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A rapid method to simultaneously separate bacterial and eukaryotic RNA during infections reveals increased intracellular expression of Staphylococcus aureus and Shigella flexneri virulence factors
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DOI：10.1128/spectrum.03745-25.png)
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Abstract
En 中文
Transcriptome analysis has become an increasingly reliable method to assess the response of microorganisms to the environment inside the host cell. However, to maximize the reading depth of the pathogen and host transcriptomes, physical separation of the RNA pools is preferred, particularly to determine intracellular gene expression of the pathogen. Here, we set out to determine the intracellular gene expression of two important pathogens, the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Shigella flexneri. For accurate determination, we developed a rapid method to physically separate bacterial from eukaryotic RNA with a high level of purity. Analysis by RT-qPCR demonstrated that bacterial and eukaryotic RNA could be separated efficiently, enriching the bacterial RNA pool >20-fold. Comparing gene expression of RNA extracted from different purification fractions by RNAseq showed an upregulation of different S. aureus genes. Among these was vraX, which encodes a secreted peptide that binds the C1q protein in the classical complement pathway.
Keywords:
intracellular bacteria
Staphylococcus aureus
Shigella flexneri
macrophages
RNA separation
RT-qPCR
RNAseq
gene expression
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