pckA-deficient Mycobacterium bovis BCG shows attenuated virulence in mice and in macrophages

Abstract

Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the reversible decarboxylation and phosphorylation of oxaloacetate (OAA) to form phosphoenolpyruvate (PEP). In this study, the regulation of the PEPCK-encoding gene pckA was examined through the evaluation of green fluorescent protein expression driven by the pckA promoter. The results showed that pckA was upregulated by acetate or palmitate but downregulated by glucose. Deletion of the pckA gene of Mycobacterium bovis BCG led to a reduction in the capacity of the bacteria to infect and survive in macrophages. Moreover, mice infected with DeltapckA BCG were able to reduce the bacterial load much more effectively than mice infected with the parental wild-type bacteria. This attenuated virulence was reflected in the degree of pathology, where granuloma formation was diminished both in numbers and degree. The data indicate that PEPCK activity is important during establishment of infection. Whether its role is in the gluconeogenic pathway for carbohydrate formation or in the conversion of PEP to OAA to maintain the TCA cycle remains to be determined.