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.
Highlights
Brucella is a genus of gram-negative bacteria of the α-2 subdivision of the class Proteobacteria [1] that includes the causal agents of brucellosis, a zoonosis producing important economical loses and human suffering in many developing countries [2]
(which we had proven to be active) under the control of its own promoter. When we introduced this plasmid into B. abortus 292 and 544, the pRH001Ba2308WCAII constructs were able to grow in a normal atmosphere (Figure 3)
If we define carbonic anhydrases (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 C O2-dependent B. abortus biovars or in B. ovis PA
Summary
Brucella is a genus of gram-negative bacteria of the α-2 subdivision of the class Proteobacteria [1] that includes the causal agents of brucellosis, a zoonosis producing important economical loses and human suffering in many developing countries [2]. 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, 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 [9]. Even though these methods are being rapidly replaced by molecular tests, antigenic structure and C O2-dependence remain of immediate practical interest as these properties affect the conditions for primary isolation and. B. abortus vaccines have been developed on CO2-independent backgrounds [10]
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