Polymorphisms inBrucellaCarbonic anhydrase II mediate CO2dependence and fitnessin vivo

Abstract

AbstractSomeBrucellaisolates are known to require an increased concentration of CO2for growth, especially in the case of primary cultures obtained directly from infected animals. Moreover, the differentBrucellaspecies and biovars show a characteristic pattern of CO2requirement, 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-independentBrucellastrains we have confirmed that carbonic anhydrase II (CA II), is the enzyme responsible for this phenotype in all theBrucellastrains tested.Brucellaspecies contain two carbonic anhydrases of the β 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 CO2requirement 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 easilyin vitro, although with a low frequency ranging from 10−6to 10−10depending 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, growthin vivoin a mouse model of infection provides a significant advantage to the CO2-dependent strain. This could explain why someBrucellaisolates are CO2-dependent in primary isolation. The polymorphism described here also allows thein silicodetermination of the CO2requirement status of anyBrucellastrain.