Identification Of Host-targeted Targets For The Control Of Brucella Infection By Metabolomic Analysis

Purpose: Brucella causes more than half a million infections each year.Because chronic Brucella infections are extremely difficult to treat,effective treatments are urgently needed.As a specialized intracellular parasitic bacterium,Brucella abortus is strictly parasitic in host cells.Here,we performed metabolomic analyses in Brucella-infected patients to gain a detailed understanding of the metabolic changes caused by Brucella infection and to identify the metabolic pathways essential for Brucella growth.On this basis host-targeted targets for the control of Brucella infection were identified,and these results highlight the potential of functional screens at the histological scale for the discovery of drug targets against bacterial infections.Methods: The metabolite changes in B.abortus-infected patients were analyzed using ultra performance liquid chromatography-mass spectrometry(UHPLC-QE-MS)technique to explore the metabolomic characteristics of B.abortus infection,the modulation of differential metabolites and metabolic pathways,and to validate relevant targets that may affect metabolic pathways;B.abortus 544 was used to infect mouse macrophages with an infection plural of100 RAW264.7 cells,to establish a macrophage infection model;MTT assay to detect the cell viability of mycophenolate ester-treated RAW264.7;c.f.u smear counting method to measure the total number of colonies of mycophenolate ester-treated RAW264.7 cells after co-incubation with B.abortus;real-time fluorescence quantitative PCR to detect IMPDH2 m RNA in RAW264.7 cells.The expression of IMPDH2 m RNA in RAW264.7 cells was measured by real-time fluorescence PCR;the concentration of TNF-α and IFN-γ in RAW264.7 cells was measured by double antibody sandwich ELISA;the protein expression of IMPDH2 in RAW264.7 cells was analyzed by Western Blot.Results: In this study,we have identified possible metabolic pathways of Brucella infection by means of metabolomics.The up-and down-regulation of these metabolic pathways suggests that the pathogen utilizes a wide range of host-derived compounds.It was shown that differential metabolites were significantly enriched for the regulation of host cell energy and the biosynthesis of guanosine-containing compounds.Among the enriched metabolic pathways,the ab initio nucleotide biosynthesis pathway was identified as the significantly altered metabolic pathway.Through the analysis of key host factors,we concluded that IMPDH2 is a promising target for the targeted inhibition of Brucella proliferation.The expression of IMPDH2 m RNA in RAW264.7 cells treated with B.abortus 544 increased significantly compared to the control group,while the expression of m RNA in the mycophenolate ester group decreased significantly(P < 0.05);ELISA experiments showed significantly higher levels of pro-inflammatory factors TNF-α and IFN-γ in the infected group compared to the blank group,and significantly lower levels of TNF-α and IFN-γ after administration(p<0.01).The Western blot confirmed that at the protein level,the expression level of IMPDH2 in the infected group increased significantly compared with that in the blank group,while that in the mycophenolate ester group decreased significantly(P < 0.001);in the experiment to determine the in vitro bacterial load,the intracellular viable bacteria count in the mycophenolate ester group was significantly lower than that in the infected group after 24 h of infestation(P < 0.001).Conclusion: We demonstrate that Brucella increases nucleotide synthesis and metabolism in the host and also identify IMPDH2,a key regulatory complex controlling nucleotide metabolism during Brucella infection.pharmacological targeting of IMPDH2 in guanine nucleotide biosynthesis can effectively inhibit Brucella growth in vitro.