A rapid method to simultaneously separate bacterial and eukaryotic RNA during infections reveals increased intracellular expression of <i>Staphylococcus aureus</i> and <i>Shigella flexneri</i> virulence factors

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 <i>Staphylococcus aureus</i> and the Gram-negative bacterium <i>Shigella flexneri</i>. 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 <i>S. aureus</i> genes. Among these was <i>vraX</i>, which encodes a secreted peptide that binds the C1q protein in the classical complement pathway.IMPORTANCEInfectious diseases are one of the largest causes of deaths world-wide despite access to antimicrobials and vaccines. To develop new strategies to defeat microbial infections, a greater understanding of the infection process is needed such as analyzing the microbial and host responses during different stages of infection. Several microbes can invade host cells and being able to accurately monitor their and the host cells' gene expression is critical. Here, we have developed an easy and inexpensive method to reliably enrich for and separate bacterial and eukaryotic RNA after an intracellular infection. Our method is applicable to both Gram-negative and Gram-positive bacteria. Using this method, we have identified several genes to be upregulated during S. aureus infection of macrophage cells. Our data could prove useful to obtain new strategies for developing antimicrobial drugs.