A study on some aspects of brucella intracellular lifecentral carbon metabolism and trafficking

Abstract

Brucellosis is a disease of terrestrial and marine mammals and an important zoonosis. The ability of brucellae to multiply intracellularly in a variety of cells is the cornerstone of the biology of this pathogen and requires both controlling the intracellular trafficking and using the nutrients available in the replicative niche. This work has been devoted to study both aspects in two brucellae, B. abortus biovar 1 and B. suis biovar 5. During intracellular trafficking, phosphatidylinositol phosphates and calcium levels play an important role and are thus the target of several intracellular pathogens. Although Brucella is able to control its own intracellular trafficking along the phagocytic pathway, the mechanisms involved in this process are not completely understood. In this work, we studied B. abortus proteins with putative inositol-phosphatidylinositol phosphatase or calcium binding domains and we found that deletion of these genes does not generate attenuation. Therefore, an interference of Brucella with cell signaling molecules related to inositol phosphates either does not take place or needs cooperation of unknown factors. Likewise, interference with calcium levels as a means to divert intracellular trafficking is unlikely to be relevant in Brucella intracellular life. With reference to metabolism in B. abortus, inositol has been related to Brucella intracellular lifestyle. However, our results showed that mutants in genes mosC, iolG and rpiR, putatively involved in inositol catabolism, were not attenuated in macrophages and mice. Concerning the glyoxylate cycle, we found that this pathway was not essential for Brucella virulence, in contrast to other intracellular pathogens. Regarding gluconeogenesis, our results indicated that phosphoenolpyruvate was synthesized via pyruvate dikinase (Ppdk) but not via phosphoenolpyruvate carboxykinase (PckA), and that the first enzyme was essential for Brucella virulence. Besides, synthesis of fructose 1,6 bisphosphate by Fbp or GlpX was not required in vivo. However, similar experiments suggested a different metabolic picture for B. suis biovar 5. Finally, vaccination experiments indicated that the B. abortus ppdk mutant induces a protective immune response similar to the one induced by the reference vaccine S19 but with a lower residual virulence. Attenuation by ppdk disruption was reproducible in B. suis biovar 5, suggesting that ppdK related attenuation is useful for the production of new vaccines against brucellosis.