Construction And Biological Characterization Of Brucella Abortus Pyk And Ppdk Mutants

Brucella abortus is a Gram-negative, facultative intracellular bacterium which causes brucellosis in humans and animals characterized by undulant fever in humans and abortion in animals. Compared to other pathogenic bacteria, Brucella has no classic virulence factors, such as cytolysins, exotoxins, or capsules. Its virulence depends on the ability of invision into and survival within host cells. Recently,one research hotpot is about links between carbon metabolism and virulence for intracellular bacteria.Intracellular pathogenic bacteria may regulate its carbon metabolism to adapt to the environmental conditions encountered in its intracellular replicative niche including low pH, low levels of nutrients starvation and reactive oxygen intermediates. Several studies have been reported in model intracellular pathogenic bacteria like Listeria monocytogenes, Shigella and Mycobacterium tuberculosis, however,little is known for Brucella.In our previous study, we found that two metabolism related genes, pyruvate kinase(pyk, locus tag BAB_RS24320) and pyruvate phosphate dikinase(ppdk, locus tag BAB_RS18420), may be associated with Brucella virulence. In this study, we constructed the pyk and ppdk mutants by homologous recombination and analyzed several important biological characteristics of the mutants in order to validate their roles on Brucella virulence.According to our results, Pyk plays an important role in Brucella growth especially under the nutrition deprivation condition and resistance to oxidative stress rather than resistance to low pH,bovine serum and polymyxin B. Additionally, Pyk is necessary for Brucella intracellular survival and the pyk mutant cannot efficiently exclude the lysosomal degradation within RAW264.7 cells compared with S2308. Espect a markedly reduced survival within macrophages, the pyk mutant has a significant defect in establishing chronic infection in BABL/c mouse. While the ppdk mutant was more sensitive to polymyxin B and natural bovine serum in comprasion with the S2308 strain. Like the pyk mutant, the ppdk mutant showed a reduced intracellular survival and a defect in establishing survival in mouse.In conclusion, we constructed the pyk and ppdk mutants and analyzed their biological characteristics, which suggest that Pyk and Ppdk play crucial roles on Brucella virulence. This research provides important theoretical research materials for carbon metabolism and Brucella virulence, and lays the groundwork for study of Brucella metabolic defect live attenuated vaccine.