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Transcriptome analysis reveals the effect of STAT3 gene of JAK-STAT signaling pathway on apoptosis of Brucella macrophages

ABSTRACT: Brucella is a significant intracellular parasitic bacterium that establishes an infection within a host cell to facilitate its proliferation. While the survival of Brucella in cells is an important manifestation of its virulence, the research on this aspect is not yet complete. Transcriptome sequencing techniques were employed for the analysis of transcript levels of various genes in RAW 264.7 macrophages infected with M5 and wild strains. siRNA can be employed to silence differentially expressed genes, with the subsequent validation of their functions conducted through a variety of techniques, including RT-qPCR, Western blot, flow cytometry, and others.In the study, a significant up-regulation of the STAT3 gene was observed in the vaccine strain when compared with the wild strain. The design of the relevant siRNA to silence STAT3 resulted in the promotion of apoptotic gene expression and the inhibition of apoptosis-inhibiting gene expression, thereby facilitating cell apoptosis.The results demonstrated that the discrepancy in STAT3 gene transcription between the vaccine and wild strains resulted in a reduction in cell damage and a decline in the virulence of the vaccine strain M5. This finding offers novel insights into the intracellular survival mechanism of Brucella and the underlying cause of M5 weakness.

ORGANISM(S): Mus musculus

PROVIDER: GSE284112 | GEO | 2024/12/13

REPOSITORIES: GEO

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Project description:Brucella spp. are gram-negative, facultative intracellular pathogens that cause brucellosis in humans and animals. Iron is an essential element required for all organisms. Iron response regulator (Irr) is a crucial transcriptional regulator and can affect the growth and iron uptake of Brucella. The growth rate of Brucella melitensis M5-90 irr mutant was significantly lower than that of B. melitensis M5-90 under normal or iron-sufficient conditions, however, the growth rate of the B. melitensis M5-90 irr mutant was significantly higher than that of B. melitensis M5-90 under iron-limited conditions. In addition, irr mutation significantly reduced iron uptake under iron-limited conditions. Currently, bhuA (a heme transporter), bhuQ (heme utilization oxygenase Q), and RirA (iron-responsive regulator) have been identified as iron metabolism genes and are regulated by the Irr protein. These results suggest that the Irr protein has multiple target genes in the Brucella genome that are involved in iron metabolism. Therefore, a combination of RNA-seq and Dap-seq was used to investigate the other iron metabolism genes that are also regulated by the Irr protein in Brucella. A total of seven genes were identified as target genes for Irr in this study and the expression levels of these seven genes were similar in both the RNA-seq and qRT-PCR results. Furthermore, the DNA bases C and G and amino acids asparagine, arginine, histidine, and serine were predicted as predominant docking sites for DNA and Irr using the HDOCK server. The electrophoretic mobility shift assay confirmed that six out of the seven genes, namely rirA (BME_RS13665), membrane protein (BME_RS01725), hypothetical protein (BME_RS09560), Iron transporter (BME_RS14525), cation-transporting P-type ATPase (BME_RS10660), and 2Fe-2S binding protein (BME_RS13655), interact with the Irr protein. In summary, our results identified six genes regulated by the Irr protein that may participate in iron metabolism. In addition, from the RNA-seq results, the mutation of irr resulted in the down/upregulation of many genes that are associated with the bacterial secretion system, lipopolysaccharides, and flagellar assembly, and these three components are the main virulence factors for Brucella. Collectively, these results provide valuable insights for the exploration of Brucella iron metabolism and its effects on virulence.

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Transcriptome analysis reveals the effect of STAT3 gene of JAK-STAT signaling pathway on apoptosis of Brucella macrophages

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