these polymorphisms does not allow the determination of CA I functionality, while the polymorphisms in CA II consist of small deletions that cause a frameshift that changes the C-terminus of the protein, probably affecting its dimerization status, essential for the activity. CO2-independent mutants arise easily in vitro, although with a low frequency ranging from 10-6 to 10-10 depending on the strain. These mutants carry compensatory mutations that produce a full-length CA II. At the same time, no change was observed in the sequence coding for CA I. A competitive index assay designed to evaluate the fitness of a CO2-dependent strain compared to its corresponding CO2-independent strain revealed that while there is no significant difference when the bacteria are grown in culture plates, growth in vivo in a mouse model of infection provides a significant advantage to the CO2-dependent strain. This could explain why some Brucella isolates are CO2 dependent in primary isolation. The polymorphism described here also allows the in silico determination of the CO2 requirement status of any Brucella strain.

Dataset Information

The CO₂-dependence of Brucella ovis and Brucella abortus biovars is caused by defective carbonic anhydrases.

Publications

The CO<sub>2</sub>-dependence of Brucella ovis and Brucella abortus biovars is caused by defective carbonic anhydrases.

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